Immunolocalization of scaffoldin, a trichohyalin-like protein, in the epidermis of the chicken embryo.

Recent comparative genomic studies have identified a chicken gene that codes for a trichohyalin-like protein rich in arginine and glutamic acid termed scaffoldin. Immunocytochemistry and immunoelectron microscopy show that this protein is predominantly localized in periderm granules, subcellular structures present in the periderm of the embryonic epidermis of chick scales, beak, claw, and in the sheath of developing and regenerating feathers. This suggests that scaffoldin contributes to the formation of periderm granules and to the soft cornification of the embryonic epidermis before the definitive epidermis is formed. Scaffoldin is absent from the definitive and adult epidermis generated underneath the periderm in scales and in inter-follicular regions. Scaffoldin mixes with corneous beta-proteins (beta-keratins) synthesized in keratinocytes of the transitional layers formed beneath the periderm in the subunguis of the developing claws. Immunoreactivity for scaffoldin is absent in keratinocytes that accumulate corneous beta-proteins such as those of scales, claws, and barbule-barb cells of feathers. Corneous beta-proteins represent the prevalent type of proteins present in adult epidermis of claws, scales, and feathers. These observations indicate that scaffoldin is a protein of transitional epidermal cells of the avian integument and might represent an important component of periderm granules.

Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model.

BACKGROUND: Defects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis. METHODS: Human primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule. RESULTS: The addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80-95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls. CONCLUSIONS: Our findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases.

Impact of filaggrin mutations on Raman spectra and biophysical properties of the stratum corneum in mild to moderate atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is associated with null mutations in the filaggrin (FLG) gene. OBJECTIVE: To assess the impact of FLG null mutations on biophysical properties and the molecular composition of the stratum corneum (SC) in healthy individuals and AD patients. METHODS: A total of 196 French adults, including 97 with a history of mild to moderate AD, were genotyped for the three major European FLG mutations. Components of the natural moisturizing factor (NMF), lipids and water content in the SC were determined using Raman spectroscopy. In addition, trans-epidermal water loss, capacitance and pH of the SC were measured. RESULTS: Stratum corneum concentrations of total NMF, water, ornithine and urocanic acid (UCA) were significantly lower in AD patients than in healthy controls. Null mutations of FLG were detected in 4% of controls and 10% of AD patients. FLG mutations were associated with increased SC levels of lactate, reduced concentrations of most other NMF components and higher disease severity in AD patients. In AD patients without FLG mutations, the content of NMF constituents decreased with increasing disease severity. The concomittant presence of low concentrations of histidine, alanine and either glycine or pyrrolidone-5-carboxylic acid (PCA) in the SC was associated with FLG mutations with 92% specificity. CONCLUSIONS: Our findings suggest a low prevalence of FLG mutations in mild AD and support an important role for filaggrin in determining the physicochemical parameters of the SC. The combined measurement of several filaggrin breakdown products in the SC may be useful to specifically predict the presence of FLG mutations.

Psoriasin (S100A7) is a major Escherichia coli-cidal factor of the female genital tract.

The female urogenital tract requires an efficient defense against bacteria, potentially derived from the adjacent intestinal tract. We have thus sought to identify the factors that protect against Escherichia coli (E. coli) in the female genital tract. Vaginal fluid from healthy human donors consistently killed E. coli in vitro and vaginal epithelium strongly expressed and secreted psoriasin. Psoriasin was constitutively produced in an organotypic vaginal epithelium model, and exposure of these cells to supernatants of E. coli cultures led to an enhanced psoriasin expression. Secreted psoriasin in vaginal fluids accounted for approximately 2.5-3% of total protein. Fractionation of vaginal fluids by high performance liquid chromatography (HPLC) showed that psoriasin co-eluted with a peak of E. coli killing activity. Our data show that normal vaginal fluid contains a powerful intrinsic antimicrobial defense against E. coli and that psoriasin contributes to the innate immune response of the female genital tract.

DNase1L2 suppresses biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus.

