Structure and transcriptional regulation of the human cystatin A gene. The 12-O-tetradecanoylphorbol-13-acetate (TPA) responsive element-2 site (-272 to -278) on cystatin A gene is critical for TPA-dependent regulation.

Cystatin A, a cysteine proteinase inhibitor, is one of the precursor proteins of cornified cell envelope of keratinocytes and is expressed during the late stage of keratinocyte differentiation. We have isolated and characterized the human cystatin A gene. The cystatin A gene consists of three exons and two introns. The first, the second, and the third exons consist of coding sequences that are 66, 102, and 126 base pairs in length, respectively. The first and the second introns consist of 14 and 3.6 kilobase pairs, respectively. The transcription initiation site was located 55 base pairs upstream from the first translation site. The fragment, +77 to -2595 in the 5'-flanking region of the human cystatin A gene, was subcloned into a chloramphenicol acetyltransferase (CAT) reporter vector. The expression vector, p2672CAT, produced a significant CAT activity in transiently transfected SV40-transformed human keratinocytes (SVHK cells), that were further stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent protein kinase C activator. Sequence analysis of the gene detected three TPA responsive elements (TRE-1, TRE-2, and TRE-3) and one AP-2 site on the 5' upstream promoter region. Deletion analyses of the p2672CAT vector demonstrated that TRE-2, which was located between -272 and -278, was critical for the regulation by TPA. Gel shift analyses revealed that c-Jun, JunD, and c-Fos bound to the TRE-2 region and that the p2672CAT activity level was elevated by co-transfection with c-Jun and c-Fos or with JunD and c-Fos expression vectors. Furthermore, co-transfection of SVHK cells with the protein kinase C-alpha expression vector and the p2672CAT expression vector also resulted in an increased CAT activity. These results indicate that the 5'-flanking region of the human cystatin A gene confers promoter activity and contains a TRE (TRE-2) that mediates, at least in part, the enhanced expression of this gene by TPA.

Cloning of human keratolinin cDNA: keratolinin is identical with a cysteine proteinase inhibitor, cystatin A, and is regulated by Ca2+, TPA, and cAMP.

Keratolinin has been described as one of the precursor proteins of cornified cell envelope of keratinocytes. Using rabbit polyclonal anti-human keratolinin antibody, we isolated a cDNA clone of human keratolinin gene from a human Agt11 cDNA expression library that was constructed by random priming from poly(A)+RNA extracted from cultured normal human keratinocytes. Screening by rabbit anti-human keratolinin antibody detected one positive clone (HKL-1). The recombinant 12.5-kDa protein constructed from the clone reacted specifically with the anti-human keratolinin antibody. DNA sequence analysis revealed that HKL-1 clone was 448 bp long, and its putative amino acid sequence was identical with that of a human cysteine proteinase inhibitor, cystatin A. Western blot analysis showed that the commercially available recombinant cystatin A also reacted specifically with the anti-human keratolinin antibody. Northern blot analysis indicated that HKL-1 clone hybridizes with mRNA of about 0.5 kb, consistent with the size of the HKL-1 clone. The keratolinin mRNA was highly expressed in cultured human keratinocytes in high Ca2+ (1 mM); in low Ca2+ (0.05 mM), the keratolinin mRNA expression was significantly lower. Using SV40-transformed human keratinocytes (SVHK cells), we further analyzed the regulation of keratolinin mRNA. In low Ca2+ (0.05 mM), keratolinin mRNA in SVHK cells was marginally detectable. Upon shift to 1 mM calcium, keratolinin mRNA was markedly increased. The upregulation of keratolinin mRNA was also observed by the treatment of SVHK cells with 10 ng TPA per ml or 100 microM forskolin under low calcium conditions (0.05 mM). Our results indicate that keratolinin is identical with cystatin A, a cysteine proteinase inhibitor, and its expression is positively regulated by Ca2+, TPA, and forskolin.

Gene for familial psoriasis susceptibility mapped to the distal end of human chromosome 17q.

A gene involved in psoriasis susceptibility was localized to the distal region of human chromosome 17q as a result of a genome-wide linkage analysis with polymorphic microsatellites and eight multiply affected psoriasis kindreds. In the family which showed the strongest evidence for linkage, the recombination fraction between a psoriasis susceptibility locus and D17S784 was 0.04 with a maximum two-point lod score of 5.33. There was also evidence for genetic heterogeneity and although none of the linked families showed any association with HLA-Cw6, two unlinked families showed weak levels of association. This study demonstrates that in some families, psoriasis susceptibility is due to variation at a single major genetic locus other than the human lymphocyte antigen locus.

