Ultraviolet light induces binding of antibodies to selected nuclear antigens on cultured human keratinocytes.

Antibodies which bind to different nuclear antigens in tissue sections or in permeabilized cell cultures are useful markers of subsets of connective tissue disease, especially of lupus erythematosus (LE), but whether these antibodies are able to react with these intracellular sequestered antigens in vivo and cause immunologic tissue damage has been a matter of much debate. We report experiments which show that ultraviolet light-irradiated, cultured human keratinocytes bind IgG antibodies from the sera of LE patients with either monospecific anti-SSA/Ro, anti-RNP, or anti-Sm activity, which implies that these antigens have been made accessible on the cell surface by ultraviolet irradiation. Normal human sera or LE patient's sera with anti-double-stranded DNA, anti-single-stranded DNA, or antihistone activity do not bind to the surface of irradiated human keratinocytes. In control experiments, all antisera produced the expected patterns of nuclear and cytoplasmic staining of fixed permeabilized, unirradiated keratinocytes. Careful study of the viability and permeability of irradiated keratinocytes by several techniques showed that this apparent cell membrane expression of extractable nuclear antigens (SSA/Ro, RNP, and Sm) following irradiation was seen on injured keratinocytes whose cell membranes were intact, but not on dead cells. It is particularly significant that all six monospecific anti-SSA/Ro sera bound to irradiated keratinocytes, since this antibody antigen system is highly associated with photosensitive cutaneous LE.

Autoimmune-associated congenital heart block: demographics, mortality, morbidity and recurrence rates obtained from a national neonatal lupus registry.

OBJECTIVES: The present study describes the demographics, mortality, morbidity and recurrence rates of autoimmune-associated congenital heart block (CHB) using information from the Research Registry for Neonatal Lupus. BACKGROUND: Isolated CHB detected at or before birth is strongly associated with maternal autoantibodies to 48-kD SSB/La, 52-kD SSA/Ro and 60-kD SSA/Ro ribonucleoproteins and is a permanent manifestation of the neonatal lupus syndromes (NLS). Available data are limited by the rarity of the disease. RESULTS: The cohort includes 105 mothers whose sera contain anti-SSA/Ro or anti-SSB/La antibodies, or both, and their 113 infants diagnosed with CHB between 1970 and 1997 (56 boys, 57 girls). Of 87 pregnancies in which sufficient medical records were available, bradyarrhythmia confirmed to be CHB was initially detected before 30 weeks of gestation in 71 (82%) (median time 23 weeks). There were no cases in which major congenital cardiac anatomic defects were considered causal for the development of CHB; in 14 there were minor abnormalities. Twenty-two (19%) of the 113 children died, 16 (73%) within 3 months after birth. Cumulative probability of 3-year survival was 79%. Sixty-seven (63%) of 107 live-born children required pacemakers: 35 within 9 days of life, 15 within 1 year, and 17 after 1 year. Forty-nine of the mothers had subsequent pregnancies: 8 (16%) had another infant with CHB and 3 (6%) had a child with an isolated rash consistent with NLS. CONCLUSIONS: Data from this large series substantiate that autoantibody-associated CHB is not coincident with major structural abnormalities, is most often identified in the late second trimester, carries a substantial mortality in the neonatal period and frequently requires pacing. The recurrence rate of CHB is at least two- to three-fold higher than the rate for a mother with anti-SSA/Ro-SSB/La antibodies who never had an affected child, supporting close echocardiographic monitoring in all subsequent pregnancies, with heightened surveillance between 18 and 24 weeks of gestation.

The effect of epicutaneous glucocorticosteroids on human monocyte and neutrophil migration in vivo.

