Immunohistochemical demonstration of age-related deposition of vitronectin (S-protein of complement) and terminal complement complex on dermal elastic fibers

Immunoreactivity of vitronectin was investigated in 100 skin specimens from different body regions in 87 individuals of different ages using monoclonal and polyclonal anti-vitronectin antibodies in an avidin-biotin-peroxidase complex technique. Vitronectin immunoreactivity was found in conjunction with dermal elastic fibers in all subjects older than 13 years. No vitronectin immunostaining was detected in subjects younger than six years, suggesting deposition of vitronectin during late childhood or early adolescence. Using an immunogold staining procedure, vitronectin immunoreactivity was ultrastructurally localized to the periphery of elastic fibers. The blood level of vitronectin in 20 healthy newborns was 67% of the adult level, suggesting active biosynthesis already in the fetus. To investigate whether vitronectin is deposited as part of the SC5b-9 complex or as uncomplexed protein, the immunoreactivity of vitronectin was compared with that of C9, using monoclonal and polyclonal antibodies against the C9 neoantigen. Distinct C9 neoantigen immunoreactivity was demonstrated in association with dermal elastic fibers in human skin in adults but only in subjects older than 30 years. The intensity of C9 neoantigen immunoreactivity appeared to increase with age and was found to be stronger in sun-exposed skin than in sun-protected skin. These findings indicate that uncomplexed vitronectin is deposited during childhood or early adolescence and that terminal complement complexes (C5b-9 and/or SC5b-9) are deposited on elastic fibers later on in life. Hypothetically, the tissue form of vitronectin may be involved in the prevention of tissue damage in proximity to local complement activation. In addition, it may be physiologically important as substratum for cells, stimulating cell migration and anchorage.     

Fibrillin immunoreactive fibers constitute a unique network in the human dermis: immunohistochemical comparison of the distributions of fibrillin, vitronectin, amyloid P component, and orcein stainable structures in normal skin and elastosis.

Fibrillin, a 350-kD glycoprotein, was recently localized to elastin-associated 10 nm microfibrils. Here, the distribution of fibrillin immunoreactivity was determined in normal skin in individuals of different ages and in lesions of solar elastosis or anetoderma. It was compared with the distribution of orcein-stainable fibers and with the immunoreactivities of vitronectin and amyloid P component. These glycoproteins are known to occur in conjunction with the orcein-stainable elastic fibers in adults, but not in the young. Fibrillin immunoreactivity was associated with orcein-stainable fibers in normal skin of both adults and the young. In addition, the fibrillin immunoreactive fiber network comprised fine fibers that were unstainable by orcein, anti-vitronectin, or anti-amyloid P component. Such fine fibers were especially abundant close to the dermal-epidermal junction zone. Immunoreactivities of anti-vitronectin and anti-amyloid P component were not always associated with fibrillin immunoreactivity but were consistently found to co-localize with orcein-stainable fibers in adults. This suggests vitronectin and amyloid P component to be associated with the amorphous elastin rather than with the microfibrils, although alternative interpretations are possible. In elastotic lesions, fibrillin immunoreactivity was generally fainter than that obtained using anti-vitronectin or anti-amyloid P component. In contrast, an extensive network of dermal fibers stained by anti-fibrillin, but not by anti-amyloid P component, anti-vitronectin, or orcein, was seen in an anetoderma lesion. In conclusion, fibrillin immunoreactivity is associated with a unique dermal network, which ultrastructurally is composed of microfibrils. These fibers are proposed to have an important structural and functional role in anchoring the dermal elastic fibers in the extracellular matrix and to the lamina densa.     

