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.     

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.     

Observations on the in vivo reaction of antinuclear antibodies with epidermal cells

In evaluating the normal skin of five patients with mixed connective tissue disease, four patients were noted to have both basement membrane zone and speckled epidermal nuclear immunofluorescence. The positive epidermal nuclear reaction was found to be associated with IgG, and no evidence of complement involvement was seen. In vitro testing demonstrated that normal control skin incubated with high-titred antisera to nuclear ribonucleoprotein reporduced the findings observed in direct staining, but incubation with high-titred antisera to other nuclear antigens such as Sm and single-stranded deoxyribonucleic acid did not cause positive epidermal nuclear staining. The association of antibodies to ribonucleoprotein and speckled epidermal nuclear immunofluorescence is discussed. Also considered is the possibility of other factors affecting nuclear membrane permeability.     

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.     

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.     

Clinicopathological significance of cutaneous epidermal nuclear staining by direct immunofluorescence

Epidermal antinuclear antibody (ANA) staining was noted during routine direct immunofluorescence (DIF) of skin biopsies from 22 cases at St John's Dermatology Centre over a 2-year period. We have reviewed the clinical, serological and immunopathological features of these patients. They comprised 13 cases of lupus erythematosus (LE), 3 dermatomyositis, 1 morphoea, 1 systemic sclerosis, 1 CREST syndrome, 1 mixed connective tissue disorder and 1 probable cutaneous sarcoidosis. Five (38.4%) patients with LE had moderate to severe oral mucosal involvement. Epidermal nuclear staining (ENS) was seen following IgG deposition in 21 cases and IgA in only 1 case. Complement C3 staining was an additional feature in 1 patient. Circulating ANA was absent in 7 cases at the time of biopsy, confirming that this pattern of staining does not occur as a result of tissue con-lamination during processing. The presence of ENS by IMF corroborates a diagnosis of a connective tissue disorder, and our results suggest that it may also be associated with oral involvement in E.E.     

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.     

A prospective immunofluorescence study of immune deposits in the skin of primary Sj?gren's syndrome

There are conflicting opinions concerning the epidermal immunofluorescence pattern in primary Sj?gren's syndrome. In a prospective study of 12 patients we found a characteristic pattern of epidermal nuclear/cytoplasmic IgG deposits in 8 (67%). This pattern was associated with the presence of antibodies against SSA/Ro and SSB/La in the serum and was also found in 2 out of 5 LE patients with monospecific antibodies against SSA/Ro. There is a resemblance to the pattern of dust-like particles described in the diseased skin of patients with subacute cutaneous LE. In one patient with primary Sj?gren's syndrome, IgG deposits were confined to epidermal cell nuclei (in vivo ANA). Instead of antibodies against SSA/Ro or SSB/La, this particular patient had nRNP-antibodies. From this study, we conclude that the epidermal IgG deposits in primary Sj?gren's syndrome may represent antibody binding to the sites within epidermal cells where the respective antigens are located.     

Urticaria. An immunofluorescence and histopathology study.

Fourteen randomly chosen patients with "garden variety" urticaria were studied for the presence of vasculitis and immunoglobulins and complement. Results of direct immunofluorescence (DIF) of the involved skin were negative, although two patients had immunoglobulins and complement demonstrable in the cytoplasm of the epidermal cells. Results of DIF of uninvolved skin were also predominantly negative. Findings from serum samples tested by indirect immunofluorescence (IIF) were negative, except for one positive in low titer (1:10, the basement membrane zone). Serum C3 and C4 levels were normal in five patients, both levels were low in two, and the C4 level was low in one patient. No skin-reactive immunoglobulins were found in these three patients by DIF or IIF. The ESR was measured and found to be elevated in four patients. Results of immunofluorescence proved negative in these cases. Of the 12 patients studied by hematoxylineosin staining to determine histology, none exhibited vasculitis. We believe that vasculitis with antigen-antibody reactions is not the rule in "garden variety" urticaria.     