Psoriasis vulgaris: a genetic approach.

Evidence for a genetic contribution in psoriasis comes from direct examination of a large segment of the population in an isolated island environment, epidemiologic and questionnaire studies presented to psoriatic patients, twin studies collected from the literature and from twin registries, and splitsibship analysis. The concordance of psoriasis in monozygotic twins was 65-72%, whereas psoriasis in dizygotic twins was 15-30%. Determination of concordance in older twin pairs from a national twin registry in Denmark revealed nearly 90-100% heritability. In order to link psoriasis with known markers within the human genome, serologic studies have been carried out with a variety of blood group and polymorphic protein antigens. A weak association with the MNS and Lewis Blood Groups Systems (relative risk, 3.5) has been identified. Stronger associations with class I B locus and class II D locus genes (relative risk, 8-12) have also been determined by studies of the human lymphocyte-antigen system. Finally, a strong association with HLA Cw6 has been determined; this marker is thought to be in linkage disequilibrium with B and D locus genes previously associated with psoriasis. The relative risk of developing psoriasis in HLA Cw6 positive individuals is about 24. A few large kindred have been reported in the dermatology literature. These support the hypothesis of autosomal dominant inheritance with penetrance of approximately 60%. In cooperation with The National Psoriasis Foundation, we have now identified over 90 families with psoriasis in three generations. We have begun the process of ascertainment, the construction of family trees, and the collection of leukocyte DNA for linkage analysis with established restriction fragment polymorphisms (RFLP). Our initial assessment is being directed to four RFLP that span approximately 30 centiMorgans of the short arm of human chromosome 6. Although karyotyping is uncommonly done in patients because of psoriasis, we now seek evidence of translocation of chromosome 6 in association with psoriasis.

Human mononuclear leukocyte transglutaminase activity is enhanced by streptococcal erythrogenic toxin and a staphylococcal mitogenic factor associated with toxic shock syndrome.

Transglutaminase activity in human peripheral lymphocytes is enhanced after incubation of the cells with concanavalin A. Streptococcal proliferative factor toxin (erythrogenic toxin) from Streptococcus pyogenes and Toxic shock syndrome toxin from Staphylococcus aureus were purified and tested for their ability to enhance transglutaminase activity. Mononuclear leukocyte transglutaminase activity was enhanced 3-5-fold 30 min after incubation with either toxin. Enhancement occurred only when toxin was incubated with intact cells; addition of toxin to cell lysates was without effect. Transglutaminase was not measurable extracellularly. Histamine and dansyl cadaverine, competitive substrates for transglutaminase, inhibited [3H]putrescine incorporation into casein and [3H]thymidine incorporation into DNA. Incubation of lymphocytes with cycloheximide and either toxin or concanavalin A did not inhibit enzyme activity. These bacterial toxins, like phytomitogens, may perturb the cellular membrane and mediate their effect by transglutaminase-mediated cross-linking of membrane proteins.

Isolation and purification of a pemphigus vulgaris antigen from human epidermis.

Pemphigus vulgaris is an autoimmune disease associated with an autoantibody directed against a keratinocyte membrane antigen. The purpose of this study was to purify the human pemphigus vulgaris antigen, to produce an antibody to this antigen, and to use the antibody to induce pemphigus in newborn mice. Various techniques to extract the membrane-rich pellet from human epidermal homogenate were compared; 1% sodium dodecyl sulfate (SDS) and 1% dimethylsulfoxide proved to be superior to extract the pemphigus vulgaris antigen. This antigen was identified by transfer blotting to nitrocellulose paper, incubated with pemphigus vulgaris serum, or 20 control sera, and detected with fluorescein labeled antisera to human IgG. Since concanavalin A inhibits the binding of pemphigus vulgaris antibody to tissue sections, we studied the binding of the extracted proteins to concanavalin A covalently coupled to Sepharose. Pemphigus vulgaris antigen bound to the concanavalin A column and was released by 0.02 M methyl alpha-D-mannopyranoside. The proteins thus recovered were subjected to AcA 54 gel permeation chromatography, and the pemphigus antigen was detected by the transfer blot assay. The antigen corresponded to a discrete peak at 66,000 D by gel permeation and gave one homogeneous band at 33,000 D in urea-SDS-polyacrylamide gel electrophoresis. Monospecific antibody to the antigen raised in rabbits stained human epidermis in the same manner as the pemphigus vulgaris autoantibody and induced pemphigus vulgaris in newborn mice when injected intraperitoneally. A pemphigus vulgaris antigen has been purified from adult human epidermis. It is a 66,000-D membrane glycoprotein that is composed of two apparently identical subunits of 33,000 D each.

