Purified Der p1 and p2 patch tests in patients with atopic dermatitis: evidence for both allergenicity and proteolytic irritancy.

Atopic dermatitis has many similarities with allergic contact dermatitis. Previous studies have revealed delayed-type allergic reactions indicating specific cell-mediated immune reactions in subgroups of patients. It has recently been recognized that purified house dust mite major allergens, Der p1 and Der p2 from Dermatophagoides pteronyssinus, exhibit a proteolytic enzyme activity similar to papain and maybe serine proteases (e.g. trypsin), respectively. This opens the possibility that house dust mites apart from an allergic epitope could elicit irritant reactions in atopic skin. We examined cutaneous reactivity to the purified proteins of house dust mite antigens, Der p1 and Der p2, in 36 consecutive patients with atopic dermatitis. We also patch-tested with trypsin and papain, in order to see if these proteolytic enzymes could induce irritant reactions. Twelve patients had type 1 allergy to Der p1 and two of these had type IV reactivity to D. pteronyssinus extract. Positive reactions were observed in another four patients, but they had also irritant reactions to papain and trypsin, indicating that the enzymatic activity may have elicited the reactions. The cutaneous reactivity was not linked to total serum IgE, but the patients with specific allergic patch tests had type I reactions to D. pteronyssinus extract. Our observations indicate that allergic patch tests towards Der p1 and p2 are rare and that irritant reactions from D. pteronyssinus proteolytic activity may be a more common phenomenon when patch-testing atopic dermatitis patients with house dust mite antigen extract.

A polymerase chain reaction-based method for the semiquantitative study of interleukin-8 mRNA in human basophil leukocytes.

We examined a potential method for quantitative analysis of cytokine expression patterns in purified human basophil leukocyte preparations. Basophil mRNA was reverse transcribed and cytokine cDNA levels determined by competitive polymerase chain reaction (PCR) with internal standard cDNAs constructed by site-directed mutagenesis. Co-reverse transcribed glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) cDNA levels were used as an internal control to correct for unequal efficiencies in RNA isolation and reverse transcription. The method was subjected to a statistical validation giving the within series precision of the analysis. This method was used to examine interleukin-8 expression patterns in basophils from different donors. The results from this study revealed increased interleukin-8 mRNA levels after in vitro challenge with a variety of stimuli.

The effect of selection for high-level vector expression on the genetic and functional stability of a single transcript vector derived from a low-leukemogenic murine retrovirus.

Single-gene murine leukemia virus-based retroviral vectors carrying the G418-resistance gene (neo) under transcriptional control of the long terminal repeat were used to study the effect of selection on long-term vector expression in a murine lymphoid cell line, L691. We used two isogenic vectors carrying either a strong or a weak transcriptional enhancer from low-leukemogenic Akv and high-leukemogenic SL3-3 murine leukemia virus, respectively. Effects of G418 selection were studied at the level of vector-transduced cell populations and at the level of single-vector-transduced cell clones obtained without selection for vector expression. Selection for vector expression prior to isolation of cell clones changed the range of vector expression for the two populations of cell clones. Cell clones harboring the Akv enhancer, isolated without selection and then subjected to prolonged growth under selective conditions, exhibited no mutations in the enhancer region or major vector rearrangements although showing increased vector expression in some cases. Our results are discussed in terms of retrovirus-mediated gene transfer strategies employing selection for expression of a selective marker in single-gene or bicistronic vectors with a low- or nonleukemogenic virus-derived backbone.

Lack of correlation between basal expression levels and susceptibility to transcriptional shutdown among single-gene murine leukemia virus vector proviruses.

Integrated retroviruses or retroviral vectors may be transcriptionally inactive although their promoter-enhancer regions are able to direct transcription in the host cell. We have used single-gene retroviral vectors with a long terminal repeat-directed neo marker gene to determine if the level of transcription relates to the susceptibility of a provirus to inactivation. We used two isogenic vectors, carrying long terminal repeats with a strong and a weak transcriptional enhancer derived from SL3-3 and Akv murine leukemia viruses, respectively. Nonselected cell clones of the murine lymphoid cell line L691 with single integrated vector proviruses exhibiting a 20-fold range of initial expression levels were studied. The basal expression level of a given cell clone with a single provirus did not show any pattern of correlation with the long-term stability of expression, as monitored for periods up to 150 days. Our results thus indicate that the inactivation mechanism operates independently of the initial transcriptional activity of the provirus.

A correlation between dexamethasone inducibility and basal expression levels of retroviral vector proviruses.