Lupus erythematosus associated with C1 inhibitor deficiency

We report here a patient with skin lesions of lupus erythematosus (LE) associated with a type 1 hereditary C1 inhibitor deficiency. She had not experienced any episodes of angioedema. A histological examination of the affected skin lesions demonstrated liquefaction of the basal cell layer in the perifollicule. Direct immunofluorescence staining revealed the granular deposition of IgM along the dermo-epidermal junction. Blood laboratory examinations revealed low levels of CH50, C1q, C4, C2 and C1 inhibitor, but the C3 and C5 levels were within normal limits. Similar reductions in the C1 inhibitor levels were observed in 2 out of 3 sisters. Although one sister has been asymptomatic until now, the other has suffered from SLE. The antinuclear antibody titer was negative initially, but has changed to positive. The skin lesions became pigmented following topical corticosteroid therapy, but the deficient complement component levels remained unchanged. We also reviewed 23 cases in the literature of hereditary C1 inhibitor deficiency associated with SLE, DLE, LE-like eruption, and SCLE and discussed several common characteristics such as a female predominance, a high incidence of antinuclear antibodies, cutaneous manifestations, and photosensitivity.     

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.     

Mixed immunofluorescence findings in mixed connective tissue disease

An adult female with mixed connective tissue disease is described who had non-scarring alopecia, telangiectases, Raynaud's phenomenon and swollen hands. No clinical signs of SLE and only modest signs of myositis were present. High levels of RNP antibodies were detected in serum and direct immunofluorescence showed IgG staining of epidermal nuclei with a speckled pattern. Elution techniques performed on circulating lymphoid cells showed mixed features of both LE and scleroderma, namely antibodies to basal zone, to epidermal basal cells and to endothelial cells in mid-dermis.     

Fc gamma-receptor as a functional marker on epidermal Langerhans' cells in situ

Fc gamma-receptors (FcR) in cryostat sections of normal human skin were detected with soluble immune complexes of horseradish peroxidase (HRP) and rabbit IgG anti-HRP (HRP-anti-HRP). The binding of HRP-anti-HRP to Langerhans' cells (LC) was demonstrated using a double immunofluorescence staining in which LC were identified with a CD1a specific monoclonal antibody (Leu 6). The immune complexes gave granular staining of CD1a+ epidermal cells in sections of all specimens from normal skin. The mean percentage of CD1a+ cells that were FcR+ was 49 +/- 11 (n = 8). The FcR+/CD1a+ cells had a clearly defined dendritic pattern. The staining intensity of LC with HRP-anti-HRP was weaker than the intense staining of CD1a-macrophages in the dermis. Results of inhibition experiments indicate that human epidermal LC express low affinity FcR, but the presence of high affinity FcR as well cannot be excluded. The demonstration of FcR expression on normal LC clarifies previous uncertainty on LC membrane receptors, though the functional significance of these receptors is still not well understood.     

The presence of anti–basement membrane zone antibodies in the sera of patients with non–bullous lupus erythematosus

We performed indirect immunofluorescence (IF) studies using 1 mol/1 sodium chloride split skin to determine whether or not a positive IF is specific to patients with bullous lupus erythematosus (LE). We examined the sera from 21 patients with systemic LE (SLE), three of which were obtained from two SLE patients and one subacute cutaneous LE (SCLE) patient with bullous eruptions. As a comparison, we also studied the sera from patients with discoid LE (DLE,n= 7). SCLE (n= 1), systemic sclerosis (SSc, n= 20), bullous pemphigoid (n= 2) and normal individuals (n= 10). Sera from 16 SLE, four DLE and two SSc revealed a linear deposition of IgG isotype antibody at the epidermal side and/or the dermal side on indirect IF of split skin. The sera from three patients with bullous eruption and from 12 patients of SLE, SCLE, DLE without bullous eruption or SSc were further analysed by immunoblotting using five defined antigens, i.e, dermal extract, epidermal extract, three fusion proteins of 230 kDa bullous pemphigoid antigen (BPAG), 180 kDa BPAG, and human epidermolysis bullosa acquisita (EBA) antigen. Two SLE sera as well as one of the SCLE and the DLE serum reacted with 230 kDa BPAG in epidermal extract, and one of the SCLE and the DLE serum also reacted with the fusion protein of 180 kDa BPAG, No serum reacted with the dermal extract or the fusion protein of 230 kDa BPAG or EBA antigen. There was no consistent correlation between split–skin IF results and immunoblotting results. These results may suggest that even non–bullous LE patients often have autoantibodies to the basement membrane zone antigens, most of which are less pathogenic. Although we rarely examine the sera from non–bullous LE patients, we should keep this phenomenon in mind to avoid overestimating the results of split–skin test and immunoblotting.     

