Phenotypic characterization of skin lesions in urticaria pigmentosa and mastocytomas

In order to identify possible cellular abnormalities in human mastocytosis, sections from 13 urticaria pigmentosa lesions and 5 mastocytomas were compared with 5 normal skin specimens using histochemical, enzyme histochemical and immunohistochemical techniques. All toluidine blue-positive mast cells also reacted with FcRI and c-kit antibodies, almost all stained for tryptase, many for chymase and the myeloid workshop mast cell antibodies, few for FcRII and none for the proliferation marker Ki-67. Urticaria pigmentosa lesions contained fewer epidermal Langerhans cells and a lower percentage of avidin-positive mast cells than mastocytomas and normal skin. Mastocytomas exhibited generally weaker staining for mast cell markers and mostly lacked FcRI-bound IgE on mast cells and Langerhans cells, although the receptor was able to bind IgE in tissue sections. Most of the mast cell antibodies also reacted with other cell types. Only toluidine blue, avidin, tryptase and chymase stains were mast cell specific. Mast cells in mastocytosis thus differed only to a minor degree from normal mast cells, although distinct pathomechanisms may play a role in urticaria pigmentosa and mastocytosis.     

Comparative immunoreactivity of the eosinophil constituents MBP and ECP in different types of urticaria

In order to elucidate the role of eosinophil constituents in urticaria, we investigated major basic protein expression immunohistologically in comparison with that of eosinophilic cationic protein and the low-affinity IgE receptor in lesional and uninvolved skin of different types of urticaria. Eosinophil activation was studied with the markers EG1 and EG2. Different eosinophil constituents were found in all urticarial lesions except those of urticaria pigmentosa. MBP staining tended to be distributed diffusely throughout the tissue, whereas EG1 and EG2 antibodies were located at or close to individual cells. Staining with the low affinity IgE receptor antibody was rare. In uninvolved skin, major basic protein and particularly eosinophilic cationic protein reactivity was found in chronic recurrent urticaria, delayed pressure urticaria and, to a minor degree, in cholinergic urticaria. No correlation was found between antibody reactivity and eosinophil counts. Reactivity with either of the eosinophil constituents is thus a better marker for eosinophil involvement than routine H&E staining of the cells. The demonstration of eosinophil constituents in nonlesional skin of some urticaria patients suggests generalized eosinophil activation in certain subtypes of the disease.     

High affinity IgE receptor (FcεRI) expression on eosinophils infiltrating the lesions and mite patch tested sites in atopic dermatitis

Expression of the high affinity IgE receptor (FcRI) on eosinophils has recently been reported. This led us to evaluate FcRI expression on eosinophils in atopic dermatitis (AD). Double immunofluorescence stainings with an anti-FcRI monoclonal antibody (mAb) and a polyclonal antieosinophil cationic protein (ECP) antibody were performed on lesional biopsy specimens from patients with AD and from patients with bullous pemphigoid (BP) as controls. In AD and BP lesions, 77% and 70% of eosinophils expressed FcRI, respectively. However, the intensity of FcRI staining in AD was much stronger than in BP, suggesting upregulation of FcRI expression on eosinophils in AD. In addition, the eosinophils infiltrating AD lesions were stained strongly with anti-CD23 mAb and anti-IgE antibody. At the sites of mite patch testing in AD, FcRI-, CD23- and IgE-positive eosinophils were observed to the same degree as in the lesions, and a considerable number of mite antigen-bearing eosinophils were detected. FcRI and CD23 were both upregulated on the skin-infiltrating eosinophils in AD and bound IgE molecules.     

PUVA-induced erythema and changes in mechanoelectrical properties of skin. Inhibition by tocopherols

Summary Influence of antioxidants on two phototoxic effects of 8-methoxypsoralen (8-MOP) was studied: erythema and changes in mechanoelectrical properties of skin. -Tocopherol and its analogs with shortened lateral hydrocarbon chains at C₂-atoms of chromane groups (chromanols) were used as antioxidants. -Tocopherol and its analogs inhibited both phototoxic effects of 8-MOP. Inhibition was observed only if antioxidants were present in skin during irradiation. When applied after irradiation these antioxidants produce no inhibitory effect. The antioxidant antierythemal action depends greatly on their concentration. The protective effect is maximal at antioxidant concentrations 2.5·10^-10 – 5·10^-9mol·cm^-2 of skin, at concentrations higher than 5·10^-9mol·cm^-2 the protective action is decreased. The protective effect of antioxidants depends on the irradiation dose.     

