Amyloid P components in normal human skin and skin with lesions

Amyloid P component (AP) is a glycoprotein which is found in tissue deposits of all types of amyloid and is identical to and derived from serum amyloid P component (SAP). SAP binds in a calcium-dependent fashion to various ligands, such as agarose, desoxyribonucleic acid, fibronectin, C4-binding protein, glycosaminoglycans and isolated amyloid fibrils. Tissue AP (TAP) is also a constituent of the normal human renal glomerular basement membrane and is, in adult humans, invariably associated with elastic fiber microfibrils in connective tissue throughout the body, including that of blood vessels. In normal human skin anti-AP antibody binding was localized to the microfibrils of oxytalan fibers in the papillary dermis and to the peripheral microfibrillar mantle of elaunin and mature elastic fibers in the reticular dermis. Since SAP binds to fibronectin and glycosaminoglycans, which in turn bind to collagen fibers, TAP on elastic fiber microfibrils may play an important role in the maintenance of the normal dermal architecture and in dermo-epidermal adhesion. Under pathological conditions, AP is found in all forms of cutaneous amyloidosis, including primary localized cutaneous amyloid (PLCA); it is also detectable on keratin bodies, which represent precursor structures for PLCA. The association of AP with elastic fiber microfibrils and amyloid fibrils and their close anatomical relationship in vivo may reflect the significance of AP in the deposition of cutaneous amyloid.     

Amyloid elastosis: analysis of the role of amyloid P component.

We report the second case of amyloid elastosis. Our patient had an underlying primary systemic amyloidosis with lambda light chain paraproteinemia. Salient clinical features included a sclerodermatous facial appearance, cordlike thickening of superficial blood vessels, neck skin resembling that in pseudoxanthoma elasticum, livedo reticularis-like changes on the trunk, Raynaud's phenomenon, arterial and venous thromboses, and the nephrotic syndrome. Amyloid deposits were present in the dermis, around appendages, in blood vessel walls, and in a striking distribution surrounding individual elastic fibers, that appeared shortened and fragmented. Immunofluorescence, electron microscopic, and immunoultrastructural studies with antibodies to lambda light chain, localized the amyloid deposits to the region of the elastic fiber microfibrils, with which amyloid P component (AP) is invariably associated in normal tissues. Because AP binds amyloid fibrils, codistribution of amyloid deposits and AP in amyloid elastosis strongly supports the theory that elastic fiber-associated AP may act as a nidus for amyloid deposition.     

Immunohistochemical studies of amyloid P component distribution in normal human skin

The distribution of amyloid P component (AP) in normal human skin was investigated by a light and electron microscopic immunoperoxidase technique, using antibodies to serum amyloid P component (SAP). AP, or an immunologically cross-reactive protein, was found to be specifically localized to the microfibrils of papillary oxytalan fibers and to the peripheral microfibrillar mantle surrounding the elastin core of mature elastic fibers in the reticular dermis; collagen fibers were not stained with anti-SAP. AP was not detected in the dermal-epidermal basement membrane or in the basement membranes surrounding dermal papillary blood vessels and eccrine structures. These findings, which establish the detailed distribution of normal tissue AP in the skin, provide a basis for further studies of the function and behavior of this protein in health and disease.     

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.     

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.     

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.     

Elastic globes: electron microscopic and immunohistochemical observations

Specimens from a patient with epidermolysis bullosa contained many elastic globes in the dermis. Ultrastructurally they were composed of (i) medium electron-dense amorphous substances, (ii) electron-dense round structures, and (iii) fine filaments. These various elements were seldom organized into typical normal elastic fiber and, therefore, it was difficult ultrastructurally to recognize them as such or components thereof. Immunohistochemically, elastic globes were strongly reactive with NKH-1, which stains elastic microfibrils, and antibody to serum amyloid P component (anti-SAP), which binds to elastic fiber microfibrils. However, elastic globes were negative with EKH-4 which recognizes 50 kd keratin of amyloid keratin and cytoid bodies. These findings suggested that elastic globes have a close immunologic profile to elastic fiber microfibrils, but not that of epidermal or epithelial keratin.     

Tissue amyloid P component in normal human dermis is non-covalently associated with elastic fiber microfibrils