Vitronectin shows complement-independent binding to isolated keratin filament aggregates

Keratinocyte cell death, whether produced by skin disease or by physiologic apoptosis in normal skin, may result in formation of dermal keratin bodies, consisting mainly of keratin intermediate filament aggregates. Vitronectin, a multifunctional plasma and tissue glycoprotein, which inhibits the complement membrane attack complex and promotes cell attachment and spreading, is, like amyloid P component, associated with keratin bodies in vivo. To investigate a potential role for vitronectin in the removal of keratin bodies, we studied the interaction of vitronectin with keratin intermediate filaments in normal human skin and in Hep-2 cells, as well as with isolated keratin intermediate filament aggregates in vitro. Following pre-incubation of skin sections and Hep-2 cells with normal human serum (as a source of vitronectin), cytoplasmic staining of keratinocytes and of cytoskeletal filaments in Hep-2 cells was observed by immuno-fluorescence staining with polyclonal and monoclonal anti-vitronectin antibodies. Vitronectin binding to keratin intermediate filament aggregates extracted from normal human epidermis was demonstrated by immunofluorescence and by immunoblotting, and was not dependent on complement activation, because it occurred even when heat-inactivated human serum or C4-deficient serum was used as a source of vitronectin. Amyloid P component shows Ca++- dependent binding to keratin intermediate filament aggregates. does not involve amyloid P component because it occurred when binding of the latter protein was inhibited by EDTA buffer. Moreover, purified vitronectin also bound to keratin intermediate filament aggregates in immunofluorescence studies. Vitronectin binding to keratin intermediate filaments may play a role both in limiting complement-mediated tissue damage (because keratin bodies may activate complement) and in promoting removal of keratin bodies by fibroblasts and/or macrophages.     

Tissue vitronectin in normal adult human dermis is non-covalently bound to elastic tissue.

Vitronectin is a multifunctional human plasma glycoprotein that is also found in constant association with elastic tissue fibers in normal adults. We have investigated the nature of the association of vitronectin with elastic tissue, and compared it to that of other elastic fiber-associated proteins, namely fibrillin and amyloid P component. Samples of normal human dermis were incubated with a variety of extraction agents, including high molar salt solution, non-ionic detergent (Nonidet P-40), the reducing agents dithiothreitol or 2-mercaptoethanol, and the chaotropic agents sodium dodecyl sulfate or guanidine hydrochloride. Vitronectin purified from serum typically migrates as two bands of 75 and 65 kD. By contrast, immunoblotting studies of residual dermal material after extraction with the various agents revealed only lower molecular weight (58, 50, 42, 35, and 27 kD) anti-vitronectin reactive bands. Although these bands may represent degradation products of vitronectin generated as a result of the extraction procedure, we cannot exclude the possibility that tissue vitronectin is distinct from plasma vitronectin. Anti-vitronectin reactive polypeptides co-migrating with the 58-, 50-, and 42-kD bands were solubilized following extraction with sodium dodecyl sulfate or guanidine hydrochloride, but not with the other extraction agents. Immunofluorescence studies using residual dermal material after extraction with guanidine hydrochloride demonstrated a marked reduction in elastic fiber staining intensity with anti-vitronectin and anti-amyloid P component, but not with anti-fibrillin. Thus the majority, if not all of dermal vitronectin, is, like amyloid P component, non-covalently associated with, and not an integral constituent of, elastic fibers.     

Vitronectin in normal human skin and in skin lesions]

Vitronectin (S-protein of complement, serum spreading factor) is a multifunctional glycoprotein present in human plasma and in the elastic tissue of various organs. It belongs to the group of adhesion proteins and is of importance in the terminal stages of both the coagulation and complement system and in fibrinolysis. In human skin it is localized on dermal elastic fibers and on pathologically altered elastic material (solar elastosis, pseudoxanthoma elasticum) as well as on keratin filament material such as keratin bodies in lichen planus or amyloid deposits in localized cutaneous amyloid. It is also found in the abnormally thickened cutaneous blood vessels in erythropoietic protoporphyria and porphyria cutanea tarda. Late-stage inhibition of the complement cascade in bullous disorders in which activation of the complement system is of pathogenetic significance may be an additional important function of vitronectin in skin diseases.     

Immunohistochemical studies on fibrillin in amyloidosis, lichen ruber planus and porphyria.

