Cellular, immunologic and biochemical characterization of topical retinoic acid-treated human skin.

Histologic and clinical improvement of sun-exposed skin following topical treatment with retinoic acid has been reported. Daily application of retinoic acid typically results within 2-5 d in an erythematous scaling reaction, which lessens with continued usage. The cellular, immunologic, and biochemical basis of this retinoid reaction and its role in the repair of photodamaged skin are not known. To investigate the retinoid reaction in man, we have treated non-sun-exposed skin with 0.1% retinoic acid cream (Retin-A, Ortho Pharmaceutical Corporation, Raritan, NJ) under occlusion for 4 d to induce erythema and then examined changes in 1) histology, 2) expression of cell-surface molecules, 3) the enzymes and second messengers of the phospholipase C/protein kinase C signal-transduction system, 4) levels of eicosanoids, and 5) levels of interleukin-1 protein and mRNA. These parameters were chosen for measurement both because they are indicators of epidermal function and previous studies suggest they may be responsive to retinoic acid treatment. Epidermal cell growth as judged by increased epidermal thickness and mitotic figures was significantly increased in retinoic acid-treated skin compared to vehicle-treated controls. Increased numbers of CD4+ T cells accompanied by prominence of dermal dendrocytes in the papillary dermis and focal keratinocyte expression of intercellular adhesion molecule-1 were observed in retinoic acid-treated biopsies. Phosphoinositide-specific phospholipase C activity and 1,2-diacylglycerol content were also elevated in retinoic acid-treated epidermis. Protein kinase C activity was reduced by one third in both the soluble and membrane fraction, suggesting down-regulation. Surprisingly, in view of the inflammatory nature of the retinoid reaction, no increases were observed in arachidonic acid, its metabolites, interleukin-1 alpha, or interleukin-1 beta. To examine the specificity of the retinoid reaction, subjects were treated with the irritant sodium lauryl sulfate, under conditions that resulted in a reaction clinically similar to that observed with retinoic acid. The histologic alterations induced by sodium lauryl sulfate were found to be indistinguishable from those induced by retinoic acid. These data indicate that, although a wide range of cellular and molecular alterations occur in retinoic acid-treated skin, these changes may not be necessarily specific or unique for retinoic acid.

Detection of interferon-gamma mRNA in psoriatic epidermis by polymerase chain reaction.

Psoriatic skin lesions contain HLA-DR positive T lymphocytes, and other activation antigens, which suggest that the T cells may be producing lymphokines. Gamma interferon is produced by activated T cells, and its presence in psoriasis has been inferred by the lesional keratinocyte expression of 3 gamma interferon-inducible proteins i.e. HLA-DR, intercellular adhesion molecule-1, and gamma-IP-10. To determine whether gamma interferon is being produced directly in psoriatic lesions, punch biopsies of normal and diseased skin were separated into epidermal sheets and dermal fragments. Total cellular RNA was isolated from each epidermal and dermal compartment, and reverse transcribed followed by amplification of the resultant DNA by polymerase chain reaction. The amplification process involved the use of 5' and 3' primers for gamma interferon, and tumor necrosis factor-alpha, with beta-actin serving as a control. Gamma interferon mRNA, but not tumor necrosis factor alpha mRNA, was detectable in 4 of 5 psoriatic epidermal specimens. Neither mRNA was detectable in any normal skin dermal/epidermal specimens. Gamma interferon mRNA was also detectable in a single psoriatic dermal specimen. If reverse transcriptase was omitted, no polymerase chain reaction products were detected, indicating that the fragments detected were not derived from contaminating genomic DNA. These results indicate that gamma interferon mRNA can be extracted and successfully detected from human psoriatic lesional skin biopsies, using polymerase chain reaction technology. This molecular approach can easily be expanded to measure many other cytokines in both epidermal and dermal locations. The detection of gamma interferon in this clinical setting may be of particular pathophysiological significance because injection of gamma interferon has been reported to induce psoriatic lesions.

Cellular localization of interleukin-8 and its inducer, tumor necrosis factor-alpha in psoriasis.

The importance of immunologic mechanisms in psoriasis has been deduced from the ability of immunosuppressive therapies to ameliorate this common and chronic skin disease. Certainly the histology of psoriatic lesions suggests a dialogue between the hyperplastic keratinocytes and infiltrating T lymphocytes and macrophages. To begin dissecting the cytokine network involved in the pathophysiology of psoriasis, the location, in both epidermal and dermal compartments, of tumor necrosis factor-alpha, interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha at the protein and/or mRNA levels were identified. Tumor necrosis factor-alpha was selected as a potentially key regulatory cytokine, first because it induces cultured keratinocyte interleukin-8, intercellular adhesion molecule-1, and transforming growth factor-alpha production, and second because intercellular adhesion molecule-1 expression by keratinocytes in psoriatic epidermis had been identified previously. Using immunohistochemical localization, tumor necrosis factor-alpha was identified in 12 psoriatic lesions as intense and diffuse expression by dermal dendrocytes (macrophages) in the papillary dermis (without significant staining of endothelial cells, mast cells, or dermal Langerhans cells), and focally by keratinocytes and intraepidermal Langerhans cells. Functional interaction between the dermal dendrocytes and keratinocytes was suggested by the presence of interleukin-8 expression of suprabasal keratinocytes immediately above the tumor necrosis factor-alpha-positive dermal dendrocytes. Interleukin-8 mRNA and transforming growth factor-alpha mRNA were detectable in the epidermal roof of psoriatic lesions, but neither was detectable at the protein or mRNA levels in any normal skin specimens. Treatment of cultured human keratinocytes with phorbol ester (which experimentally produces psoriasiform changes on mouse skin) or tumor necrosis factor-alpha also increased interleukin-8 and transforming growth factor-alpha mRNAs. Further elucidation of the cellular and molecular basis for the genesis and evolution of psoriasis will provide the framework for a better evaluation of the cause and treatment of this skin disease.