Tissue amyloid P component (TAP), a protein that crossreacts immunohistochemically with the normal plasma glycoprotein serum amyloid P component (SAP), is invariably associated with elastic fiber microfibrils in adult humans. We have investigated the nature of this association. Aliquots of minced, homogenized dermis, obtained following ethylenediamine tetraacetic acid (EDTA) separation of whole adult human skin, were extracted with different reagents, and the presence or absence of TAP in the pellet and in the supernatant following centrifugation was determined by SDS-PAGE and immunoblotting using anti-SAP antibodies. TAP was extractable from dermis using reagents which disrupt non-covalent bonds, including sodium dodecyl sulfate (SDS) and guanidine hydrochloride. TAP was not extracted by high molarity salt solutions, non-ionic detergents, or the reducing agents dithiothreitol and 2-mercaptoethanol. EDTA solution was similarly unsuccessful at eluting TAP from the dermal preparation, indicating that the association of TAP with elastic fiber microfibrils is not simply the result of Ca++-dependent binding. Collagenase solubilized some TAP, but this does not prove covalent linkage to elastic tissue of part of the TAP, because the apparent Mr of TAP extracted was identical to that of normal SAP subunits. We cannot completely exclude the possibility that a few subunits in each multimeric TAP molecule are covalently attached to the microfibrils. However, our findings that denaturing agents alone extracted most of the TAP from normal human dermis strongly suggest that the great majority of the dermal TAP is non-covalently bound to elastic fiber microfibrils. Thus TAP is not an integral constitutent of elastic fiber microfibrils.     

Cutis laxa in hereditary gelsolin amyloidosis

BACKGROUND: Hereditary gelsolin amyloidosis (AGel amyloidosis) is an age-associated systemic disease with global distribution, caused by a G654A or G654T gelsolin gene mutation. Cutis laxa is a principal clinical manifestation of this disease. However, only few data on the dermatological involvement are available, and the pathogenesis of this amyloidosis-associated form of cutis laxa has remained unknown. OBJECTIVES: To elucidate the pathomechanism of this less well-known genodermatosis. METHODS: We performed systematic clinical, histological, immunohistochemical and ultrastructural skin biopsy studies in 12 patients with a G654A gelsolin gene mutation. For comparison, skin specimens from 10 control subjects were analysed. RESULTS: All patients had clinically characteristic cutis laxa, and frequently other signs of symptomatic skin disease such as increased fragility and risk for intracutaneous bleeding. All patients showed cutaneous deposition of gelsolin amyloid (AGel), mainly attached to basement membranes or basal laminae of various cutaneous structures, dermal nerves and blood vessel walls, and elastic fibres, particularly in the lower reticular dermis. AGel often encircled the elastic fibres, and colocalized with amyloid P component (AP), an elastic fibre microfibrillar sheath-associated protein. Fragmentation and loss of elastic fibres, epidermal atrophy, and reduction of dermal appendages were also common. Antibodies to wild-type gelsolin bound to S-100-positive epidermal dendritic cells, a previously unrecognized immunoreaction. Patients had fewer gelsolin-positive dendritic cells than controls. CONCLUSIONS: Widespread skin involvement with AGel deposition and elastic fibre involvement are essential pathological features in AGel amyloidosis, and contribute to the characteristic cutis laxa, dramatic in old age. Codistribution of AGel and AP, with demonstrated specific binding affinity for amyloid fibrils, suggests that elastic fibre-associated AP acts as a matrix for cutaneous amyloid deposition in AGel amyloidosis.     

Keratinocytes express fibrillin and assemble microfibrils: implications for dermal matrix organization

Fibrillin–containing microfibrils are key architectural structures of the upper dermis and integral components of the dermal elastic fibre network. Microfibril bundles intercalate into the dermal—epithelial junction and provide an elastic connection between the dermal elastic fibre network and the epidermis. Immunohistochemical studies have suggested that they are laid down both at the dermal—epithelial junction and in the deep dermis. While dermal fibroblasts are responsible for deposition of the elastin and microfibrillar components that comprise the elastic fibres of the deep dermis, the cellular origin of the microfibril bundles that extrude from the dermal—epithelial junction is not well defined. We have used fresh tissues, freshlyisolated epidermis and primary human and porcine keratinocyte cultures to investigate the possibility that keratinocytes are responsible for deposition of these microfibrils. We have shown that keratinocytes in vivo and in vitro synthesize both fibrillin-1 and fibrillin-2, and assemble beaded microfibrils concurrently with expression of basement membrane collagen. These observations suggest that keratinocytes co-ordinate the secretion, deposition and assembly of these distinct structural elements of the dermal matrix, and have important implications for skin remodelling.     

Middermal elastolysis. Report of a case and immunohistochemical studies on the dermal distribution of fibrillin, vitronectin and amyloid P component.

A 39-year-old woman with demarcated wrinkled areas, histologically characterized by absence of elastic fibers in the middle and upper reticular dermis, is described. Immunoreactivity of vitronectin and amyloid P component, present at the periphery of elastic fibers in normal skin in adults, was absent from the middermis of lesional skin as were orcein stained fibers. C9 neoantigen immunoreactivity, associated with elastic fibers in sun-exposed skin of middle-aged and elderly individuals, was present in conjunction with elastic fibers in papillary and lower reticular dermis in lesional skin but was absent in the middermis. In contrast, a fibrillin immunoreactive network was present throughout the dermis, indicating that the elastin-associated microfibrils are retained in the absence of amorphous elastin in lesional skin of middermal elastolysis.     