The pathogenesis of macular amyloidosis and lichen amyloidosis remains unsolved and the primary amyloid fibril protein(s) has not yet been identified. Ultrastructural association of skin amyloid with elastin associated microfibrils has been noted earlier. The presence of fibrillin in conjunction with such microfibrils was recently demonstrated immunohistochemically. The presence of fibrillin immunoreactivity in the amyloid deposits in skin biopsies from 3 patients with macular amyloidosis and 3 patients with lichen amyloidosis was studied, using monoclonal anti-fibrillin antibodies. For comparison, skin specimens were studied from five patients with lichen ruber planus, four patients with erythropoietic protoporphyria and from a patient with myeloma-associated cutaneous amyloidosis. Renal specimens from two cases of the amyloid A type of renal amyloidosis also were investigated. There was no immunostaining either of the keratin bodies in specimens of lichen ruber planus, the cutaneous PAS-positive vascular deposits in patients with erythropoietic protoporphyria, or the amyloid deposits in specimens of systemic amyloidosis and it was faint or absent in amyloid deposits in the specimens from patients with lichen amyloidosis. In contrast, distinct fibrillin immunoreactivity could be demonstrated in amyloid deposits in specimens from patients with macular amyloidosis. It was sometimes absent in deposits located in the upper part of the papillary dermis, close to the dermal epidermal junction zone, while consistently strong in deposits located lower down in the dermis. The results suggest that fibrillin or part of the fibrillin molecule may be present in some of the amyloid deposits in specimens of macular amyloidosis.     

Vitronectin colocalizes with Ig deposits and C9 neoantigen in discoid lupus erythematosus and dermatitis herpetiformis, but not in bullous pemphigoid

C9 neoantigen immunoreactivity has been found to colocalize with C3 immunoreactivity at the dermal-epidermal junction zone (DEZ) in skin specimens from patients with bullous pemphigoid, lupus erythematosus and dermatitis herpetiformis. The present study was designed to elucidate whether the C9 neoantigen immunoreactivity represents deposition of membrane attack complexes or non-lytic SC5b-9 complexes. Skin specimens from 11 patients with pemphigoid, five patients with discoid lupus erythematosus and from nine patients with dermatitis herpetiformis were studied with immunofluorescence using both monoclonal and polyclonal antibodies against C9 neoantigen and against vitronectin (S-protein), an inhibitor to the membrane attack complex of complement. Specimens from the pemphigoid patients demonstrated C9 neoantigen reactivity along the DEZ without detectable colocalized vitronectin. This suggests deposition of membrane attack complexes in the pemphigoid lesions. Immunoreactivity of both C9 neoantigen and vitronectin was detected in the DEZ in specimens of discoid lupus erythematosus and in the tips of dermal papillae in specimens of dermatitis herpetiformis. The combined presence of C9 neoantigen- and vitronectin immunoreactivity may indicate deposition of C9 as part of the non-lytic SC5b-9 complex. The finding reported here of differential deposition of vitronectin and C9 in different diseases indicates that the presence of C9 neoantigen immunoreactivity in tissue per se does not represent the deposition of membrane attack complexes, but that it may also be C9 deposited as part of the nonlytic SC5b-9 complex.     

Generalized acquired cutis laxa associated with coeliac disease: evidence of immunoglobulin A deposits on the dermal elastic fibres

Summary Acquired cutis laxa (ACL) is an uncommon elastolytic disorder of unknown aetiology. In rare instances. ACL has been reported in association with autoimmune diseases and dermal deposit of immunoglobulins, suggesting that destruction of elastic tissue may be immunologically mediated. We report a 35-year-old man with generalized acquired cutis laxa (GACL) associated with a persistent papular erythematous eruption that histopathologically showed some resemblance to dermatitis herpetiformis. A marked reduction and degeneration of dermal elastic fibres was noted in biopsies from loose-hanging skin. Direct immunofluorescence from non-inflammatory loose skin revealed granular immunoglobulin A (IgA) deposits at the basement membrane zone and fibrillar IgA deposits in the dermal papillae. IgA deposits were also observed on the elastic fibres of the reticular dermis. Electron microscopy of skin from the submammary fold revealed fragmented elastic fibres, partial absence of peripheral microfibrils and abundant neutrophils, some of which were degranulated and adjacent to elastic fibres. Immunoelectron microscopy of an erythematous papule revealed IgA deposits around dermal elastic fibres. Antigliadin, antireticulin and antiendomysium antibodies were present. Jejunal biopsies showed a gluten-sensitive enteropathy. A possible IgA-mediated immune mechanism for the development of GACL in our patient is suggested.     