The effect of epicutaneous methyl prednisolone (MP) at 10-4, 10-5, and 10-6 molar concentration was studied in 54 normal, healthy volunteers using a new, in vivo microchemotaxis technique. Significant inhibition of monocyte chemotaxis occurred at all concentrations studied and persisted over a 24-hr period with 10-4 molar MP. Neutrophil chemotaxis was significantly inhibited only with 10-4 MP. The inhibitory effect of MP on neutrophil and monocyte chemotaxis occurred earlier and at lower concentrations if the skin sites were pretreated with steroid. Thus, when corticosteroids are applied on abraded skin in concentrations achievable in vivo, monocyte chemotaxis into tissue is inhibited for longer periods and at lower drug concentrations than is neutrophil chemotaxis. By avoiding the significant systemic effects of corticosteroids on circulating monocyte and neutrophil populations, these experiments establish that local inhibition of chemotaxis is an important anti-inflammatory effect of corticosteroids, with differential effect on monocytes and neutrophils.

Serum-free serial culture of adult human keratinocytes from suction-blister roof epidermis.

Coating cell culture flasks with natural extracellular matrix (ECM) enhanced the culture of adult human keratinocytes from suction-blister roof epidermis in an environment without fetal calf serum (FCS), bovine pituitary extracts or cellular feeder layers. A higher incidence of cell attachment on natural ECM was observed than on collagen and human fibronectins (HFN)-coated plastic dishes, and natural ECM was necessary for growth and proliferation of attached cells under the culture conditions used. Cells in primary culture grew to confluency on natural ECM-coated surfaces within about 14 days, and subsequent serial passage could be made up to fourth passage in collagen- and HFN-coated plastic flasks. Cultured keratinocytes in this serum-free environment formed colonies of small cuboidal, healthy cells with little keratinization or stratification and demonstrated antigenic characteristics of human basal cells.

Detection of herpes simplex virus DNA in cutaneous lesions of erythema multiforme.

The association between erythema multiforme (EM) and herpes simplex virus (HSV) infection has long been appreciated, although the exact role which HSV may play in the pathogenesis of this herpes-associated EM (HAEM), is unknown. Previous studies have suggested, but not definitively demonstrated, the presence of HSV in lesions of HAEM. The presence of HSV would support the hypothesis that an immune-mediated response directed against HSV-specific antigens in the skin is central to lesion development in HAEM. The purpose of this study was to examine lesions of EM for the presence of HSV DNA by using the polymerase chain reaction (PCR). In addition, in situ hybridization using an HSV-specific RNA probe was performed to further localize the HSV nucleic acids within the skin. DNA was extracted from formalin-fixed, paraffin-embedded specimens of cutaneous lesions of HAEM and also from EM for which no precipitating factor could be documented, otherwise known as idiopathic EM (IPEM). DNA from lesions of bullous pemphigoid served as a negative control. Using PCR to specifically amplify HSV sequences which might be present, and then performing Southern analysis, we demonstrated HSV DNA in 9/13 HAEM and 6/9 IPEM biopsies. No HSV was detected in six lesions of bullous pemphigoid. In situ hybridization of three cutaneous HAEM lesions using an 35S-labeled HSV-specific RNA probe localized the HSV nucleic acids predominantly to the epidermis. Three biopsies of chronic dermatitis, used as negative controls, did not demonstrate this specific hybridization. These findings confirm the presence of HSV in lesions of HAEM and are consistent with the concept of an HSV-specific immune-mediated pathogenesis for this disease. In addition, most cases of IPEM appear to be herpes associated despite the absence of clinically apparent HSV infection.

Human monocyte chemotaxis: a quantitative in vivo technique.

This report describes a new quantitative technique for evaluating monocyte chemotaxis to a site of superficial epidermal abrasion. Micro-acrylic chambers containing 50% Zymosan activated autologous serum were separated from a 5-mm diameter epidermal abrasion by 2 Nucleopore filters which entrapped migrating monocytes but allowed free neutrophil migration. Monocytes were specifically identified by alpha napthyl acetate esterase activity. Monocytes accumulated within the filters by 4 hr and maximized at 16 and 20 hr. This technique is superior to previous skin chamber techniques in the high yield of monocytes and in specific histochemical identification of monocytes. In contrast to the Rebuck window, it does not generate attractants and has greater reproducibility. This technique will be useful in the study of diseases characterized by monocytic infiltrates, in contrasting the function of peripheral blood monocytes to those available in the skin, and in testing the effects of drugs, immunodeficiency and infection on monocyte function in vivo.