BACKGROUND: The formation of biofilms, which is an important step in bacterial colonization, can be inhibited by deoxyribonuclease (DNase)-mediated breakdown of extracellular DNA. We have recently demonstrated that epidermal keratinocytes strongly express DNase1-like 2 (DNase1L2) in a differentiation-associated manner. OBJECTIVES: To determine whether enzymatically active DNase1L2 is present in human stratum corneum and whether it is able to suppress bacterial biofilm formation. METHODS: DNase1L2 was extracted from normal human stratum corneum, immunocaptured and incubated with plasmid DNA. DNA hydrolysis was monitored by gel electrophoresis and ethidium bromide staining. The effect of DNase1L2 on biofilm formation was assayed by cultivation of Pseudomonas aeruginosa and Staphylococcus aureus in the presence or absence of purified recombinant DNase1L2 in microtitre plates and subsequent quantification of biofilm-forming bacteria by crystal violet staining. RESULTS: DNase1L2 was found to be present in an enzymatically active form in the stratum corneum of human skin. In an in vitro assay, purified recombinant DNase1L2 efficiently suppressed the formation of biofilms by P. aeruginosa and S. aureus. CONCLUSIONS: Our data suggest that DNase1L2 is a novel component of the innate antimicrobial defence of the epidermis.

Alternative splicing of caspase-8 mRNA during differentiation of human leukocytes.

Caspase-8 is a key initiator of death receptor-induced apoptosis. Here we provide evidence that caspase-8 expression is subject to posttranscriptional regulation in human leukocytes. Resting peripheral blood lymphocytes preferentially use a distant splice donor site at the 3'-end of caspase-8 exon 8 to generate mRNAs with a truncated open reading frame. When lymphocytes were activated, the expression of caspase-8 variants was shifted to caspase-8/a and b which lack the extension of exon 8. The opposite change of the splicing pattern was found in a neutrophil differentiation model. Promyelocytic HL-60 cells mainly expressed caspase-8 mRNAs with the normal exon 8, but the splicing pattern was changed to the distant exon 8 splice site during DMSO-induced differentiation of HL-60 cells. In spite of the presence of these novel mRNAs, the corresponding translation products were not detectable in either cell type. Our findings suggest that leukocyte differentiation and alternative splicing of caspase-8 pre-mRNA are inter-dependent processes.

Evidence that caspase-13 is not a human but a bovine gene.

Caspase-13 was reported to be a member of the human caspase family of proteases (Humke, E., et al., J. Biol. Chem. 273, 15702-15707, 1998). By contrast, a recent study (Lin, X., et al., J. Biol. Chem. 275, 39920-39926, 2000) could not confirm caspase-13 expression in human tissues. When we searched the GenBank database we found several expressed sequence tags (ESTs) from bos taurus completely matching the published caspase-13 sequence. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that bovine but not human peripheral blood mononuclear cells express caspase-13. From these cells we cloned two bovine caspase-13 splice variants and found that the sequence of the larger variant was identical to the mRNA published by Humke et al. Our findings strongly suggest that the previously published caspase-13 sequence is not of human origin but represents a bovine gene.

Terminal differentiation of human keratinocytes and stratum corneum formation is associated with caspase-14 activation.

Programmed cell death of epidermal keratinocytes (KC) results in the formation of cornified cells, which constitute the outermost skin layer, the stratum corneum. Here we show by reverse transcription-polymerase chain reaction, western blot, and immunohistochemistry that epidermal KC express caspase-14, a member of the caspase family of pro-apoptotic proteases, in a tissue-specific manner. Caspase-14 protein abundance strongly increases during terminal differentiation of KC in vivo and in vitro. Under conditions that lead to stratum corneum formation caspase-14 cleavage products, which indicate proenzyme activation, appeared in the KC lysates. Cleavage of the enzyme was also detected in lysates from normal human epidermis and in extracts of stratum corneum. Our findings demonstrate that caspase-14 is activated during KC differentiation and strongly suggest that it is involved in the formation of the human skin barrier.J Invest Dermatol 115:1148-1151 2000

Caspase-14: analysis of gene structure and mRNA expression during keratinocyte differentiation.

