The speckled epidermal nuclear immunofluorescence of mixed connective tissue disease seems to develop as an in vitro phenomenon

The pathogenesis of speckled epidermal nuclear immunofluorescence in patients with mixed connective tissue disease (MCTD) was studied by reproducing this reaction in guinea-pigs, using serum samples containing high litre antibody to ribonucleoprotein (RNP). Immunofluorescence studies on specimens obtained from guinea-pig skin into which scrum samples containing high titre RNP antibody had been injected intradermally, revealed positive epidermal nuclear staining for IgG. This speckled immunofluorescence was demonstrable immediately after injection and remained so for 24 or 48 h. The pattern of fluorescence was similar in all cases, and there was no penetration of RNP antibody through the cell membrane. The epidermal nudear fluorescence was not detected with sera at a dilution of 1:100 or more. These results provide strong evidence that the epidermal nuclear immunofluorescence observed in patients with high titre antibody to RNP develops as an in vitro phenomenon.     

直接免疫荧光检查在诊断及鉴别硬皮病与混合结缔组织病上的应用

50例PSS、10例MCTD患者前臂伸侧皮肤活检DIF检查发现:10%PSS与60% MCTD出现有表皮细胞核Ig着色,MCTD均为IgG大斑点型,PSS则以均质为主,无1例为IgG大斑点型.10%PSS与30%MCTD患者BMZ出现Ig带状沉积.作者结论为:(1)在患有手部皮肤不典型硬化、雷诺氏现象、关节炎的患者,若DIF出现表皮细胞核大斑点IgG着色,即可诊断为MCTD.(2)BMZ带状Ig沉积不能做为区分二者的主要依据.     

In-vivo epidermal nuclear reactions: a selective process

Epidermal in-vivo nuclear reactions of IgG occur primarily in patients with mixed connective tissue disease or systemic lupus erythematosus and have been associated with high titres of circulating antibodies to ribonucleoprotein (RNP). This study was carried out to examine whether these epidermal nuclear reactions are true or simply an excision artefact. We observed the epidermal nuclear reactions for IgG only and not for other immunoglobulins in both in-vivo and in-vitro organ-culture studies, despite the presence of antinuclear antibodies (ANA) of all immunoglobulin classes. The association of the in-vitro epidermal nuclear reactions with serum RNP antibodies, although not absolute was statistically significant. The absorption of the serum with extractable nuclear antigen (ENA) preparation diminished the nuclear reactivity on tissue explants. In addition, the penetration of ANA into the nuclei of skin explants was both time and temperature dependent and was inhibited by sodium azide and by oligomycin. We conclude that the epidermal nuclear staining reactions observed by direct immunofluorescence on skin biopsies is selective and that the penetration of IgG into the epidermal cell nuclei is an active process and not an artefact.     

Speckled (particulate) epidermal nuclear IgG deposition in normal skin. Correlation of clinical features and laboratory findings in 46 patients with a subset of connective tissue disease characterized by antibody to extractable nuclear antigen.

Clinical and laboratory findings were correlated from 46 patients with IgG localization in epidermal nuclei in a speckled (particulate) pattern on direct immunofluorescence of normal skin. Cutaneous manifestations included lupus erythematosus (LE), swollen hands or sclerodactyly, alopecia, vasculitis, and dyspigmentation. Systemic manifestations included arthritis or arthralgia, Raynaud's phenomenon, serositis, vascular headaches, mild renal disease, myositis, and sicca syndrome. High titer (mean = 1:142, 800) serum antibody to extractable nuclear antigen (ENA) was found in 81%. Eighty-six percent had antibody to an RNase-sensitive antigenic component of ENA (ribonucleoprotein or RNP); 14% had antibody to an RNase-resistant ENA termed Sm. Deposition of IgG in a speckled pattern in epidermal nuclei is an immunopathologic marker for a subset of connective tissue disease characterized by antibody to ENA. Those with Sm specificity had systemic LE (SLE); Those with RNP specificity had Raynaud's phenomenon usually associated with overlapping features of SLE, scleroderma, and/or dermatomyositis.     