Identical transcription units inserted at different positions of mammalian chromosomes may vary widely in transcriptional activity. We have used a set of ten cell clones with random unselected single integrations of retroviral vectors to study such position effects. The vector used carries a neo gene driven by the Akv murine leukemia virus long terminal repeat that has only a weak promoter-enhancer activity in the target cell, the lymphoid cell line L691. Under transient expression conditions, the strength of the Akv promoter-enhancer in the L691 cells is increased by dexamethasone. In cell clones with single vector integrations, a correlation is observed between the non-induced expression levels and the degree of dexamethasone induction. The strongest relative induction is found for the integrated vectors with the lowest non-induced expression levels and approaches the inducibility under transient expression. These results indicate that expression levels are composed of distinct contributions from the integrated vector and from the site of integration and are best explained in terms of a model in which the sites of chromosomal integration exert variable positive enhancer effects upon vector transcription.

Expression of monocyte chemotactic and activating factor (MCAF) in skin related cells. A comparative study.

A significant proportion of the infiltrating cells in several inflammatory skin diseases, including psoriasis and allergic contact dermatitis, are monocytes. Additionally, it is known that the cytokine monocyte chemotactic and activating factor (MCAF) can be produced by several cell types present in the skin, suggesting a significant role for MCAF in the accumulation of monocytes during immunological and inflammatory skin reactions. We have recently developed a precise method for quantification of the amount of a specific mRNA species in a given sample and have used this technique to compare specific MCAF mRNA amounts in cultures of human keratinocytes, dermal fibroblasts, endothelial cells, and monocytes, after stimulation with interleukin 1 alpha (IL-1 alpha) for 6 h. Endothelial cells produced very high, monocytes and fibroblasts intermediate, and keratinocytes low amounts of MCAF mRNA. We have also performed time course studies of MCAF mRNA levels in the four cell types. Our findings suggest that the regulation of MCAF mRNA expression in these cells parallels the regulation of the lymphocyte and neutrophil chemotactic factor interleukin 8.

ETH615, a synthetic inhibitor of leukotriene biosynthesis and function, also inhibits the production of and biological responses towards interleukin-8.

ETH615 (4-(2-quinolylmethoxy)-N-(3-fluorobenzyl-phenyl-amino-methyl -4- benzoic-acid), a synthetic inhibitor of leukotriene B4 production and activities, was tested for its effect on the production of and biological responses towards human interleukin-8. We found that ETH615 inhibits lipopolysaccharide-induced (LPS-induced) expression of interleukin-8 messenger-RNA (mRNA) and interleukin-8 production in human peripheral blood mononuclear cells. We also observed that ETH615 completely inhibited interleukin-8 as well as leukotriene B4 directed chemotaxis of human neutrophils in a dose-dependent manner. A moderate effect on fMLP-directed neutrophil chemotaxis was observed. Further, no significant effect on either interleukin-8, leukotriene B4 or fMLP-directed T-cell migration was observed. These results further support the concept of a cytokine-leukotriene regulatory circuit and encourage the establishment of clinical trials testing the effect of ETH615 on inflammatory skin diseases, which are characterized by high levels of interleukin-8 and leukotriene B4 in lesional skin.

The polymerase chain reaction and dermatology. A new technique with important implications for the study of skin inflammation and for diagnostic tests of dermatological disorders.

The polymerase chain reaction is a powerful and versatile tool for the analysis of nucleic acids. Through a reaction imitating in vivo DNA replication, a defined fragment of DNA is repeatedly replicated by a DNA polymerase in an exponential manner. Such selective amplification of a sequence of interest has created new possibilities in molecular biology and related sciences. The basic principle and some of its variations and applications for both qualitative and quantitative analyses of potential interest to dermatologists are described.

Use of the polymerase chain reaction in quantification of interleukin 8 mRNA in minute epidermal samples.

Quantitative studies of cytokine gene expression in vivo are necessary in order to properly describe the cytokine network and to elucidate its role in skin inflammation. Ideally, one should be able to follow cytokine gene expression in epidermal, dermal, and blood compartments. However, such studies are limited by small amounts of available material. Here we report a polymerase chain reaction (PCR) cDNA amplification protocol useful for quantification of specific mRNAs in small skin samples. We found that analysis of dilution series of each sample permitted establishment of quantitative PCR amplification conditions using only picogram to nanogram amounts of total RNA. Cytokine mRNA amounts could then be measured relative to an internal standard species, co-reverse transcribed, and co-amplified with the cytokine species as a measure of cDNA input. Large numbers of samples can be screened rapidly with initial short dilution series identifying cytokine-positive samples and the correct dilution range for each, followed by closer analysis in this range. Epidermal samples obtained through curettage of a small skin area, 2-mm dermal biopsies from the scraped sites, and a few blood drops from the biopsy sites all yielded sufficient RNA for analysis by this protocol. Any mRNA of known sequence can be studied. We analyzed interleukin 8 mRNA levels in more than a hundred epidermal samples from patients and normal test persons and found a variation over several orders of magnitude that seemed to follow the degree of inflammation of the skin.