Wheat protein antibodies in dermatitis herpetiformis.

An enzyme-linked immunosorbent assay was used to detect class-specific antibodies to wheat protein antigens. Antibodies which we detected by this technique reacted indistinguishably with antigens prepared from crude gluten, crude gliadin, alpha-gliadin, Frazer fraction III, and subfraction B and B3 of Frazer fraction III. No sera reacted with a human serum albumin control antigen. The prevalence of IgG antibodies to wheat protein antigens was significantly greater in patients with gluten sensitive enteropathy, 12 of 17, (p = .00011) and in patients with dermatitis herpetiformis, 5 of 14, (p = .046) than in normal control subjects. Strongly positive reactions for IgG antibodies were present only in patients with gluten sensitive enteropathy or dermatitis herpetiformis. IgA antibodies to wheat protein antigens were found only in gluten-sensitive enteropathy patients. We have found this to be a sensitive, precise technique for measurement of antibodies to wheat protein antigens and feel that it will prove useful in evaluation of the role of immune complexes involving wheat protein antigens and their antibodies in the pathogenesis of dermatitis herpetiformis.

Enhancement of specific immunofluorescent findings with use of a para-phenylenediamine mounting buffer.

A recently described immunofluorescence mounting buffer containing para-phenylenediamine prevents fading of specific staining in skin sections during microscopic examination, and allows better appreciation of morphological detail. Examination of slides at high powers with intense illumination, as well as improved photomicrographs, are possible with this reagent.

Detection of gluten in human sera by an enzyme immunoassay: comparison of dermatitis herpetiformis and celiac disease patients with normal controls.

We have developed a triple sandwich enzyme immunoassay to detect circulating gluten in human sera. With human sera containing known amounts of added gluten as controls, the assay was sensitive in the range of 0.75 to 75 micrograms of gluten per ml of serum. Forty-one control subjects were compared to 21 patients with dermatitis herpetiformis and 11 patients with celiac disease. The dermatitis herpetiformis and celiac disease patients had significant elevation of serum gluten values over the control subjects. Circulating gluten antigenemia is a previously unrecognized feature which may be important in understanding the pathogenesis of dermatitis herpetiformis and celiac disease.

Interleukin-1 receptor antagonist production by human keratinocytes.

Human keratinocytes produce biologically active pro-IL-1 alpha and inactive pro-IL-1 beta with most protein remaining intracellular. IL-1 receptor antagonist (IL-1ra) is a newly described member of the IL-1 family that is secreted by stimulated monocytes and binds competitively to IL-1 receptors without stimulating target cells. We examined the characteristics of IL-1ra production by cultured human keratinocytes. By ELISA, keratinocyte lysates contained 390 ng IL-1ra/mg total protein with little IL-1ra detected in supernatants. In contrast, monocytes produced 297 ng IL-1ra/mg total protein during 24 h of culture on adherent IgG with about half of the IL-1ra detected in supernatants. By Western blot analysis, keratinocyte IL-1ra was approximately 20 kD in size and was slightly larger than recombinant monocyte IL-1ra. In contrast to monocytes, human keratinocyte IL-1ra was not secreted in 22-25-kD molecular weight glycosylated forms. Affinity-purified keratinocyte IL-1ra exhibited identical biologic activity to recombinant monocyte IL-1ra, each inhibiting IL-1-dependent augmentation of murine thymocyte proliferation to the same degree per amount of protein. An IL-1ra mRNA of 1.8 kb was detected by Northern blot analysis in RNA extracted from keratinocytes. In order to determine the effect of differentiation on IL-1 and IL-1ra production, human keratinocytes were cultured for 72 h in low (0.03 mM), medium (0.15 mM), or high (1.0 mM)-calcium concentrations. The absolute amounts of IL-1ra increased twofold and the ratio of IL-1ra to IL-1 alpha in keratinocyte lysates increased from approximately 12:1 to 25:1 during differentiation. These results indicate that keratinocytes constitutively produce large amounts of a biologically active intracellular variant of IL-1ra that increase with differentiation. IL-1ra released during keratinocyte damage may be important in modifying the inflammatory effects of IL-1 alpha in human skin.

