In situ hybridization analysis of CRABP II expression in sebaceous follicles from 13-cis retinoic acid-treated acne patients.

The aim of this study was to investigate the effects of 13-cis retinoic acid treatment on cellular retinoic acid binding protein II (CRABP II) mRNA expression in sebaceous follicles from acne patients, using in situ hybridization. Biopsies were taken from uninvolved skin areas in close juxtaposition to inflamed comedos before therapy, and at 2-4 or 14-16 weeks of treatment. Paraffin sections were used for in situ hybridization study with riboprobes transcribed from human CRABP II cDNA. After oral treatment with 13-cis retinoic acid, sebaceous glands were reduced in size and atrophic, and the ratio of sebum-free to fully differentiated (sebum-producing) sebocytes was dramatically increased. The CRABP II expression in the sebaceous gland, and to some extent in infundibular structures, was strongly increased compared with the level of expression in the epidermis. The maximum signal was always found in layers of suprabasal sebocytes lacking lipid droplets, but never in the basal layers. These findings indicate a selective activity of 13-cis retinoic acid on CRABP II mRNA expression in the sebaceous glands of acne patients.

Cell kinetics in skin disorders with disturbed keratinization.

A relatively simple immunohistochemical method was developed and used on cryostat sections. The monoclonal antibody Ki67 was used as marker for actively cycling cells and Pab601 for germinative cells. Counts were expressed as Ki67- or Pab601-positive cells/mm. In order to improve our understanding of the pathogenetic mechanisms in skin disorders with disturbed keratinization we have measured cell kinetic values in dyskeratosis follicularis, pemphigus benigna familiaris chronica, autosomal dominant ichthyosis vulgaris, X-linked recessive ichthyosis, atopic dermatitis and psoriasis and compared them with previous values derived with autoradiography using tritiated thymidine. The results showed that microscopical acanthosis is related to an increase of the germinative population, while the increased epidermal turnover is associated with increased numbers of cycling cells. The cell kinetic changes seem to be all secondary except in psoriasis where a dysregulation in the epidermal growth may cause the epidermal changes. This simple method allows quick evaluation of drug efficacy which might be useful in atopic dermatitis and psoriasis.

Pharmacological studies on the role of protein kinase C in signal transduction of human basophils.

Recent investigations have demonstrated that activation of basophils involves the activation of protein kinase C (PKC). In the present study the effects of different nonselective and selective PKC inhibitors on IgE-mediated histamine release from human basophils were investigated. While potent but nonselective inhibitors such as staurosporine exerted a dose-dependent inhibition of Fc epsilon-receptor-mediated histamine release, staurosporine derivatives with high selectivity for PKC potentiated the IgE-mediated response. The results provide evidence that the histamine release-inhibiting activity of protein kinase inhibitors is inversely correlated with their specificity for PKC. This may confirm the hypothesis that PKC exerts a negative modulatory role during the process of stimulus secretion-coupling following receptor aggregation in basophils. Moreover, investigations with phorbol esters and diacylglycerol derivatives as potent PKC activators show that direct cellular PKC activation and antigen-stimulated mediator release are not closely correlated.

Flow cytometric analysis of epidermal subpopulations from normal and psoriatic skin using monoclonal antibodies against intermediate filaments.

Keratin-type intermediate filament proteins show characteristic expression in normal and pathologic epidermis. Some keratins are restricted to the basal cell layers, and others occur exclusively in the suprabasal compartment. SDS-gel-electrophoresis and immunohistochemistry are generally used for the assessment of keratin profiles and their localizations. In the present investigation, flow cytometric analysis of four different monoclonal antibodies (MAb) against intermediate filament-type proteins, in addition to measurement of relative DNA content, was performed on cell suspensions derived from lesional and clinically uninvolved skin of psoriatic patients and from skin of healthy controls. MAb Ks8.12, reacting with keratins 13 and 16, was used as a marker for hyperproliferation. Pab601 recognizes the basal cell layer(s) of human epidermis. Keratin 10 expression as a marker of keratinization was quantified with RKSE60 and the anti-vimentin MAb MVI was used as a marker for non-keratinocytes. Psoriatic skin showed significantly reduced numbers of RKSE60-positive cells and MVI-positive cells compared with normal skin. In contrast to normal skin and uninvolved skin of psoriatic patients in which only a minority of the cells were Ks8.12 positive, up to 60% of the cell population in psoriatic lesions bound with MAb. Simultaneous measurement of relative DNA content and MAb binding showed that Pab601 binding was associated with cells in S-phase and G2M-phase of the cell cycle, whereas RKSE60 and Ks8.12 binding were associated with diploid cells. Multiparameter flow cytometry allows quantitative population analysis that could lead to a better understanding of the complex mechanisms of epidermal growth control under normal and pathologic conditions.