In vitro complement activation by antinuclear antibodies in the epidermis from patients with systemic sclerosis

Skin biopsies from twenty-one systemic sclerosis patients with significant serum fluorescent antinuclear antibody (FANA) titres (more than 1:40) were examined by direct immunofluorescence (DIF) and by in vitro immunofluorescent complement (C) activation (C + DIF). Eight patients showed epidermal nuclear ANA deposits by DIF. In vitro complement activation was achieved in epidermal nuclei from all eight patients. In addition, one patient with diffuse scleroderma revealed a dense speckled, and two patients with the CREST syndrome a discrete speckled, epidermal nuclear staining pattern by C + DIF. The latter two patients showed high titres of anticentromere antibody in their sera. We consider that the C + DIF findings of these two patients with the CREST syndrome reflect binding of serum anticentromere antibodies to their antigens in vivo.     

Nucleolar, homogeneous and peripheral fluorescence of epidermal nuclei in direct immunofluorescence: 4 cases (author's transl)]

Epidermal nucleolar IgG deposition on direct immunofluorescence of covered normal skin was found in two patients with scleroderma and high serum concentrations of antibody to nucleolar antigen. Epidermal homogeneous and peripheral IgG deposition was also observed in two patients with systemic lupus erythematosus, antinuclear antibody of homogeneous staining pattern, but without antibody to extractable nuclear antigen. Epidermal nuclear IgG deposition in a speckled, nucleolar, homogeneous or peripheral pattern appears to correlate with high titer serum antinuclear antibody giving on immunofluorescence the same staining pattern. These immunopathologic findings cannot be considered as being specific of a subset of connective tissue disease.     

Mixed connective tissue disease syndrome.

Fifteen patients with epidermal nuclear staining on direct immunofluorescence of normal skin and high titer serum antibody to ribonuclease-sensitive extractable nuclear antigen (ENA) had diffuse nonscarring and focal alopecia, abnormal pigmentation, swollen hands with sclerodactyly, and chronic cutaneous lupus erythematosus (LE) as the most common dermatologic features. Direct immunofluorescence of normal, unexposed skin revealed a particulate ('speckled') epidermal nuclear staining pattern in all 15 patients and subepidermal immunoglobulin deposits in 5. Ribonucleoprotein antibodies in high titer are associated with this characteristic type of epidermal nuclear staining. These findings provide easily detectable markers for a less aggressive subset of LE characterized by distinctive clinical and laboratory features consistent with mixed connective tissue disease.     

Mixed connective tissue disease characterized by speckled epidermal nuclear IgG deposition in normal skin

Four African female patients are described, who presented with the features of systemic sclerosis. Overlapping features of lupus erythematosus or dermatomyositis were present in three cases but were not prominent. Direct immunofluorescence of uninvolved skin revealed a particulate (or speckled) epidermal nuclear staining, with specificity for IgG. In view of the reported association between this finding and mixed connective tissue disease, these patients were treated with corticosteroids and marked improvement occurred in all cases. The usefulness of this investigation in making the distinction between systemic sclerosis and mixed connective tissue disease and in indicating a potentially effective form of therapy is discussed.     

Immunofluorescence studies in progressive systemic sclerosis (scleroderma) and mixed connective tissue disease

Immunofluorescence (IF) investigations of the skin were performed in thirty patients with progressive systemic sclerosis (scleroderma) and eight patients with mixed connective tissue disease (MCTD). The results show that speckled epidermal nuclear immunoglobulin deposition occurs not only in MCTD but also in true scleroderma. Granular IgM deposition at the dermo-epidermal junction of light-exposed skin was detected in both groups of patients, but six of eight MCTD patients also showed a granular IgM band in non-exposed skin. Antinuclear antibodies (ANA) were demonstrated in the sera of 96% and 100% of patients with scleroderma and MCTD respectively. The pattern of nuclear IF staining in scleroderma included dense fine speckles, large coarse speckles, threads, nucleolar and centromere staining. In MCTD, by contrast, the ANA staining pattern consisted of threads. The significance of ANA titres and immunological specificities for the in vivo reaction of serum ANA with epidermal nuclear antigens is discussed.     