Effect of various metals on intercellular adhesion molecule-1 expression and tumour necrosis factor alpha production by normal human keratinocytes

Nickel, cobalt and chromium are metals very often implicated in allergic contact dermatitis. In vivo, keratinocytes, which are the first target cells, can be directly activated to participate in the local reaction, especially through the expression of the membrane antigen ICAM-1, a ligand of the leucocyte antigen LFA-1, and the production of cytokines. Our aim was to assess the effects of sensitizing metal haptens (nickel, cobalt and chromium) compared with the toxic metal cadmium on the induction of ICAM-1 and the production of TNF by epidermal cells. For this purpose, normal human keratinocytes obtained during plastic skin surgery were cultured in low-calcium defined medium (MCDB153) and the metals were used in non-toxic concentrations. Using FACS analysis, ICAM-1 expression was found to be induced only by nickel. This stimulation appeared as early as 24 h after stimulation. All the metals induced a low expression of TNF detectable by immunocytochemistry correlating with the induction of the nuclear stress protein Hsp72 which is closely linked genetically with the TNF locus. However, only Ni²⁺, Co²⁺ and Cr²⁺ induced a significant release of TNF detectable by ELISA after 48 h stimulation. This secretion was lower than that observed with known stimulants such as lipopolysaccharide. These results indicate that the metals studied are able to induce an aggressive cellular effect, and that nickel, by its ICAM-1 induction, may play a major role in the keratinocyte activation state during allergic contact dermatitis.     

Effect of tumour necrosis factor alpha in vivo on human granulocyte oxidative metabolism

Summary Tumour necrosis factor alpha (TNF) effectively stimulates the oxidative metabolism of human PMN in vitro. Moreover, preincubation of PMN with TNF has been shown to result in an altered response of the target cells to subsequent stimulation. In the present study the response of PMN to stimulation in vitro was investigated in patients with metastasizing malignant melanoma receiving bolus injections of recombinant human TNF as therapy. TNF was given daily for 5 days. Blood samples were taken prior to TNF administration on days 1 to 4 and on day 8. Lucigenin-enhanced chemiluminescence (CL) was used as a sensitive measure of granulocyte oxidative metabolism. PMN were stimulated with TNF, TNF, GM-CSF, PMA, opsonized zymosan and f-met-leu-phe. A significant increase in CL responses was detected upon stimulation with TNF, TNF and PMA from day 1 to day 3, whereas no significant changes were observed for the background activity or when GM-CSF or opsonized zymosan were used as stimuli. On day 4 all CL responses returned to the day 1 starting level. A further significant decrease was observed on day 8 upon stimulation with TNF, TNF and GM-CSF. In contrast, the effect induced by f-met-leu-phe reached a maximum on day 4, but the CL response was found to be at the starting level on day 8. The results indicate that TNF induces significant changes in PMN response to distinct stimuli in vivo. Moreover, it may be possible that continous daily infusions with TNF induce a hyposensitization of PMN oxidative metabolism.     

In vitro activation of immune lymph node cell proliferation by photohapten-modified cells in murine contact photosensitivity

Summary Painting of 3,3,4,5-tetrachlorosalicylanilide (TCSA) plus ultraviolet A (UVA) irradiation to the same site induces contact photosensitivity (CPS), but at the same time results in death of the photohapten-modified cells. Using an in vitro immune lymph node cell (LNC) proliferation system, we investigated the mechanism of induction and elicitation of CPS by TCSA painting plus UVA irradiation. The proliferation of LNC from TCSA-photosensitized mice was not augmented by the addition of TCSA-photocoupled syngeneic spleen cells (SC) or epidermal cells (EC), whereas the picryl chloride immune LNC proliferation was activated by trinitrophenyl-coupled (TNP-coupled) SC or EC. While the viability of SC and EC was unchanged even after TNP haptenization, cells showed very low levels of viability after TCSA photohaptenization. This suggests that the inability of photoTCSA-modified cells to activate LNC proliferation is because of their low viability. Nylon wool column purified lymph node T cells from TCSA-photosensitized mice were activated by photohapten-conjugated SC or photohaptenized EC fragments only in the presence of peritoneal macrophages (M). The function of live M was not replaced by interleukin-1 (IL-1), suggesting that M were required for processing and/or presentation of photohapten rather than simply providing IL-1. Our in vitro study implies that photoTCSA-modified cells generated in vivo require intact antigen-presenting cells to effectively induce and elicit the CPS reaction.     