Immunohistochemical studies of amyloid P component in disorders of cutaneous elastic tissue

Distinctive abnormalities in the immunofluorescence/immunoperoxidase staining pattern of dermal elastic tissue were observed with antibodies to serum amyloid P component (SAP) in solar elastosis, lupus erythematosus, porphyria and pseudoxanthoma elasticum, and resembled those seen with conventional elastic tissue stains. Electron microscopy of elastotic skin revealed the presence of vacuotated disintegrating elastic fibres. Islands of amorphous microfibrillar material were surrounded by a rim of specific staining with anti-SAP, indicating an elastic tissue origin; there was no evidence for the involvement of collagen fibres in the formation of elastotic material. Immunohistochemical staining with anti-SAP, a marker for elastic fibre microfibrils, is a useful method for the investigation of cutaneous elastic tissue disorders.     

Immunohistochemical demonstration of amyloid P component in skin of normal subjects and patients with cutaneous amyloidosis

The presence of amyloid P component (AP) in dermal deposits of cutaneous atnyloidosis was demonstrated by a direct immunofluorescence technique using an antibody to serum amyloid P component (SAP). AP was also shown, for the first time, to be a constituent of normal human skin. It was present at the periphery of dermal elastic tissue fibres, in basement membranes of dermal blood vessels and surrounding eccrine sweat glands but was absent from the dermo-epidermal basement membrane. The staining pattern in cutaneous amyloidosis was morphologically distinctive and readily distinguishable from staining of thickened vascular basement membranes in porphyria. Itnmunofluorescence with anti-SAP is simple and specific and may become the procedure of choice in the differential diagnosis of amyloidosis.     

IgA-binding structures in dermatitis herpetiformis skin are independent of elastic-microfibrillar bundles

Dermatitis herpetiformis (DH) is characterized in part by the presence of granular deposits of IgA in the papillary dermis just beneath the dermal-epidermal junction. The nature of the structures to which IgA binds in DH skin, however, has not been clearly demonstrated. Previous immunoelectron-microscopy studies using the peroxidase-antiperoxidase technique have concluded that the IgA may bind to abnormal elastic microfibrillar bundles. Recently, antibodies have been developed against a major component of the elastic microfibril bundles, fibrillin. In addition, another dermal matrix protein, hexabrachion, has been characterized and found in normal human skin in a distribution similar to the IgA deposits of DH. Utilizing antibodies against fibrillin, hexabrachion, and human IgA and immunoelectronmicroscopy with immunogold staining techniques, we have examined the skin from patients with DH in order to localize the IgA deposits. Normal-appearing skin from five patients with DH exhibited electron-dense patches within the dermis, which were not seen in skin from normal subjects. These structures were sometimes adjacent to the basement membrane zone, but appeared amorphous and without a well-defined fibrillar structure. The electron-dense patches were labeled with anti-human IgA, but not with antibodies to fibrillin or hexabrachion. The anti-IgA antibody did not label the normal basement membrane. These studies confirm the presence of abnormal electron-dense, amorphous structures in the skin of patients with DH. Due to this lack of association with the elastic microfibril bundles and the lack of labeling with antibodies against fibrillin, we suggest that these deposits are distinct from the microfibrillar bundles of elastic tissue and may represent IgA bound to degraded basement membrane or isolated dermal deposits of IgA.     

A direct immunofluorescence study in elastosis perforans serpiginosa

Two cases of elastosis perforans serpiginosa were studied by direct immunofluorescence. In one case, homogeneous deposits of IgM, C3 and C4 on the abnormal elastic fibres in the papillary dermis were demonstrated. In addition, in both cases, along the basement membrane zone, coarse linear deposits of fibrinogen and properdin were observed inside and near the transepidermal channels. Groups of cytoid bodies stained for IgM and occasional weak linear deposits of IgG, IgA, IgM and C3 were also observed. The immune deposits on the abnormal elastic fibres might indicate antigenic alteration and activation of humoral immunological mechanisms in the process of their elimination. The other findings may reflect a non-specific binding by damaged tissue structures.     