Leukotriene C4 and TGF-alpha are stimulators of human melanocyte migration in vitro.

Human vitiligo is a disease of melanocyte destruction that leads to areas of depigmentation in the skin. The major form of treatment for vitiligo is photochemotherapy using psoralens and UVA radiation (PUVA), which induces the slow migration of pigment cells from hair follicles and normal skin into the depigmented areas. Our hypothesis is that immune cytokines and inflammatory mediators released as a result of the photochemotherapy are signals for melanocyte migration. We have developed an in vitro assay that quantitates the movement of individual cultured melanocytes over a 72-h period using time-lapse photography. Using this assay we found that both LTC4 and TGF-alpha were stimulators of melanocyte migration in vitro. The LTC4 effect was greater and lasted for the entire 72-h experimental period, whereas the TGF-alpha effect was significant only during the first 24 h of the experiment.

Characterization and practical benefits of keratinocytes cultured in strontium-containing serum-free medium.

Strontium (Sr2+) can substitute for Ca2+ and stimulate a variety of functions of numerous types of cells. The purpose of this study was to investigate the details of the biologic effects of Sr2+ on human keratinocyte growth, cell cycle, viability, and differentiation and to compare these effects with Sr2+ effects on cultured skin melanocytes. Cultured keratinocytes stimulated with 1.0-3.0 mM Sr2+ showed higher viability and almost a twofold increase in cell number compared with those grown in a standard calcium concentration. Time course studies revealed that 2.0 mM Sr2+ had no effects on growth of cultured melanocytes or fibroblasts. Sr2+ increased the percentage of cultured keratinocytes in G2/M phase, with a decrease in cells in G0/G1 phase. This effect of Sr2+ on the cell cycle was not seen in cultured melanocytes or fibroblasts. A 2 mM concentration of Sr2+ produced an increase in low-density keratinocytes separated by a Percoll gradient. In addition, increased expression of human fibronectin was observed in the cytoplasm and on cell membranes of keratinocytes cultured in Sr2+. Sr2+ can be of practical benefit in the culture of human keratinocytes in serum-free medium, increasing the viability and proliferative rate and producing a more uniform population of basaloid cells with increased expression of cell surface fibronectin.

Class-specific antibodies to gluten in dermatitis herpetiformis.

An immune reaction to wheat protein has been previously proposed to explain the pathogenesis of dermatitis herpetiformis. In order to detect and characterize antibodies to gluten in human sera, we developed an enzyme immunoassay for class-specific antibodies. Results of this assay in 49 patients with dermatitis herpetiformis were compared with those of 38 normal control subjects, 11 patients with celiac disease, and 6 small-bowel bypass patients. IgA antibodies to gluten were significantly more frequent in dermatitis herpetiformis sera (28/49) than in normal control sera (4/38). IgG antibodies to gluten were significantly more frequent in both celiac disease (10/11) and dermatitis herpetiformis (16/49) sera than in control (5/38) sera. Dermatitis herpetiformis sera also had an increased prevalence of IgM antibodies to gluten (19/49). Small-bowel bypass patients demonstrated no antibody to gluten. Antibodies to gluten in dermatitis herpetiformis objectively mark a state of immune reactivity to wheat protein and may be involved in the genesis of the cutaneous IgA immune deposits and the skin disease.

Transcutaneous leukocyte migration in vivo: cellular kinetics, platelet and C5a dependent activity.

A simple quantitative method for the measurement of leukotaxis in vivo is described. Duplicate skin chambers are placed over tape-stripped skin with 50% autologous serum--50% Hank's balanced salt solution as the attractant. Neutrophils predominate throughout 24 hr in this method with no change to mononuclear cells. A recommended modification of our original method is that chambers are sampled and removed after 8 hr rather than 24 hr since the majority of leukocyte migration occurs within the first 8 hr. Analysis of serum factors showed that heat-inactivation of the serum (56degreesC for 30 min) had no effect. However, depleting platelets or C5 from the serum removed approximately 90% of chemotactic activity for human neutrophils in vivo. Platelets, presumably through activity of their granules, enzymatically cleaves C5a from C5 in plasma. We conclude that C5a, after cleavage from C5, accounts for the majority of chemattractant activity in vivo.