A sequential double immunoenzymic staining procedure to obtain cell kinetic information in normal and hyperproliferative epidermis.

A sequential double immunoenzymic staining procedure was developed using the monoclonal antibody anti-BrdUrd and Ki67 in order to determine whether hyperproliferative skin disorders, such as psoriasis, are characterized by an increased growth fraction rather than a much shorter cell cycle time of all germinative cells. Ki67 binds to a proliferation-associated nuclear antigen in a variety of human cell types, and anti-BrdUrd can be used to identify DNA-synthesizing cells. Although in hyperproliferative epidermis the absolute numbers of BrdUrd-positive cells as well as Ki67-positive cells were grossly increased, the ratio of these values was not changed compared to the ratio found in the epidermis of the clinically uninvolved skin of psoriatic patients and in normal epidermis. This suggests an increased growth fraction in hyperproliferative epidermis. Our data show that immunohistochemical double-staining techniques can be a valuable tool in the study of cell cycle kinetics in epithelial tissues.

Topical corticosteroids delay the proliferative response to sellotape stripping.

Tape stripping of normal human skin results in a hyperproliferative response brought about by a recruitment of resting (G0) cells. The effect of diprosone and hydrocortisone on this response was studied by flow cytometric techniques. Both corticosteroids were found to delay the appearance of the cohort of recruited cells in the S-phase, the more potent antipsoriatic (diprosone) giving the greatest delay. These data indicate that corticosteroids cause a temporary block in late G1 or an inhibition of the recruitment process itself. The model may serve for the evaluation of (future) antipsoriatic drugs.

Two binding sites for Ki67 related to quiescent and cycling cells in human epidermis.

The monoclonal antibody Ki67 (Ki67) binds to a nuclear antigen expressed by cycling cells of several human tissues and to the cytoplasm of the basal layer cells of squamous epithelia. We have used an immunohistochemical method to visualize the binding sites of Ki67 in normal and hyperproliferative epidermis. Cytoplasmic staining was present in the basal layer cells of normal epidermis, but was decreased in psoriatic and post-tapestripping epidermis. In sections of normal epidermis only a small minority of nuclei were positive, but sections of psoriatic epidermis and epidermis 40 and 48 h after tapestripping showed large numbers of positive nuclei in the basal and suprabasal layers. Since recent reports strongly suggest that the cell production rate is regulated by changes in the number of cycling cells, the hypothesis that Ki67 binds also in human epidermis to the nuclei of cycling cells is supported by the present findings.

A rapid flow cytometric method for bivariate bromodeoxyuridine/DNA analysis using simultaneous proteolytic enzyme digestion and acid denaturation.

This report describes an immunocytochemical procedure for the simultaneous quantification of bromodeoxyuridine (BrdUrd) incorporated into cellular DNA and total DNA content in individual cells in suspension. Improvement of existing methods was achieved by combining acid denaturation and proteolytic enzyme digestion (0.2 mg/ml pepsin in 2N HCl for 30 min at room temperature). Acid denaturation preceded by enzyme digestion resulted in a large amount of debris and the occurrence of naked nuclei. In contrast, the simultaneous denaturation/protein digestion procedure did not damage the cellular structure, is rapid and reproducible, and has cell recoveries of more than 85%. Although experimental conditions were tested on human cultured keratinocytes, this method also appeared applicable to bone marrow cells and cells obtained from solid tissues.