Pro-inflammatory cytokines upregulate the skin immunoreactivity for NGF,NT-3, NT-4 and their receptor,p75NTR in vivo: a preliminary report

Skin and hair follicles are both source and target of various cytokines and neurotrophins (NTs). While several pro-inflammatory cytokines are recognized to alter the expression of NTs and their receptors (NTRs), for example, on brain cells and fibroblasts in vitro, it is unknown whether this also occurs in normal mammalian skin in vivo. As a first step toward exploring this, we studied in murine back skin (C57BL/6) whether intradermally injected interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and interferon- (IFN-) altered the cutaneous immunoreactivity patterns of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), Trk-A, Trk-B, Trk-C and p75NTR and their receptors (TrkA, TrkB, TrkC, p75NTR) on the protein level in situ. By immunohistology, IFN, IL-1, and TNF- as well as a cocktail of all three cytokines increased NGF immunoreactivity (IR) in the proximal outer root sheath and hair matrix of anagen VI pelage hair follicles. The cytokine cocktail upregulated NT-3 and NT-4 IR in the epidermis, increased NT-4 IR in selected cells of the proximal outer root sheath, and also enhanced IR of p75NTR, in the follicular dermal papilla. Therefore, this pilot study provides the first preliminary indications that proinflammatory cytokines upregulate the cutaneous immunoreactivity of NGF, NT-3, NT-4 and their receptor p75NTR in vivo. This raises the question to which extent several of the recognized cutaneous effects of IFN, IL-1 and TNF- are mediated indirectly via modulating the expression of selected NTs and/or NTRs.Holger Bläsing and Sven Hendrix have contributed equally     

Fcγ receptors on keratinocytes in psoriasis

Summary Epidermis in cryostat sections of skin biopsy specimens from patients with psoriasis and from healthy individuals bound bovine erythrocytes (E) sensitized with rabbit IgG antibodies (A)(EA). No binding occurred using E or E sensitized with IgM or F(ab)₂ fragments of IgG. The binding of EA was inhibited by human IgG and by Fc fragments of IgG, whereas human IgA, IgM, albumin, and F(ab)₂ fragments of IgG did not inhibit the binding, indicating the presence of receptors for the Fc part of IgG (FcR).EA bound mainly to stratum spinosum and most strongly above FcR-positive cell infiltrates in dermal papillae. The binding of EA to sections from patients with active psoriasis was stronger than to sections from patients with stationary psoriasis vulgaris. Sections of unaffected skin from patients with psoriasis and healthy individuals also bound EA, but the binding was weaker than to sections of psoriatic lesions. The receptors were sensitive to periodic acid, formaldehyde, and heat.Using immune complexes of horseradish peroxidase (HRP) and rabbit IgG antibodies to HRP, the receptors were localized to the outer aspect of the keratinocytes and to the inflammatory cells in the microabscesses. The strongest binding occurred in the same areas which adhered EA most strongly. FcR on dendritic epidermal cells could not be demonstrated in situ. A monoclonal antibody against FcR also stained the outer aspect of most keratinocytes throughout the epidermis.FcR on keratinocytes support the assumption that these cells contribute to immune reactions in the skin.     

Side gland of Suncus murinus as a new model of sebaceous gland: 5α-reductase,androgen receptor,and nuclear androgen content in male and female animals

Summary The side gland of Suncus murinus is composed of well-developed sebaceous glands in both males and females. We measured the 5-reductase activity, androgen receptor content, and the intranuclear concentrations of testosterone and dihydrotestosterone in this side gland taking it as a new experimental model of human sebaceous glands. Lineweaver-Burk plot analysis suggested the presence of two classes of 5-reductase with different Km for testosterone in the homogenate. The enzyme activity was slightly higher in males than in females in the presence of a high concentration of testosterone. The levels of androgen receptors were approximately 40 and 30 fmol/mg protein in the cytosol and nuclei, respectively. The values did not differ significantly between males and females. The intranuclear concentration of dihydrotestosterone in the side gland was higher than that of testosterone in each sex. The intranuclear level of each of these two androgens in the female side gland was comparable to that in the male side gland despite the fact that the serum level of testosterone was much lower in the female. These data clearly indicate that the side gland is a typical target tissue for androgens in the female as well as in the male. Androgens other than testosterone may serve as precursors of dihydrotestosterone in the female side gland.This work was supported by grant no. 61770752 from the Ministry of Education of Japan     

Receptor-linked adenylate cyclase in the membranes of cultured human epidermal keratinocytes