The effect of second-line antirheumatic drugs on interleukin-8 mRNA synthesis and protein secretion in human endothelial cells.

Interactions between interleukin 8 (IL-8) and endothelial cells play an important role in the emigration of mononuclear cells from the blood into areas of inflammation. We examined the ability of specific second-line antirheumatic drugs to regulate (IL-8) gene expression and protein secretion in interleukin 1 (IL-1) stimulated human umbilical vein endothelial cells and peripheral blood mononuclear cells. The drugs sodium aurothiomalate, D-penicillamine and sulphasalazine were all able to modulate IL-8 mRNA synthesis in and protein secretion from endothelial cells. A bimodal effect was observed: at low concentrations IL-8 was suppressed, whereas higher concentrations resulted in an increased IL-8 production. In endothelial cells, treatment with hydrocortisone led to a linear suppression of IL-8 production in concentrations ranging from 0.5 micrograms/ml up to 500 micrograms/ml. Sulphapyridine, auranofin, hydroxychloroquine and methotrexate, had no effect on IL-8 secretion in endothelial cells. By contrast, 5-aminosalicylic acid induced a threefold increase in the IL-8 release. In peripheral blood mononuclear cells it was only possible to suppress the IL-8 production by hydrocortisone treatment. These results indicate that suppression of IL-8 production in endothelial cells could be an important factor in the mode of action for a number of second-line antirheumatic drugs.

Measurement of hygromycin B phosphotransferase activity in crude mammalian cell extracts by a simple dot-blot assay.

Hygromycin B (Hy) resistance, encoded by the prokaryotic gene hph, is commonly used as a dominant selectable marker for gene transfer experiments in mammalian cells. We describe a simple, quantitative dot-blot assay for measuring the activity in crude mammalian cell extracts of Hy phosphotransferase, the product of the hph gene. The assay shows no cross interference with substrates for neomycin phosphotransferase II, the product of the commonly used marker gene neo; hph and neo may thus be useful as a set of two non-interfering selectable marker and reporter genes for gene transfer experiments in mammalian cells.

Quantitative determination of IL-1 alpha-induced IL-8 mRNA levels in cultured human keratinocytes, dermal fibroblasts, endothelial cells, and monocytes.

The presence of the leukocyte chemotactic cytokine interleukin 8 (IL-8) in psoriatic scales and in epidermal tissue overlying allergic patch test reactions suggests a role for this cytokine in certain inflammatory skin diseases. IL-8 can be produced by several cell types present in the skin. Their relative potentials for IL-8 expression has, however, not yet been studied, due to the lack of convenient methods for quantitative comparison of specific mRNA amounts in different cell types. Using a new method for quantification, we compared specific IL-8 mRNA amounts in cultures of keratinocytes, dermal fibroblasts, endothelial cells, and monocytes, stimulated with interleukin 1 alpha (IL-1 alpha). Endothelial cells produced very high, fibroblasts and monocytes intermediate, and keratinocytes low amounts of IL-8 mRNA. We also studied the time course of IL-8 mRNA levels in the four cell types following IL-1 alpha stimulation, and found a clear difference both in onset and stability of the response. We discuss the different strength of the response at different time points in the cell types analyzed in relation to their possible role in regulation of the normal response to stimulation.

1,25(OH)2-D3 is a potent regulator of interleukin-1 induced interleukin-8 expression and production.

Interleukin 8 (IL-8) is a potent leukocyte chemotactic and activating cytokine produced by keratinocytes, fibroblasts, peripheral blood monocytes (PBMC) and endothelial cells. IL-8 is believed to play an important role in the development of inflammation and is thus an obvious target for therapeutical modulation. We studied the possible effect of an endogenous immune modulator 1,25(OH)2-cholecalciferol (1,25(OH)2-D3) on the IL-1-induced IL-8-production by several types of cells. 1,25(OH)2-D3 inhibited the IL-1-alpha induced IL-8 production and mRNA expression in keratinocytes, fibroblasts and PBMC, but not in endothelial cells. Optimal vitamin concentrations varied between 10(-10) and 10(-11) M. These results suggest a potential role of this hormone in the regulation of chemotactic cytokine production.