Intracellular expression of type VII collagen during wound healing in severe recessive dystrophic epidermolysis bullosa and normal human skin

The ability of keratinocytes to synthesize basement membrane components in vivo during wound healing in normal human skin and in severe recessive dystrophic epidermolysis bullosa (RDEB) was investigated. Indirect immunofluorescence using anti-type VII collagen (VIIc, recognizing the globular non-helical component of the molecule), anti-type IV collagen, anti-laminin and bullous pemphigoid antisera, was performed on biopsies of intact skin and of healing skin taken between 7 and 14 days after dermatome injury (upper to mid-dermal wounding) in eight patients with severe RDEB and in seven normal subjects. Baseline anti-type VIIc immunofluorescence showed completely absent staining of the epidermis, dermis and dermo-epidermal junction in severe RDEB samples, and bright linear dermo-epidermal junction fluorescence in normal human skin. In 5/5 normal human skin samples taken 9-12 days post-wounding, some type VIIc expression was noted within basal cells as well as in a continuous or interrupted linear distribution at the basement membrane zone. In all the severe RDEB biopsies sampled between days 10 and 13 (5/5), anti-VIIc fluorescence was also seen with varying intensity within basal and lowermost suprabasal cells, and in one day 14 sample at the dermo-epidermal junction. Low levels of intracellular type IVc were seen in both groups, but only in those samples taken 7-9 days after injury; later biopsies showed only continuous dermo-epidermal junction staining. Linear basement membrane zone labelling with laminin and bullous pemphigoid antisera was seen in all samples in both sets of subjects, even at day 7, but there was no detectable intracellular antisera staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

A specific defect in glycosylation of epidermal cell membranes. Definition in skin from patients with epidermolysis bullosa simplex

Why blister formation occurs within the epidermis in epidermolysis bullosa (EB) simplex is not known. One possibility is that there are diminished amounts, absence, or biochemical alterations of one or more structural components of epidermal cell membranes, thereby leading to increased skin fragility. In order to test this hypothesis, biopsy specimens were obtained from clinically normal-appearing skin of patients with simplex, junctional, and dystrophic forms of EB and normal adult volunteers. Immunofluorescence studies were performed on each specimen using eight fluorescein-labeled affinity-purified lectins shown to bind uniformly to epidermal cell membranes of normal human adult skin and neonatal foreskin. To examine the epidermal cytoskeleton, each tissue specimen was also examined using two antikeratin monoclonal antibodies. Irregular and focally granular epidermal membrane staining was noted in each EB simplex specimen examined with the lectin-peanut agglutinin. In contrast, uniformly crisp membrane staining was seen in each specimen from patients with junctional or dystrophic EB and from normal volunteers. This epidermal cell membrane glycosylation defect appears to have the restricted carbohydrate specificity of peanut agglutinin since staining of EB simplex skin with each of the remaining seven lectins was indistinguishable from that seen in skin from patients with the other forms of EB and normal adult skin. Furthermore, the epidermis in EB simplex skin appears to be selectively abnormal since the same tissue specimens demonstrated normal keratin cytoskeleton staining.     

Cutaneous immunofluorescence study of erythema multiforme: correlation with light microscopic patterns and etiologic agents

Direct immunofluorescence microscopy was positive in 88% of forty-one skin biopsy specimens from thirty-four patients with the clinical diagnosis of erythema multiforme. The most common finding, present in 67% of positive specimens, was the cytoid body, or fluorescent keratinocyte, which stained most often with IgM (homogeneously) or with C3 (speckled). Other findings included basement membrane zone (BMZ) fluorescence, primarily with fibrinogen and C3, and vascular fluorescence, most commonly C3 in a granular pattern. Correlation of direct immunofluorescent and light microscopic findings revealed that (1) the fluorescent keratinocyte was prevalent only in epidermal and mixed patterns, correlating with the eosinophilic necrotic keratinocyte by light microscopy, and (2) vascular fluorescence was most prominent in dermal forms. Herpes simplex-associated erythema multiforme showed exclusively a mixed histologic pattern, whereas the drug-related form was primarily epidermal.     