Keratolinin: the soluble substrate of epidermal transglutaminase from human and bovine tissue.

Substrates of human and bovine epidermal transglutaminase (glutaminyl-peptide gamma-glutamyltransferase, R-glutaminyl-peptide:amine-gamma-glutamyltransferase, EC 2.3.2.13) were isolated and purified by ion exchange chromatography and preparative zone electrophoresis. These substrates of Mr 36,000, which we propose to call keratolinin, incorporated dansylcadaverine and were precipitated by antibody. Keratolinin is ultimately polymerized on the inner leaflet of the keratinocyte membrane to form the cornified envelope. Each Mr 36,000 substrate was dissociated by chaotropic agents or detergents into noncovalent subunits; the Mr of these subunits was 6,000-6,200 on electrophoresis in 15% acrylamide/1% NaDodSO4/6 M urea gels. Isoelectric focusing of human or bovine keratolinin revealed two moieties separated by 0.3-0.4 pH unit (human, 5.4/5.0; bovine, 6.3/6.0). The two proteins were readily resolved by chromatofocusing and each isoelectric moiety of bovine keratolinin incorporated dansylcadaverine by epidermal transglutaminase and calcium and reacted with identity to antiserum to soluble Mr 36,000 keratolinin. Antiserum to human keratolinin failed to crossreact with its bovine counterpart. Antiserum to involucrin did not crossreact with either keratolinin or epidermis by immunodiffusion. Human and bovine epidermal keratolinins are biochemically similar but immunochemically distinct proteins from the epidermis. Involucrin appears only in significant quantities in cell culture.

Dermatitis herpetiformis: effects of sulfones and sulfonamides on neutrophil myeloperoxidase-mediated iodination and cytotoxicity.

The effects of sulfones and sulfonamides on neutrophil myeloperoxidase-mediated iodination and cytotoxicity were studied using in vitro assays to measure these parameters. Leukocyte iodination was documented using a quantitative assay to measure the iodination of protein by human neutrophils undergoing phagocytosis. Cytotoxicity for the tumor cell line LSTRA by human neutrophils activated by exposure to phorbol myristate acetate was measured by a 51Cr release assay. Dapsone, diasone, and sulfapyridine, at concentrations comparable to serum levels obtained by therapeutic doses of drug, effectively inhibited iodination and cytotoxicity mediated by human neutrophils. Other sulfonamides showed little inhibition of either iodination or cytotoxicity. The amount of inhibition was comparable to that seen with the inhibitors azide or cyanide and occurred in a dose dependent manner with all three drugs. A cell-free cytotoxic system using myeloperoxidase, iodide, a H2O2 generating system, and target cells also showed inhibition by dapsone, diasone and sulfapyridine in a similar fashion. The active drugs inhibited both the intra- and the extracellular myeloperoxidase-H2O2-halide cytotoxic systems. Serial iodination studies of four dermatitis herpetiformis patients, evaluated while taking dapsone or sulfapyridine, showed inhibition of iodination by either drug. Levels of IgA immune complexes, as measured by the Raji cell radioimmune assay adapted for IgA, did not change when medication was withheld. These studies demonstrate that dapsone, diasone, and sulfapyridine inhibit both neutrophil iodination and cytotoxicity for tumor cells, while other sulfonamides have no effect. This confirms previous studies showing inhibition by myeloperoxidase mediated iodination by dapsone. Furthermore, the effect on neutrophils is quickly reversible; in vivo administered drug has no effect on in vitro function. The active drugs inhibit both intra- and extracellular cytotoxic systems. This may represent an important mechanism by which these drugs produce their therapeutic effects when used to treat inflammatory skin diseases.

Epidermal and hair follicle transglutaminases and crosslinking in skin.