Cytokeratin expression in lichen amyloidosus and macular amyloidosis

AIM: To understand the role of epidermal cells in the pathogenesis of lichen amyloidosus (LA) and macular amyloidosis (MA). METHODS: We carried out immunohistochemical investigations on cytokeratins (CKs) in amyloid deposits in formalin-fixed and paraffin-embedded tissue specimens from eight persons with LA and 12 with MA. The primary antibodies of CK1-8 (AE3), CK10 (DEK-10), CK14 (LL002), CK17 (E3), CK18 (DC10), CK19 (KS19.1), CK5/6/18 (LP34) and CK8/18 (5D3) were used in the study. RESULTS: In amyloid deposits, immunoreactivity with only two monoclonal antibodies (CK1-8 and CK5/6/18) was observed in 14 cases (eight LA and six MA), confirming the hypothesis that epidermal cells participate in amyloid formation of LA and MA. COMMENTS: All of the CKs detected in amyloid deposits were basic type (type II). It seems plausible either that acidic CKs might be degraded faster than basic types in amyloidogenesis or that paraffin-embedded tissue specimens are less sensitive than frozen tissue sections. The results of our study suggest that when paraffin-embedded specimens are investigated by immunohistochemical methods, CK5 antibody is useful in the diagnosis of LA and MA.     

Pseudoxanthoma elasticum-like papillary dermal elastolysis: immunohistochemical study using elastic fiber cross-reactivity with an antibody against amyloid p component

Pseudoxanthoma elasticum (PXE)-like papillary dermal elastolysis is a rare disorder clinically characterized by multiple, asymptomatic nonfollicular papules, yellow or flesh colored, grouped to form plaques with cobblestone appearance. These plaques are arranged symmetrically on the neck and supraclavicular areas of adult or elderly women, closely resembling the lesions of PXE. Histopathologically, the lesions are characterized by a band-like area of elastolysis along the papillary dermis. We report 2 cases of PXE-like papillary dermal elastolysis, in which the anomalies of the elastic tissue were immunohistochemically investigated with an antibody to the amyloid P component. This immunostaining demonstrated a band-like loss of elastic tissue along the papillary dermis. No clumping, fragmentation, or calcification of the elastic tissue was seen. We also review the literature about this rare process and discuss the differential diagnosis with other elastic tissue disorders that may show similar clinical and/or histopathologic findings.     

Immunoglobulin and complement deposits in the skin and circulating immune complexes in scabies

Sixteen patients with papulovesicular, 6 with nodular and one with a Norwegian scabies were studied. Direct immunofluorescence (IF) examination revealed C3 deposits in the skin lesions of 13 of the 18 patients. Among them were all 6 cases with nodular scabies. C3 was found mostly in dermal vessel walls and 3 of the patients also showed IgM and 2 IgA deposits at the same site. No circulating immune complexes were found, with a solid-phase C1q radioimmunoassay (RIA), but HSV- and RSV-RIA methods detected IgM antibodies of rheumatoid factor type in 5 of the 15 sera examined. These results suggest that local complement activation and perhaps also immune complex deposition may by important in the pathogenesis of the papular and nodular skin lesions of human scabies.     

Diffuse dermal angiomatosis: A previously undescribed pattern of immunoglobulin and complement deposits in two cases

Two cases of diffuse dermal angiomatosis are reported in middle-aged women. This rare disease of unknown origin is characterized by increased dermal angiomatosis and ulceration. The clinical and histologic presentations of the presently reported lesions were typical for this disorder. Endothelial cells exhibited a normal immunophenotype. The perivascular basement membranes showed a distribution of collagen alpha chains typical for blood vessels, but not for lymphatics. Immunohistochemistry revealed other undescribed features. At the site of the clinical lesions, linear and granular deposits of immunoglobulins A and M, and complement were found around the vessels and at the dermal-epidermal junction. The same deposits were also found restricted to the dermal-epidermal junction in the peripheral clinically intact skin. No serological signs of auto-immune disorder were detected in one patient. A monoclonal gammopathy was disclosed in the other patient. A pattern of immunoreactant deposits similar to that disclosed in the two patients was not found in the control specimens, and has not been described so far in other types of vascular hyperplasia and neoplasia. A pathogenic role of these deposits is unsettled and should be further explored.     