The demonstration of SS-A/Ro antigen in human fetal tissues and in neonatal and adult skin.

Anti-SS-A/Ro antibodies have been statistically associated with congenital heart block and cutaneous lupus in the neonatal lupus syndrome, and with photosensitive cutaneous lupus in subacute cutaneous lupus erythematosus, but it had not been demonstrated that SS-A/Ro antigen is present in fetal tissues or at any age in human skin. We examined normal fetal tissues, normal neonatal and adult skin, and keratinocytes from purified serum-free cultures by immunofluorescence (IF) and by immunodiffusion or counterimmunoelectrophoresis (CIE) to determine the presence of SS-A/Ro antigen. We found SS-A/Ro antigen to be present in fetal hearts and in other internal organs. SS-A/Ro antigen could be demonstrated in biopsies of neonatal and adult skin by IF and was confirmed to be in keratinocytes by CIE. SS-A/Ro antigen may be found on the surface of keratinocytes in culture and therefore may be present at a relevant site for antibody binding. We have shown that SS-A/Ro antigen is a normal component of tissues that may be affected in neonatal lupus (fetal myocardium, neonatal epidermis) and in subacute cutaneous lupus erythematosus (adult epidermis).

Human herpesvirus 6 is present in lesions of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is a disease characterized by Langerhans cell infiltration of skin and bone, with its most severe form manifested by multifocal infiltration of many organs. The etiology is unknown, although viral infection has been proposed as a potential pathogenic factor. Human herpesvirus 6 (HHV-6), a recently described member of the human herpesvirus family, has been associated with atypical or malignant lymphocytic processes, and immune disorders. Based on these observations, we suspected that HHV-6 may play a role in the pathogenesis of LCH. Lesional tissue of 30 patients with LCH was retrospectively examined for the presence of HHV-6 by using the polymerase chain reaction. Tissue specimens from 63 patients with other benign and malignant histiocytic and lymphocytic diseases served as controls. In addition, all specimens were examined with control primers specific for herpes simplex virus (HSV). HHV-6 DNA was detected in lesions of 14 of 30 patients with LCH (47%). On clinical subgroup analysis, HHV-6 DNA was found in 10 of 16 patients with extraosseous disease (63%) and in four of 14 patients with disease limited to bone (29%). In each case, the prevalence of HHV-6 in LCH lesions was statistically significant, when compared to the control population. HSV DNA was not found in any of the LCH or control specimens. Although the presence of a virus alone does not establish a causal role in the disease, it supports the possibility of an etiologic relationship. From this study, we emphasize the need for further investigation of the potential HHV-6-mediated pathogenesis of LCH.

Comparison of hypothalamus-pituitary-adrenal axis suppression from superpotent topical steroids by standard endocrine function testing and gas chromatographic mass spectrometry.

We evaluated 38 males who had psoriasis vulgaris for evidence of hypothalamus-pituitary-adrenal axis suppression (HPAS) during treatment with superpotent topical glucocorticosteroids. All men were treated with 49 g per week of either Betamethasone Diproprionate in an optimized vehicle or Clobetasol Proprionate ointment. Three methods used to assess HPAS were compared. Classic 8 a.m. plasma cortisol measurements, urinary-free cortisol, and 17-hydroxycorticosteroid determinations and gas chromatograph-mass spectrometry (GCMS) quantitation of urinary cortisol metabolites were compared. Values for all methods were obtained just prior to therapy and at days 4, 7, 14, and 21 during therapy and at day 28 after treatment was stopped for 7 d. Plasma cortisol measurements correlated well with other measures of HPAS. GCMS determination of urinary cortisol metabolites was slightly more sensitive at detecting HPAS than the other two methods. Persistent HPAS after day 7 was only appreciated by GCMS. Urinary-free cortisol and 17-hydroxycortisol was the least sensitive of the three methods. Analysis of urinary cortisol metabolites by GCMS may be most useful in the monitoring of HPAS resulting from use of topical glucocorticosteroid preparations.