Some further features for differential diagnosis between squamous cell carcinomas and basal cell epitheliomas

Two further methods for the characterization of epidermal skin tumors are described: the antinuclear antibody (ANA) immunofluorescent test, which consists of indirect immunofluorescence with known high titer sera containing homogenous ANAs on epidermal skin tumors, and the ammoniacal-silver cytochemical method, which specifically stains nuclear histones. Squamous cell carcinomas (SCCs), basal cell epitheliomas (BCEs) as well as control specimens from normal skin and benign epidermal hyperplasias were studied. The ANA immunofluorescent test was positive for most SCCs, mixed SCC and basal cell carcinomas and metatypical BCEs. The ammoniacal-silver method gave a characteristic staining pattern shared among SCCs, mixed carcinomas and metatypical BCEs. BCEs, besides metatypical ones, were always negative by the ANA immunofluorescent test and the same applied for the control specimens. The ammoniacal-silver method gave a characteristic staining pattern for BCEs and control sections quite different from the staining pattern of the more aggressive forms of epidermal tumors. The two methods usually yielded parallel results.     

Cytoplasmic and HL-A antigens in the human epidermis

HL-A antisera were found to react, in indirect immunofluorescence on human skin, with the cytoplasm of the upper cell layers of the epidermis. It has been established that this staining was not due to an HL-A-antigen-antibody reaction but rather to a contamination of HL-A antisera with non-HL-A epidermal cytoplasmic antibodies. These findings ruled out a previous hypothesis that HL-A antigens were expressed in the cytoplasm of the upper epidermal cell layers. The localization of HL-A antigens in the human epidermis has not been demonstrated by use of indirect immunofluorescence.     

Deposition of the membrane attack complex of complement in bullous pemphigoid

Bullous pemphigoid is associated with deposition of IgG and C3 at the dermal-epidermal junction. In order to see whether complement activation in bullous pemphigoid resulted in deposition of membrane attack complex (MAC) at the basement membrane zone, skin biopsies from patients with bullous pemphigoid were examined using a direct immunofluorescence technique. By employing a monoclonal antibody to a neoantigen of C9, the MAC was demonstrated in linear pattern at the basement membrane zone. These deposits were seen in both involved and uninvolved skin but the amount of MAC was greater in involved skin as judged by intensity of staining. Stippled deposits of MAC were also present in or around epidermal basal cells. The MAC could be generated in vitro by reaction of normal plasma with antibasement membrane antibody bound to sections of monkey esophagus. The IgG antibody activated complement and this complement activation proceeded all the way to the terminal step.     

Absorption of boric acid through human skin depending on the type of vehicle

Summary The distribution of procollagen in normal hyperplastic, preneoplastic or neoplastic human epidermal lesions has been analysed in indirect immunofluoresence tests with the antibodies to procollagen raised in sheep to extracted procollagen, synthesised by newborn rat skin explants in culture. These antiprocollagen antibodies produced indirect immunofluorescence staining only of the papillary dermis of human skin. We reacted serial dilutions of the antibody with each skin lesion and recorded the maximum dilution at which a positive reaction was observed. All lesions examined displayed essentially the same procollagen immunofluorescence pattern. The fluorescence was localised as a fibrillar/diffuse area just under the epidermis. In conditions in which the epidermis is highly convoluted, the fluorescent band was found to follow the pattern of the epidermis and to surround epidermal islands in the deeper dermis. Our observations suggest that in the neoplastic lesions a new dermal topography, resembling the stratum papillare of normal human skin, is found in the deeper dermis surrounding the epidermal islands produced as the epidermal mass increases and invaginates further into the stroma. Malignant epidermal lesions were found to show a positive procollagen immunofluorescent staining at tenfold lower concentrations of the antibody than normal human skin in subepidermal regions.     