Expression of intercellular adhesion molecule-1 (ICAM-1) and OKM5 in UVA- and UVB-induced lesions in patients with lupus erythematosus and polymorphous light eruption

Pathological skin reactions were induced with both UVA and UVB in 12 patients with lupus erythematosus (LE) and with UVA in 7 with polymorphous light eruption (PMLE) but in none of the controls. Biopsy specimens taken from UV-induced lesions showed that in dermal infiltrates of LE cases CD4-positive cells predominated, whereas in the majority of PMLE cases CD8-positive cells predominated. Keratinocytes expressed intercellular adhesion molecule-1 (ICAM-1) in 7 of the 12 UVA- and in eight of the ten UVB-induced LE lesions, and in three of the UVA-induced lesions of PMLE patients. Three different staining patterns were found. In subacute cutaneous LE (SCLE) cases staining throughout the epidermis resembled that seen in genuine SCLE lesions. In discoid LE (DLE) lesions, the staining was most prominent in and near the basal cell layer. In the one systemic LE case and in the PMLE cases, ICAM-1 expression was seen only in association with epidermal spongiosis and T-cell infiltration. Keratinocytes did not express ICAM-1 in the controls or in the non-irradiated skin of the LE patients. In five on the UVA-induced lesions, in eight of the UVB-induced LE lesions and in one of the PMLE cases, keratinocytes expressed CD36. In four of the six LE lesions with fewer CD1a-positive cells, dendritic CD36-positive cells were seen in the epidermis. In conclusion, the pattern of activated keratinocytes and immunocompetent cells in the dermis was similar to that seen in genuine LE and PMLE lesions, but dissimilar to each other and to the controls. Keratinocytes in both UVA- and UVB-induced lesions in LE patients and in UVA-induced lesions of PMLE expressed ICAM-1 with a staining pattern resembling that seen in genuine lesions. This may help to explain the pathomechanism of these skin lesions.     

Histopathologic findings in lupus erythematosus tumidus: review of 80 patients

BACKGROUND: In a recent study, we demonstrated that lupus erythematosus (LE) tumidus (LET) is a distinct subset of cutaneous LE (CLE), which is clinically characterized by erythematous, urticaria-like, nonscarring plaques in sun-exposed areas. OBJECTIVE: Our purpose was to analyze skin biopsy specimens from 80 patients with this disease and to determine whether it could be differentiated from other variants of CLE on histopathologic grounds. METHODS: Skin biopsy specimens from 53 primary and 38 UVA- and/or UVB-induced lesions of 80 patients with LET were examined and compared with skin biopsy specimens from patients with discoid LE (DLE) and subacute CLE (SCLE). RESULTS: Specimens from LET lesions showed a characteristic and diagnostic pattern of perivascular and periadnexal cellular infiltrates in the papillary and reticular dermis composed almost entirely of lymphocytes. In some cases, few scattered neutrophils were present. Furthermore, interstitial mucin deposition was observed in all specimens, as confirmed by colloidal iron staining. In contrast to discoid LE and subacute CLE lesions, epidermal atrophy or alteration at the dermoepidermal junction was not detected. CONCLUSION: Skin lesions of patients with LET present with specific histopathologic features, and the differences compared with subacute CLE and discoid LE further support the concept to consider LET as a separate entity of CLE.     

Induction and repair of UVB-induced cyclobutane pyrimidine dimers and (6-4) photoproducts in organ-cultured normal human skin

Summary To examine the induction and repair of UV-induced DNA damage, indirect immunofluorescence was performed on UVB-irradiated organ-cultured normal human skin using monoclonal antibodies specific for either cyclobutane pyrimidine dimers or (6-4) photoproducts. Nuclear immunofluorescence of cyclobutane pyrimidine dimers and (6-4) photoproducts were observed in a dosedependent manner after UVB irradiation. The intensity of nuclear immunofluorescence of the upper epidermal layers was stronger and clearer than that of the lower epidermal layers. DNA repair time-course studies showed that both types of DNA damage could be repaired within 24 h after UVB irradiation.     