Study of HLA-DR synthesis in cultured human keratinocytes.

Within the normal human epidermis only Langerhans and indeterminate cells express HLA-DR. Human keratinocytes (HK), however, may also express HLA-DR in certain disease states characterized by mononuclear cell infiltrates. Previous studies have shown that HK synthesize HLA-DR in response to stimulation by interferon gamma (INF-gamma). The purposes of this study were to define conditions under which cultured HK might express HLA-DR and to compare the HLA-DR synthesis of HK with that of monocytes. HLA-DR expression by HK as determined by indirect immunofluorescence of HK cultures was absent under standard low calcium conditions and remained absent with the addition of calcium, serum, mitogens, and supernatants from Pam-212 cells containing epidermal thymocyte-activating factor. HLA-DR expression in HK was induced by cocultivation with concanavalin A-stimulated peripheral blood mononuclear cells (PBMC), but not unstimulated PBMC. This effect was time-dependent and directly related to the number of PBMC. HLA-DR expression was also induced in a time- and dose-dependent manner by addition of supernatant from stimulated PBMC (SS) or by addition of recombinant INF-gamma but not by addition of interleukin (IL)-1 or IL-2. Induction by either SS or INF-gamma was blocked by an antiserum to INF-gamma. As determined by a semiquantitative immunoprecipitation technique, HLA-DR synthesis by HK was directly related to INF-gamma concentration. The pattern of HLA-DR peptides produced by HK was similar to that of monocytes, but the relative quantity synthesized was far less than that of monocytes.

Development of an in vitro keratinocyte model for use in the study of HSV specific cytotoxicity.

Herpes simplex virus (HSV) specific immune mediated cytotoxicity may be involved in control of HSV infections and in the tissue damage induced by HSV infection or HSV related skin disease such as herpes associated erythema multiforme. Developing an in vitro model to study this process has proved difficult due to the lack of an appropriate target cell that will express HSV antigens but is not simultaneously subject to viral induced cell death. The purpose of this study was to develop a model in which keratinocytes express cell surface HSV specific antigens but at the same time are protected from death due to viral infection. We found that keratinocytes infected with HSV in the presence of acyclovir (ACV) expressed such antigens yet remained viable for a period of time after the onset of antigen expression such that cytotoxicity studies could be successfully performed. Rabbit skin cells, a transformed keratinocyte line, or second passage human neonatal foreskin keratinocytes were grown in culture medium with or without 200 microM ACV and were infected with HSV. Examination by direct immunofluorescence with anti-HSV antibody revealed uniform HSV antigen expression by cells both with and without ACV by 8 h after infection. Cells infected without ACV exhibited marked structural abnormalities including formation of multinucleated giant cells, while cells with ACV showed fewer such changes throughout a 24-h period. An Ethidium Bromide-Acridine Orange cytotoxicity assay demonstrated significant increases in the cytotoxicity of infected cells not protected by ACV compared to that of cells with ACV (p less than .001). This in vitro model should prove useful in the investigation of HSV specific immune mediated cytotoxicity.

Cryoglobulinemia and decreased monocyte chemotaxis is malignant melanoma.

The sera fromm 13 patients with malignant melanoma were evaluated for immune complexes by cryoprecipitation and the 125I Clq binding assay. Cryoprecipitates were identified in 12/13 patients (92%) and cryoimmunoglobulins in 7/13 patients (54%). Either cryoimmunoglobulin or elevated Clq binding was identified in 8/13 patients (62%). Incubation of normal monocytes with the resuspended cryoimmunoglobulin of 7 melanoma patients produced greater than 50% reduction in the ability of th monocytes to respond to chemotactic stimuli (p < .01). Similar inhibition was seen with cryoimmunoglobulin from erythema multiforme patients, but not with 'medium alone, albumin, heat aggregated albumin or heat aggregated-IgG in similar concentrations. No soluble factors produced in vitro could be demonstrated to produce this inhibition. Inhibition of monocyte function by immune complexes may be an important component of impaired host response to malignant melanoma, or alternatively may represent an important mechanism for the accumulation of monocytes at sites of inflammation, analogous to migration inhibition factor.