Summary The -adrenergic receptors, previously shown to be present on the membranes of cultured human epidermal keratinocytes, were found to be functionally coupled to membrane-bound adenylate cyclase. Using membrane preparations, the enzyme could be activated by guanosine triphosphate (GTP), the stable GTP analog GPP(HN)p, and NaF, all of which are known to activate the adenylate cyclase without interacting with membrane receptors. Binding of catecholamine agonists (epinephrine, norepinephrine, and isoproterenol) to the -adrenergic receptors is followed by an increase in the activity of adenylate cyclase. This activation could be reversed (or prevented) by -adrenergic antagonists, but was unaffect by the presence of -adrenergic ligands (either agonists or antagonists). The activation by catecholamines appears to be directly related to receptor occupancy, since the activation constant (K _a) of adenylate cyclase for the three catecholamines was found to be very similar to the equilibrium dissociation constant (K _d) determined from competition binding experiments. The activation of adenylate cyclase under these conditions appears to be restricted to the catecholamine agonists only. The non-catecholamine -adrenergic agonists (salbutamol, terbutaline) did not show any measurable activation of adenylate cyclase, even though these agonists were shown previously to bind to the -adrenergic receptors on keratinocyte membranes with the expected affinities.     

Transforming growth factor β1 inhibits IL-3- and IL-4-dependent mouse connective tissue-type mast cell proliferation

Transforming growth factor 1 (TGF1) is a regulator of cell proliferation and differentiation. Using a mouse peritoneal cell-derived mast cell culture system, we investigated the effects of TGF1 on mast cell proliferation. TGF1 inhibited IL-3- and IL-4-dependent connective tissue-type mast cell proliferation. The effect was concentration dependent: 50% inhibition was observed with 1.0 ng/ml TGF1 and the maximal inhibitory effect (no proliferation), was observed with 10 ng/ml. Flow cytometric analysis suggested that the inhibitory effect of TGF1 was due to blocking of both G₁ and G₂ phases. Both control and TGF1-treated mast cells showed similar histamine release induced by the calcium ionophore, A23187. TGF1 seems to be an important negative regulator of connective tissue-type mast cell proliferation with apparently no appreciable effect on mast cell function.     

Spindelzelliger blauer Naevus mit Lymphknoten-“Metastasen”

Zusammenfassung Nicht nur der zellreiche blaue Naevus, sondern auch seine spindelzellig-fasciculäre Form kann, wie im vorliegenden Falle, mit Lymphknoten-Metastasen einhergehen. Sie finden sich vorzugsweise innerhalb der Lymphknotenkapsel, selten im Parenchym, und die abgesiedelton Geschwulstzellen stimmen mit den Elementen des Hauttumors gestaltlich überein. Ihr Vorkommen im Randsinus bedarf der Überprüfung.Innerhalb der Lymphknotenkapsel können auch epitheloide Gefäßwandzellen in Erscheinung treten, die mitunter Pigment enthalten und gelegentlich größere Zellverbände bilden. Andere Autoren sind der Ansicht, daß es sich bei diesen um Naevuszellen handelt. Die Histopathogenese und die klinische Bedeutung der Metastasen des blauen Naevus werden erörtert.Herrn Prof. Dr. H. Gartmann zum 60. Geburtstag     

Organ culture of psoriatic skin: effect of TGF-α and TGF-Β on epidermal structure in vitro

Summary Normal skin and uninvolved and involved psoriatic skin specimens were maintained in vitro in organ culture. The 3–4 mm punch-biopsied skin specimens were put freely into the culture medium with or without fetal calf serum, under an atmosphere of 95% O₂ plus 5% CO₂, and rotated at 60 rpm at 37C. In the serum-free culture medium (vitamin A-free) granular layers appeared in the involved psoriatic epidermis in culture. Addition of TGF- caused normal skin and uninvolved and involved psoriatic skin specimens to become acanthotic and to degenerate easily almost to the full thickness of the epidermal layer in proportion to increasing concentrations of TGF- as well as with the duration of the culture, but without disappearance of their granular layers. TGF- caused the normal skin and uninvolved psoriatic skin specimens to become thinned without disappearance of granular layers, but caused the involved psoriatic skin specimens to be thinned without appearance of granular layers in serumcontaining medium or with their disappearance in the serum-free medium. TGF- also antagonized the acanthotic and degenerative effect of TGF-. The results suggest that TGF- and TGF- may partially be related to the induction of psoriatic epidermal lesions.     

Amelanotic changes in B 16 melanoma after transplantation to ‘Yellow’ Ay/a mice

Summary B 16 mouse melanoma maintained on nonagouti a/a mice (C 57 Bl 6j subline) was transplanted to Yellow Ay/a mutants. B 16 melanoma has now been maintained for 1 year on the Yellow strain. A microscopic and ultrastructural study of transplanted tumors is described. Several enzymatic activities including tyrosinases are investigated. A marked depigmentation of the B 16 melanoma is noted after its transplantation to the Yellow strain, and melanogenic characteristics of the tumor are modified.This work was supported by I.N.S.E.R.M. Grant ATP 67.78.99