Epidermal and hair follicle transglutaminases crosslink structural proteins in the skin by epsilon-(gamma-glutamyl)-lysine bonds. This crosslinking produces protein polymers that are extremely insoluble and, until recently, difficult to characterize. Epidermal transglutaminase is localized to the granular layer of the epidermis. It catalyzes the crosslinking of a soluble cytoplasmic precursor to form the cornified envelope that lines the inner membrane of the mature keratinocyte in the stratum corneum. Hair follicle transglutaminase is localized to the inner root sheath and medulla of the hair follicle. It crosslinks a poorly characterized citrulline-rich protein. The enzymes and their substrates have been shown to be important markers of normal differentiation. Regulation of these processes is currently under investigation.

Purification of human epidermal calmodulin.

Human epidermal calmodulin was purified by phenothiazine-Sepharose affinity chromatography. The protein stimulated activator-deficient phosphodiesterase and was identified to be calmodulin by radioimmunoassay. Epidermal calmodulin demonstrated a Mr of 17,000 on gel permeation chromatography and migrated on sodium dodecyl sulfate-urea-polyacrylamide gel electrophoresis as a single band at the same location as rat testes calmodulin. The exact role of calmodulin in the epidermis remains to be defined.

Biochemistry of transglutaminases and cross-linking in the skin.

Transglutaminase is a calcium-dependent enzyme found widely in nature. It catalyzes the formation of epsilon-(gamma-glutamyl)lysine bonds that participate in processes varying from fibrin clot formation to epidermal cell envelope formation. Epidermal transglutaminase is localized to the granular layer of the epidermis. It catalyzes the covalent cross-linking of a soluble cytoplasmic substrate into large polymers to form the cornified envelope that lines the inner membrane of keratinocytes in the stratum corneum. The soluble precursor from epidermis has been named keratolinin, and from keratinocyte culture, it has been named involucrin. Hair follicle transglutaminase is biochemically and immunochemically distinct from its epidermal counterpart. It has been localized to the inner root sheath and medulla of the hair follicle. The substrate of hair follicle transglutaminase has been poorly defined but appears to be rich in the amino acid citrulline. Transglutaminase has been shown to be an important marker of normal differentiation. There is a rise in its activity at the time of keratinization, and transglutaminase activity has been shown to be greatly decreased in basal cell epithelioma and in psoriasis. Keratinocyte cell culture has proven most helpful in delineating the processes of normal differentiation and keratinization, since the formation of the cell envelope in culture appears to parallel the formation in vivo.

Immunoregulation in atopic dermatitis: functional analysis of T-B cell interactions and the enumeration of Fc receptor-bearing T cells.

Immune aberrations in atopic dermatitis (AD) are multiple and interrelated. We investigated immunoregulatory cell markers and functional interactions of purified T- and B-enriched cells in a pokeweed mitogen (PWM)-stimulated IgG production assay in patients with AD. Atopic mononuclear leukocytes and autologous recombinations of purified atopic T and B cells were hyporesponsive to PWM stimulation of IgG synthesis. When atopic B cells were cultured with normal T cells, they were still less responsive than normal B cells. Atopic T cells generated normal levels of suppression in three responder systems. Radioresistant T-cell help was also in the normal range whereas nonirradiated AD T cells produced slightly less help than normal T cells. We noted reduced levels of T lymphocytes with FcIgG receptors (T gamma) and found that T gamma reduction correlated inversely with log serum IgE. In the light of normal T suppression, we critically examined AD cell adherence and contamination at various steps in the T gamma assay to rule out technical causes of T gamma reduction in AD. Lowered T gamma cells in AD were not associated with circulating IgG immune complexes and subsequent blockade of the FcIgG receptors. Thus, we have identified defects in the numbers of an immunoregulatory T cell, and in the generation of PWM-responsive B cells. A model is proposed in which the alterations in atopic cyclic nucleotide metabolism of T-cell helpers could result in abnormalities of immunoregulatory T cells and PWM-recruitable B cells.

Herpes simplex antigen in immune complexes of patients with erythema multiforme: presence following recurrent herpes simplex infection.