Cutaneous Immunofluorescence in Dermatomyositis

ABSTRACT: Seven patients with dermatomyositis who displayed severe skin and muscle disease, and in whom coexistent systemic lupus erythematosus (LE) was excluded, were evaluated by direct immunofluorescent biopsies of skin lesions. Specimens from six showed deposits of immunoglobulins and complement in small to moderate amounts at the dermal-epidermal junction zone. These deposits were usually in the form of focal granular accumulations which lacked continuity and were not well developed as are those seen in LE, or fluorescent subepidermal hyaline bodies. One patient, however, had a more well developed hand of immunoglobulins at the dermal-epidermal junction. All normal skin specimens in these patients were negative by immunofluorescence.
These findings were helpful in clarifying differences in cutaneous immunofluorescence between dermatomyositis and LE, indicating that dermatomyositis specimens can usually be differentiated from those of LE patients. but demonstrating the possibility that confusion might rarely occur in interpreting a lesional immunofluorescent biopsy in dermatomyositis.     

IMMUNOHISTOCHEMICAL DEMONSTRATION OF POLY(ADENOSINE DIPHOSPHATE-RIBOSE) SYNTHESIS IN HUMAN SKIN

The distribution of poly(adenosine diphosphate-ribose) [poly(ADP-ribose)] synthesis in human skin was investigated by an indirect immunofluorescence technique using a specific antibody against poly(ADP-ribose). In normal skin, the specific immunofluorescence of poly(ADP-ribose) was observed in nuclei of not only epidermal cells but also cells in the dermis, including the hair follicle and sweat gland. Preincubation of tissue sections with NAD, a substrate for poly(ADP-ribose) synthesis, intensified the immunofluorescence of poly(ADP-ribose). Nuclear immunofluorescence was prominent in the lower and middle layers of the epidermis and became weaker in the upper layers in parallel with the disappearance of the nuclei, especially in tissues untreated with NAD. The immunostaining pattern of poly(ADP-ribose) synthesis in psoriatic epidermis was similar to that of normal skin except that fluorescence was observed in nuclei of the parakeratotic horny layer. In affected cancerous skin, immunofluorescence of poly(ADP-ribose) was weak in nuclei of cells in the inner layers of horn pearl of well differentiated squamous cell carcinoma, although fluorescence was prominent in the outer layer cells of horn pearl. In contrast, tumor cells of both malignant melanoma and basal cell epithelioma exhibited the nuclear immunofluorescence of poly(ADP-ribose) synthesis. These results suggest that the capacity for synthesizing poly(ADP-ribose) may be related to the differentiation of epidermal cells.     

Expression of SS-A/Ro and SS-B/La antigens in skin biopsy specimens of patients with photosensitive forms of lupus erythematosus

CONTEXT: The reason that only some patients with lupus erythematosus (LE) develop autoantibodies to SS-A/Ro and SS-B/La antigens and photosensitivity is unknown. One hypothesis is that both events are related to the level of expression of these antigens in the skin. OBJECTIVE AND DESIGN: To test this hypothesis, we measured the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in normal sun-protected and sun-exposed skin in 14 patients with LE with photosensitivity, 12 patients with LE without photosensitivity, and 4 normal individuals. The presence of circulating antibodies to these antigens was measured in all patients. SETTING: Outpatient clinic in an academic medical center. RESULTS: We found that the expression of the 52-kd SS-A/Ro, 60-kd SS-A/Ro, and 48-kd SS-B/La antigens in skin biopsy specimens obtained from the same site was 4- to 10-fold higher in patients with LE with photosensitivity than in those patients with LE without photosensitivity (P<.001). Antigen expression was highly correlated with the presence and titer of circulating anti-SS-A/Ro and anti-SS-B/La antibodies (P<.001). CONCLUSIONS: These findings indicate that photosensitivity and the presence and titer of circulating anti-SS-A/Ro and anti-SS-B/La antibodies are both directly correlated with the expression of accessible and immunoreactive SS-A/Ro and SS-B/La antigens in the skin specimens of patients with LE. Thus, the expression of these antigens in keratinocytes may be an important determinant of the development of both SS-A/Ro and SS-B/La autoantibodies and of photosensitive forms of LE.