Chloroplast gene for Mr 32000 polypeptide of photosystem II in Euglena gracilis is interrupted by four introns with conserved boundary sequences.

The gene for the Mr 32000 herbicide binding polypeptide of photosystem II has previously been mapped to the 5 kbp EcoRI fragment Eco I of Euglena gracilis chloroplast DNA. The nucleotide sequence of 3324 bp of Eco I, containing the psbA locus, has been determined. This locus encodes a polypeptide of 345 amino acids which is co-linear with, and has 86% derived amino acid sequence homology to sequences derived from four higher plants chloroplast psbA loci. The Euglena psbA gene contains four introns of size 435, 443, 434, and 617 bp. The four introns have conserved boundary sequences of the type previously described in the Euglena chloroplast gene (rbcL) for the large subunit of ribulose-1,5-bisphosphate carboxylase (Koller et al., Cell 36, 545-553, 1984).

Detection of multiple, unspliced precursor mRNA transcripts for the Mr 32,000 thylakoid membrane protein from Euglena gracilis chloroplasts.

The psbA gene is the coding locus for a polypeptide of 32 kilodaltons that is involved in electron transport through photosystem II. The 4.9 kilobasepair (kbp) EcoRI restriction endonuclease fragment EcoI from the 145 kbp Euglena gracilis chloroplast DNA was shown to encode psbA. Five transcripts of size 3.1, 2.8, 2.3, 1.8, and 1.2 kilobases were detected by hybridization of psbA probes to nitrocellulose filter blots of electrophoretically separated RNAs. This same pattern was observed when the hybridization probe consisted of only exon sequences from this split gene. A synthetic, intron specific probe hybridized to all RNA precursors except the 1.2 kb mature RNA. These results and psbA DNA sequence data lead to the conclusion that the four higher molecular weight transcripts are unprocessed precursors of the 1.2 kilobase RNA, some of which contain unspliced intervening sequences. There is an increase in psbA transcripts during light induced maturation of the chloroplasts.

Euglena gracilis chloroplast ribosomal RNA transcription units. Nucleotide sequence polymorphism in 5 S rRNA genes and 5 S rRNAs.

The three tandemly repeated ribosomal RNA operons from the chloroplast genome of Euglena gracilis Klebs, Pringsheim Strain Z each contain a 5 S rRNA gene distal to the 23 S rRNA gene (Gray, P.W., and Hallick, R.B. (1979) Biochemistry 18, 1820-1825). We have cloned two distinct 5 S rRNA genes, and determined the DNA sequence of the genes, their 5'- and 3'-flanking sequences, and the 3'-end of the adjacent 23 S rRNA genes. The two genes exhibit sequence polymorphism at five bases within the "procaryotic loop" coding region, as well as internal restriction endonuclease site heterogeneity. These restriction endonuclease site polymorphisms are evident in chloroplast DNA, and not just the cloned examples of 5 S genes. Chloroplast 5 S rRNA was isolated, end labeled, and sequenced by partial enzymatic degradation. The same polymorphisms found in 5 S rDNA are present in 5 S rRNA. Therefore, both types of 5 S rRNA genes are transcribed and are present in chloroplast ribosomes.

Euglena gracilis chloroplast transfer RNA transcription units. Nucleotide sequence analysis of a tRNAThr-tRNAGly-tRNAMet-tRNASer-tRNAGln gene cluster.

The arrangement and the nucleotide sequence of the tRNA genes in the 2.0-kilobase-pair EcoRI restriction fragment EcoQ of Euglena gracilis Klebs, strain Z Pringsheim chloroplast DNA have been determined. This fragment, cloned in pBR325 to form the plasmid pEZC300, contains five tRNA genes. The DNA insert of this plasmid, a known tRNA gene locus (Orozco, E.M., Jr., and Hallick, R.B. (1982) J. Biol. Chem. 257, 3258-3264) has been mapped by Southern gel analysis using a 32P-labeled oligodeoxynucleotide tRNA gene probe. The DNA sequence of 870 base pairs (bp) from EcoQ containing the entire tRNA gene locus was determined. The organization of this tRNA gene cluster on the E. gracilis chloroplast chromosome is tRNAUUGGln-14-BP spacer-RNAGCUSer-175-bp spacer-tRNACAUMet-12-bp spacer-tRNAGCCGly-5-bp spacer-tRNAUGUThr. The tRNAUUGGln and tRNAGCUSer gene sequences are of the opposite polarity as the other three gene sequences, but of the same polarity as the rRNA genes. The tRNAMet gene is a putative initiator tRNA. The five tRNA genes are separated and flanked by A-T-rich spacer sequences. This gene arrangement is consistent with the model that E. gracilis chloroplast tRNA genes are transcribed into multicistronic tRNA precursors. The DNA sequences have been used to deduce the primary and secondary structures of the tRNAs.