Serum samples from patients with erythema multiforme (EM) were examined for the presence of herpes simplex virus (HSV) antigen in immune complexes using the Raji cell radioimmunoassay. Raji-cell-bound immune complexes from eight of 12 patients with EM after HSV infection and four of four patients with EM of uncertain cause had increased anti-HSV binding, while patients with EM after drug exposure and patients with recurrent HSV infections without EM had binding in the same range as controls. Viral cultures for HSV of immune complexes eluted from Raji cells were negative. Sucrose density gradient ultracentrifugation studies of serum samples of patients with EM after HSV infection showed HSV antigen in large molecular weight fractions. The HLA typing of lymphocytes from 16 patients with EM after HSV infection was not different from that of controls. Immune complexes composed of antibody and HSV antigen are present in serum samples of patients with EM after HSV infection and some cases of EM of uncertain cause and may mediate the pathogenesis of these disorders.

Diet-induced systemic lupus erythematosus (SLE) in primates.

Ten adult, female cynomolgus macaques were randomly assigned to two equal groups: (1) semipurified diet (SPD); and (2) SPD with 45% ground alfalfa seed (AS). Both groups were studied at monthly intervals after 5 mo on their respective diets. Control animals had a mean hematocrit (Hct) of 43 +/- 2%, negative antiglobulin (AG), antinuclear antibody (ANA) and LE cell tests. Mean values for C3 and C4 were 309 +/- 47 mg/dl and 35 +/- 7 mg/dl, respectively. Mean serum binding to radiolabeled double stranded deoxyribonucleic acid (dsDNA) was 1.9 +/- 0.2%. Three of five animals fed AS developed signs of an SLE-like illness characterized by AG-positive anemia (lowest Hct 30%), positive ANA (highest titer greater than 1:15, 360; rim pattern) and elevated anti-dsDNA binding (highest 96%) with variable degrees of hypocomplementemia. One animal had granular deposition of immunoglobulin and complement at the dermal-epidermal junction of clinically normal skin the presence of immune complex-induced glomerulonephritis.

Purification and characterization of streptococcal proliferative factor.

Group A streptococcal infections are often associated with scarlet fever and flares of guttate psoriasis. Previous investigation has demonstrated the presence of a factor in streptococcal culture filtrates capable of stimulating proliferation of rabbit keratinocytes in vivo and human lymphocytes in vitro. This report outlines an in vivo method for the production of streptococcal proliferation factor, its purification, and characterization of its physical properties. We cultured Group A streptococci (Type 12, Strain NY5) in synthetic media by in vivo incubation within dialysis casing surgically implanted in rabbit peritoneum. Streptococcal exoproteins were isolated by centrifugation of the bacteria and millipore filtration. Purification of streptococcal proliferative factor was accomplished by differential solubility and molecular sieve was discovered in the resulting product. The relative by SDS gels and molecular sieve chromatography. The sedimentation coefficient determined by sucrose gradient ultracentrifugation is 2.7S. Isoelectric focusing showed minimal microheterogeneity with the pI of the major band being 5.0. Thus, streptococcal proliferative factor can be produced by in vivo incubation of streptococci in synthetic media. Purification entails a rapid 2-step process. The relative molecular weight, sedimentation coefficient and isoelectric points have been established.

Immune complexes in erythema multiforme and the Stevens-Johnson syndrome.

Early cutaneous lesions of erythema multiforme or mucosal lesions of the Stevens-Johnson syndrome contain delicate granular deposits of immune reactants and/or complement components lodged in the walls of vessels of the papillary dermis. Such deposits are not present in normal, unaffected skin although they can be caused to occur there by injection of substances which increase vascular permeability. Factors which cause accumulation of mononuclear cells in the papillary dermis have not been elucidated but we suggest that receptors for the C3d fragment of the third component of complement could be responsible. Factors which cause intercellular edema in the epidermis, epidermal-dermal separation, or necrotic keratinocytes are unknown. We, and others, may now measured immune complexes in the serum of patients experiencing these reaction patterns by means of Raji cell radioimmunoassay, C1q precipitation, 125I-C1q binding, cryoprecipitation and with monoclonal rheumatoid factor. Cryoimmunoglobulins have also been described and antigens of Herpesvirus hominus have been measured in the cryoprecipitate of 2 patients who experienced erythema multiforme following recurrent herpesvirus infection. Erythema multiforme commonly accompanied serum sickness reactions which occurred following injection of immune horse serum in the 1980s (von Pirquet and Schick). Such reactions are now well established as due to circulating immune complexes in antigen excess.