Hemorrhagic bullous amyloidosis. A histologic, immunocytochemical, and ultrastructural study of two patients

In patients with bullous hemorrhagic amyloidosis of the skin, the skin lesions were the first manifestations of a plasma cell dyscrasia. Both cases were characterized by similar clinical, histologic, and ultrastructural findings showing an intradermal blister within deposits of amyloid substances. Immunohistologic investigations with a panel of antibodies directed against amyloid fibril proteins showed reactivity of the amyloid deposits with an anti-A lambda serum in both patients.     

Deposition of C3, C9 neoantigen and vitronectin (S-protein of complement) in lichen planus pemphigoides

We have compared the distribution of C3, C9 neoantigen (C9n) and vitronectin at the dermoepidermal junction in lichen planus pemphigoides with that in bullous pemphigoid. Eight out of 30 biopsies from patients with lichenoid lesions had linear C3 deposition at the basement membrane zone (BMZ); four of these patients had bullae and fulfilled the criteria for lichen planus pemphigoides. C9n immunoreactivity was detected as a linear or an intermittent linear/granular band at the BMZ only in these four patients, suggesting a role for the membrane attack complex of complement (MAC) in the pathogenesis of blister formation in lichen planus pemphigoides. Faint linear deposition of vitronectin, in addition to C9n, at the BMZ was seen in two of the four cases of lichen planus pemphigoides and three of six cases of bullous pemphigoid. This suggests that vitronectin may be deposited in association with C9n not only as part of the non-lytic SC5b-9 complex, but also as a regulatory step following the lytic action of MAC. A regulatory function for vitronectin in limiting tissue damage following activation of MAC is supported by our finding of a heavy deposition of vitronectin in association with C9n in a lichen planus pemphigoides patient in whom bulla formation had ceased.     

Keratotic vascular papules over the feet: a case of Waldenström’s macroglobulinaemia‐associated cutaneous macroglobulinosis

Waldenström’s macroglobulinaemia (WM) is a plasma‐cell dyscrasia characterized by the monoclonal proliferation of lymphoplasmacytes. A 48‐year‐old man presented with a 4‐year history of multiple painful, hyperkeratotic deep‐seated papules over the pressure areas of both soles. He had a 1‐year history of Raynaud’s phenomenon, intermittent epistaxis, recurrent vomiting, tingling and numbness, and visual disturbances. Histological examination of a skin biopsy found amyloid‐like deposits in the upper and mid dermis involving dermal blood vessels, but apart from periodic‐acid–Schiff, various stains gave negative results for amyloid. Direct immunofluorescence was positive for IgM antibody. Hence, a diagnosis of WM with cutaneous macroglobulinosis was made. Immunoelectrophoresis found monoclonal IgM kappa antibody, and bone‐marrow examination revealed a lymphoplasmacytoid malignancy. The patient’s systemic systems were attributed to hyperviscosity syndrome associated with WM and the cutaneous papules were identified as deposits of excess IgM antibodies. The patient received five cycles of chemotherapy, resulting in nearly complete resolution of the skin lesions and systemic symptoms.     

Microangiopathy, connective tissue changes and amyloid deposits in chronic renal failure

Skin biopsies were taken from 59 patients with chronic renal failure (52 patients were on regular dialysis treatment, and seven patients were predialytic). The histological examination of the skin biopsies revealed microangiopathy and pericollageneous deposition of a substance with the histochemical behavior of amyloid. In electron microscopy studies these deposits were found to be fine granular, but microfibrillar structures could also be detected. The alterations of the vessel walls are obviously caused by a non-reactive deposition of immunoglobulins, complement components, and fibrinogen. The alteration of the connective tissue mainly imposes as actinic elastosis. These findings could be demonstrated in both predialytic patients and patients on regular dialysis. The extent of the observed alterations seemed to be dependent on the duration of uremia. Atrophy of sweat and sebaceous glands also characterizes the skin lesions of patients with chronic renal failure.