Synchronized growth in human epidermis following tape-stripping: its implication for cell kinetic studies.

A recent investigation of the hyperproliferative activity of human epidermis following sellotape stripping showed a wave of cell divisions with a maximal percentage of cells in mid-S phase at about 39 h after stripping. Here we present a study over the period of 52-76 h following stripping, showing a second wave of cell divisions with a maximal percentage of cells in mid-S at about 63 h. This indicates an average cell cycle time of about 23 h. Human epidermis after tape-stripping provides us with a useful model of synchronized growth, allowing us to study drug influences on cell kinetics accurately.

Recruitment of quiescent (G0) cells following epidermal injury is initiated by activation of the phosphoinositol cycle.

Under normal circumstances, the rate of production of new cells by the epidermis is rather low, but injury results in a burst of mitotic activity that continues until repair is complete. It is now recognized that most cells of the germinative population are in a resting (G0) state, and "postinjury cell renewal" is the consequence of G0 cells entering the mitotic cycle. The biochemical events triggering this process, however, are unknown. Here we show that phorbol myristate acetate (PMA) is able to induce G0 mobilization in the epidermis of the nude mouse. Further, we demonstrate that amiloride (an inhibitor of the membrane Na+-H+ pump), applied topically to human skin, abolishes almost completely the regenerative response after experimental injury. We suggest that activation of the phosphoinositol cycle may initiate recruitment of G0 cells in the epidermis.

Flow cytometric analysis of the recruitment of G0 cells in human epidermis in vivo following tape stripping.

Tape stripping of human skin elicits a proliferative response of a synchronously-dividing group of cells. The progress of this cohort of cells has been monitored using two windows in the cell cycle, one located in mid-S phase and the other centred around G2 + M. The cellular DNA is measured with flow cytometry, the windows are defined by two ranges in the DNA histogram. The cohort can be described as the recruitment of cells from a pre-existing G0 compartment which consists of 76% of all proliferative cells. The duration of the S phase is calculated to be 10.2 hr and G2 + M phase 5.1 hr. The cell cycle time of 39 hr for normal human keratinocytes derived from these figures is in line with recent values obtained by different techniques.

Monoclonal antibodies for epidermal population analysis.

Three keratin antibodies (RKSE 60, Clone 77, and a rabbit polyclonal) and 2 vimentin antibodies (Vim ab and a rabbit polyclonal) were investigated using frozen sections of normal and psoriatic skin. Of these, the monoclonals RKSE 60 and Vim ab were selected for quantitative population analysis of healthy epidermis, psoriatic uninvolved epidermis, and psoriatic lesions. Suspensions of isolated cells were prepared from biopsy specimens by trypsinization, and stained with RKSE 60 or Vim ab using an indirect immunofluorescence assay. Our results showed an increase in the germinative fraction from the normal value of 30% to almost 50% in the psoriatic lesion; in absolute terms this corresponds to a 6-fold increase in the size of the germinative compartment. More interesting, the germinative psoriatic uninvolved epidermis (38%) was also significantly higher than normal. The percentage of vimentin-positive cells (Langerhans cells and melanocytes) was nearly double that of normal in both the lesion and the uninvolved psoriatic epidermis. We conclude that, in contrast to statements frequently encountered in the literature, the "uninvolved" skin of the patient is morphologically and functionally different from that of the healthy individual.

Epidermal hyperproliferation following the induction of microabscesses by leukotriene B4.

The percentage of non-diploid epidermal cells was determined by flow cytometry following application of leukotriene B4 (LTB4) to human skin. Doses in the range 35-500 ng were shown to cause a marked increase in proliferation, the non-diploid cells reaching a maximum between 72 and 96 h after LTB4 application. No difference was observed between the response of healthy controls and the uninvolved skin of psoriatic patients. We suggest, therefore, that the hyperproliferation is a consequence of the physical disruption of the stratum corneum accompanying the rupture of microabscesses.

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.