RNA purification from epidermal suction blisters.

Certain inflammatory skin diseases are accompanied by increased cytokine concentrations in the epidermis. To determine whether these cytokines are synthesized in the epidermis or exported from underlying tissues, epidermal RNA was analysed for the presence of their messenger RNAs. We report a method for RNA extraction from pure epidermal samples isolated by the suction blister method. The yield of total RNA was sufficient for hybridization experiments (12-38 micrograms per seven blisters, 5 mm in diameter). Using RNA extracted by this method, we have demonstrated the presence of messenger RNA for glyceraldehyde-3-phosphate-dehydrogenase in 13 preparations from suction blisters obtained from tuberculin skin reactions, positive patch test reactions, or normal skin. We did not, however, observe messenger RNA for interleukin 1 alpha or 8 in these preparations.

Determination of transient or stable neo expression levels in mammalian cells.

We report an extension of the neomycin phosphotransferase II dot-blot assay to allow more rapid and sensitive quantitative determination of the neo gene product in crude mammalian cell extracts. Our procedure, based upon the dot-blot assay of Platt and Yang [Anal. Biochem. 162 (1987) 502-514], measures both the enzymatic activity and the protein content of a cell extract by scanning with an enzyme-linked immunosorbent assay reader, using the same sample rather than parallel samples for both measurements. We show this assay to be comparable to the chloramphenicol acetyltransferase assay in sensitivity. Therefore, apart from being a useful selectable marker gene, the neo gene is a convenient reporter gene in studies of stable, as well as transient, expression.

Different relative expression from two murine leukemia virus long terminal repeats in unintegrated transfected DNA and in integrated retroviral vector proviruses.

Results of transient-expression studies have suggested a correlation between tissue-specific pathogenicity of murine leukemia viruses and the relative transcriptional activities of their long terminal repeats in various cell types. To test whether transient-expression ratios are representative of those of integrated proviruses, we developed a system for generation of retroviral transmission vectors differing only in U3. Vectors with the long terminal repeats of leukemogenic SL3-3 and nonleukemogenic Akv viruses were used for infection of a lymphoid cell line. We then compared expression in infected cells with transient expression after DNA transfection. In contrast to a high SL3-3/Akv reporter gene expression ratio in the transient assays, the ratio in stably infected populations was low. Sets of random cell clones from the two infected populations showed wide variation, with a mean value ratio identical to the population ratio but a considerably higher ratio between lowest values. We suggest that the lower expression levels, like transient expression, reflect inherent enhancer strength and that the higher levels represent chromosomal influence. The different pathogenicity, despite the moderate difference in average expression, may then relate to a different capacity for insertional oncogene activation owing to the different inherent enhancer strengths revealed by the transient-expression assays and the least active proviruses.

Graduated resistance to G418 leads to differential selection of cultured mammalian cells expressing the neo gene.

The effects of Geneticin (G418) selection on the growth and survival of cultured mammalian cells expressing the neomycin-resistance gene (neo) were studied by the analysis of cell clones from two retroviral neo vector-infected populations. We found a correlation between the neo expression level and growth rates in medium containing varying G418 concentrations. This relationship permits the use of differential selection schemes for the isolation of rare cells with increased expression. Comparison, by clone sampling, of vector-positive populations before and after selection with a G418 concentration in the range usually used for selection, showed different expression level and vector copy number distributions for the population infected with the vector of lower LTR activity, but not for the other. Such biasing effects of G418 selection may be important when selected cells are used for quantitative studies of gene expression.

The chromosomal arrangement of six soybean leghemoglobin genes.

Clones containing six leghemoglobin (Lb) genes have been isolated from two genomic libraries of soybean. They encompass two independent DNA regions: a 40-kb region containing four genes in the order 5' Lba-Lbc(1)-[unk]Lb-Lbc(3) 3' with the same transcriptional polarity, and another 40-kb region containing two genes in the order 5' Lbc(4)-Lbc(2) 3' with the same polarity. The order in which the Lb genes are arranged in the soybean genome imply that they are activated in the opposite order to which they are arranged on the chromosome. There is a close similarity between corresponding DNA regions outside the Lb genes in the two clusters. Thus, a moderately repetitive DNA element is present in corresponding positions in each cluster. In addition, at least two different non-Lb genes are linked to each Lb gene cluster in corresponding positions. These genes are apparently regulated in a way which differs from that of the Lb genes. The existence of two very similar Lb gene clusters in soybean suggest that soybean may have evolved from an ancestral form by genome duplication.