Caspase-14 is expressed in a tissue-specific manner in mouse skin. Here we determined the complete caspase-14 cDNA sequence of human caspase-14 by rapid amplification of cDNA ends. Sequence comparison with a cosmid clone containing genomic DNA revealed that the human caspase-14 gene comprises seven exons. Facultative utilization of a cryptic splice acceptor site within intron 5 leads to the formation of two mRNA species. In situ hybridization of human skin showed that caspase-14 is expressed in the uppermost layer of living epidermal cells, i.e., the granular layer, in hair follicles and sebaceous glands. In vitro caspase-14 transcription was low in subconfluent cultures but strongly increased when keratinocyte differentiation was simulated by maintaining cells at confluence for several days. This transcriptional upregulation was suppressed in the presence of a high extracellular calcium concentration. Our findings show that caspase-14 is regulated at the level of transcription during keratinocyte differentiation in vitro and in vivo.

Melanin binds reversibly to thermostable DNA polymerase and inhibits its activity.

We found that both RNA and cDNA preparations derived from melanocytes contain a RT-PCR inhibitor that copurified with nucleic acids. Investigation of the candidate inhibitor melanin revealed that it potently blocks PCR at concentrations below 200 ng/ml, whereas 100 microg/ml melanin was required to inhibit reverse transcription. Melanin and thermostable DNA polymerase preferentially formed a distinct complex with reduced migration velocity as compared to pure polymerase in nondenaturating polyacrylamide gel electrophoresis. The inhibition of the enzyme by melanin could be reversed by diluting solutions of preformed complexes or by adding excess amounts of other proteins such as bovine serum albumin or dry milk. Our findings demonstrate that melanin is a potent inhibitor of thermostable DNA polymerase in vitro and that the inhibitory effect is conferred by a direct and reversible polymerase-melanin interaction.

001: hepatocyte growth factor/scatter factor inhibits UVB induced apoptosis of human keratinocytes via the PI-3-kinase pathway

UVB-irradiation induces apoptosis in primary keratinocytes (KC) and KC-derived cell-lines A431 and HaCaT. Here we report on the inhibition of UV induced KC-apoptosis by hepatocyte growth factor/scatter factor (HGF/SF). The protective effect of HGF/SF for UVB-irradiated primary KC was observed at concentrations as low as 1 ng/ml HGF and was confirmed by demonstration of the inhibition of nucleosome-release and the activation of caspase-3. In contrast to the observation with primary KC HGF/SF had no effect on the survival of A431 and HaCaT cells after UVB-irradiation, despite the fact that we could demonstrate that these cells functionally express the HGF/SF receptor c-met. When blocking signalling pathways initiated by c-met, we found that the inhibition of the phosphatidylinositol-3-OH (PI-3) kinase by wortmannin or LY294002 led to a total inhibition of the anti-apoptotic effect of HGF/SF, whereas the blockade of the MAP-kinase pathway by PD90859 had no effect. This represents the first demonstration of an involvement of the PI-3 kinase pathway in the anti-apoptotic effect of HGF/SF. In conclusion, our data demonstrate that HGF/SF is able to rescue KC but not autonomously growing KC cell lines from apoptosis induced by UVB. Since in vivo HGF/SF is produced by mesenchymal cells, this mechanism may represent an important paracrine loop in the skin supporting the survival of KC after UV-injury.

Infection with human immunodeficiency virus-1 increases expression of vascular endothelial cell growth factor in T cells: implications for acquired immunodeficiency syndrome-associated vasculopathy.

Alterations in the vascular system and the onset of angioproliferative lesions such as Kaposi's sarcoma (KS) are common traits of human immunodeficiency virus-1 (HIV-1)-infected patients. To investigate possible factors involved in acquired immunodeficiency syndrome (AIDS)-associated vasculopathy and vascular malfunction, expression of vascular endothelial cell growth factor-A (VEGF-A) was analyzed in HUT 78 T lymphocytes upon infection with HIV-1. VEGF-A was found to be increased in supernatants from infected cells as compared with uninfected cells. In addition, VEGF-A mRNA expression and protein secretion were significantly increased in HUT 78 cells incubated with conditioned medium (CM) derived from HIV-1 chronically infected HUT 78 cells (HIV-TCM) as compared with CM from uninfected cells (TCM). Increase of VEGF-A production in T cells was promoted by inflammatory cytokines (IC) present in HIV-TCM, including tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), interleukin-1beta (IL-1beta), and IL-6. These IC that have been shown to be increased in sera of HIV-1-infected patients and to be increased by HIV-1 infection or cell activation in these individuals as well as HIV-TCM also increased VEGF-A expression in primary T lymphocytes. Consistent with this, VEGF-A concentrations were found to be higher in sera of HIV-1-infected patients with (mean, 357.1 +/- 197.9 pg/mL) and without KS (mean, 256.7 +/- 137.5 pg/mL) as compared with uninfected individuals (mean, 188.6 +/- 91.7 pg/mL). These data suggest that increased secretion of VEGF-A by T lymphocytes of HIV-1-infected individuals may induce vascular leakage and stimulate proliferation of vascular endothelial cells, which are hallmarks of AIDS-associated vasculopathy and especially of KS development.