混合性结缔组织病皮肤表皮细胞核染色的价值

采用 IIF法对 2 32例 CTD患者的外观正常皮肤进行了免疫荧光研究。结果发现 10 0 %的MCTD患者的表皮细胞核有 S型 Ig G沉积 ,并显著高于 SL E患者 ( 6.6% ) ,同时血清伴有高滴度、单一的抗 RNP抗体和高滴度的 S型 ANA。因此 ,S型 Ig G ENS和高滴度单一抗 RNP抗体之间具有高度的相关性。我们认为 S型 Ig G ENS可作为 MCTD的免疫病理学特征 ,其对 MCTD具有重要的辅助诊断价值     

The presence of IgE molecules on epidermal langerhans cells in patients with atopic dermatitis

Summary Skin sections of clinically involved and clinically normal-looking skin from patients with atopic dermatitis were incubated with anti-human IgE antibodies using the indirect immunoperoxidase technique. Apart from positive dermal anti-IgE staining, positive epidermal anti-IgE staining was also observed. The morphology of the epidermal staining cells suggested the involvement of dendritic cells. This was confirmed by positive immuno-double labelling with OKT6 and anti-IgE. This phenomenon seemed to be specific for atopic dermatitis since skin sections from normal nonatopic controls, patients with allergic asthma, contact dermatitis, and schistosomiasis showed no epidermal anti-IgE staining. To further elucidate the nature of the epidermal anti-IgE staining cells, epidermal cell suspensions were prepared from clinically involved skin from patients with atopic dermatitis. These cell suspensions also showed positive anti-IgE staining cells and positive immuno-double labelling with OKT6 and anti-IgE. Immunogold electron microscopy with anti-IgE on epidermal cell suspensions from patients with atopic dermatitis showed gold particles on the cell membranes of cells containing Birbeck granules, being Langerhans' cells. Epidermal cell suspensions from normal non-atopic controls were negative. The presence of IgE molecules on epidermal Langerhans' cells, which seems to be specific for patients with atopic dermatitis, provides an explanation for the high frequency of positive patch test reactions to inhalant allergens.     

Discordance of SSA/Ro and SSB/La cellular antigens in synchronized cells

SSA/Ro and SSB/La are soluble cellular proteins to which antibodies are frequently produced in patients with Sj?gren's syndrome and systemic lupus erythematosus. In this investigation, we examined anti-SSA/Ro and anti-SSB/La staining patterns on synchronized WiL2 cells and mixed lymphocyte culture cells using monospecific antisera. In addition to its presence in the nucleoplasm, the SSB/La antigen was highly concentrated in the nucleolus of cells during the late G1 and early S phase and is thus cell cycle-related. In contrast, the SSA/Ro antigen was found to be independent of cell cycle, showing a nuclear speckled pattern in all phases. Blocking experiments indicated that free SSB/La is responsible for the nucleolar staining, whereas the combination of both SSA/Ro and SSB/La determines the nucleoplasmic speckled staining pattern.     

Epidermolysis bullosa acquisita. Incidence in patients with basement membrane zone antibodies

We examined the incidence of epidermolysis bullosa acquisita in patients with circulating basement membrane zone antibodies. Serum samples from 100 sequential patients with basement membrane zone antibodies were tested by indirect immunofluorescence against 1 mol/L sodium chloride split skin and by Western immunoblot against epidermal and dermal extracts of skin. Ninety-two (92%) serum samples stained only the epidermal side of split skin, 5 (5%) stained only the dermal side, and 3 (3%) stained both sides. Four of the 5 serum samples with dermal staining but none of the serum samples with epidermal or combined staining reacted with the 290-kd epidermolysis bullosa acquisita antigen by Western immunoblot. These results indicate that approximately 5% of unselected patients with basement membrane zone antibodies have epidermolysis bullosa acquisita or bullous lupus erythematosus rather than bullous pemphigoid.