Reverse transcription-polymerase chain reaction products of alternatively spliced mRNAs form DNA heteroduplexes and heteroduplex complexes.

Reverse transcription-polymerase chain reaction (RT-PCR) is frequently used to simultaneously detect mRNA isoforms, which are generated by alternative splicing. Here we characterize two previously unrecognized RT-PCR products of vascular endothelial growth factor (VEGF) RNA. DNA products with apparent sizes of 600 and 1200 base pairs (bp) were detected at high cycle numbers. Heat denaturation of the smaller product and subsequent reannealing revealed that it was a heteroduplex consisting of two different DNA strands. These were identified by DNA sequencing as the amplification products of two VEGF transcripts, i.e. VEGF121 and VEGF165, which differ by the presence of one exon. S1 nuclease analysis showed that this exon is bulged out as a single-stranded loop. Purified heteroduplexes in solution were found to form a 1200-bp DNA product which could be reconverted into 600-bp DNA heteroduplexes by mild denaturation at 70 degreesC. These findings suggest that this product is formed by base pairing of complementary heteroduplex loops and represents a novel four-stranded DNA structure.

Identification of a human cDNA encoding a novel Bcl-x isoform.

Alternative splicing has been shown to generate two isoforms of the apoptosis regulator bcl-x, Bcl-xL and Bcl-xS, in humans. Here we describe the identification and characterization of a third splice variant of the human bcl-x gene. It differs from previously described bcl-x transcripts in two respects: (1) a novel facultative intron is spliced out at the 5' untranslated region and (2) the open reading frame arises from a continuous genomic sequence extending over the splice donor sites utilized by the bcl-xL and bcl-xS transcripts. Since the resulting molecule has an organisation homologous to mouse and rat Bcl-x beta we suggest calling this novel protein human Bcl-x beta. Northern blot analysis revealed that bcl-x beta mRNA is expressed in numerous cell lines. Like Bcl-xL, h-Bcl-x binds to the pro-apoptotic protein Bax, suggesting a functional activity in vivo.

Immunogenic presentation of a conserved gp41 epitope of human immunodeficiency virus type 1 on recombinant surface antigen of hepatitis B virus.

In view of the high antigenic variability of human immunodeficiency virus type 1 (HIV-1), a vaccine against AIDS must induce an immune response to epitopes as invariable as possible among the various virus strains and clones. Previously the highly conserved six amino acid sequence Glu-Leu-Asp-Lys-Trp-Ala (ELDKWA) from gp41, defining the epitope of the human MAb 2F5, was shown to elicit HIV-1-neutralizing antibodies when presented on haemagglutinin of influenza virus. We investigated the immunogenic potential of the MAb 2F5 epitope and two of its major escape epitopes as internal fusions to the hepatitis B virus (HBV) surface antigen (HBsAg). Recombinant HBsAg-HIV proteins produced in the methylotrophic yeast Pichia pastoris self-assembled into 22 nm lipoprotein particles. Mice immunized with these particles developed an anti-HBsAg immune response in a range that is considered to be protective against HBV infection in humans. More importantly, antisera had extremely high titres of antibodies reactive with a structurally flexible form of the HIV-1 epitope, whereby strong cross-reactivity with the escape variants of the epitope was observed. Although HIV-1 gp 160 and the ectodomain of gp41 containing the epitope were significantly recognized, the antisera failed to neutralize HIV-1 in vitro. These data, together with those on the haemagglutinin-ELDKWAS fusion suggest that the ability of the MAb 2F5 epitope to induce neutralizing antibodies depends on the molecular context in which it is presented. Therefore, further characterization of secondary and tertiary structure requirements of the epitope is indispensable for the full exploitation of its potential as a vaccine component.