Immunohistochemical studies on vitronectin in elastic tissue disorders, cutaneous amyloidosis, lichen ruber planus and porphyria

Vitronectin, identical with serum-spreading factor and S-protein of complement, is a glycoprotein present in both plasma and tissue. It stimulates cell adhesion and spreading and affects the complement and coagulation pathways. Vitronectin immunoreactivity was recently found in conjunction with dermal and renal elastic fibres, in renal amyloid deposits in cases of AL- and AA-amyloidosis, and in sclerotic glomerular lesions. Skin specimens from lesions of patients with selected skin diseases were investigated with an avidin-biotin peroxidase technique using both monoclonal and polyclonal anti-vitronectin antibodies and an alkaline phosphatase anti-alkaline phosphatase technique using monoclonal anti-vitronectin antibodies. Vitronectin immunoreactivity was found in association with the abnormal elastic tissue in solar elastosis and pseudoxanthoma elasticum. It was also found in conjunction with dermal amyloid deposits in primary localized cutaneous amyloidosis and in Civatte bodies in cases of lichen ruber planus. In cases of erythropoietic protoporphyria and porphyria cutanea tarda, hyaline perivascular deposits also demonstrated positive vitronectin immunoreactivity. The presence of vitronectin immunoreactivity not only in normal and degenerated elastic fibres but also in various pathological tissue deposits suggests that vitronectin occurs both in elastic fibres and in different types of abnormal protein deposits.     

Altered erythrocyte membrane phosphorylation in psoriasis

Immunofluorescence staining of exposed skin from patients with erythropoietic protoporphyria (EPP) with antibodies to serum amyloid P component (SAP) and to fibronectin produced striking fluorescence of abnormal vascular structures in the upper dermis. An appearance of linear fluorescence along the dermo-epidermal junction with anti-SAP was the result of confluent staining of papillary oxytalan fibres. Amyloid P component (AP) was localized in ultrastructural immunoperoxidase studies to the peripheral (abluminal) regions of thickened dermal vessel walls, the site of maximum concentration of an amorphous matrix containing microfibrillar structures; antibodies to SAP did not bind to leaflets of the reduplicated vascular basal lamina. The characteristic thickening and reduplication of blood vessel walls seen with the electron microscope in EPP therefore involves increased local deposition of both AP and fibronectin.     

An immunofluorescent study of cutaneous amyloidosis.

Twenty-four patients with primary localized cutaneous amyloidosis were studied by direct immunofluorescent techniques. Autofluorescence of the amyloid deposits was observed in two patients with very chronic lesions. IgG, IgA, IgM and C3 were found in association with the amyloid deposits in about two-thirds of the cases. The immune pathogenesis of amyloid formation is postulated.     

Immunofluorescence and histochemical studies of localized cutaneous amyloidosis

Lichen amyloidosus (LA) and macular amyloidosis (MA) are two forms of localized cutaneous amyloidosis in which the amyloid occurs as larger and smaller deposits respectively in the papillary dermis. The histogenesis of the amyloid of these conditions is unknown. By using an indirect immunofluarescence technique we showed that LA and MA do not react with antibodies against different previously characterized amyloid fibril proteins. These results indicate that the amyloid of LA and MA is different from other known types of amyloid. Protein AP, which was demonstrated in amyloid of MA and LA, is known to be present in all forms of amyloid and is of unknown significance. Antiserum against keratin did not react with the larger homogeneous amyloid bodies, but showed a weak reaction with some small deposits. Histochemical staining failed to show keratin in any of the tissues containing LA or MA.     

The fibrillin microfibril/elastic fibre network: A critical extracellular supramolecular scaffold to balance skin homoeostasis

Supramolecular networks composed of fibrillins (fibrillin‐1 and fibrillin‐2) and associated ligands form intricate cellular microenvironments which balance skin homoeostasis and direct remodelling. Fibrillins assemble into microfibrils which are not only indispensable for conferring elasticity to the skin, but also control the bioavailability of growth factors targeted to the extracellular matrix architecture. Fibrillin microfibrils (FMF) represent the core scaffolds for elastic fibre formation, and they also decorate the surface of elastic fibres and form independent networks. In normal dermis, elastic fibres are suspended in a three‐dimensional basket‐like lattice of FMF intersecting basement membranes at the dermal‐epidermal junction and thus conferring pliability to the skin. The importance of FMF for skin homoeostasis is illustrated by the clinical features caused by mutations in the human fibrillin genes (FBN1, FBN2), summarized as “fibrillinopathies.” In skin, fibrillin mutations result in phenotypes ranging from thick, stiff and fibrotic skin to thin, lax and hyperextensible skin. The most plausible explanation for this spectrum of phenotypic outcomes is that FMF regulate growth factor signalling essential for proper growth and homoeostasis of the skin. Here, we will give an overview about the current understanding of the underlying pathomechanisms leading to fibrillin‐dependent fibrosis as well as forms of cutis laxa caused by mutational inactivation of FMF‐associated ligands.     

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.     

Amyloid P component binds to keratin bodies in human skin and to isolated keratin filament aggregates in vitro

Dermal keratin bodies, consisting mainly of keratin intermediate filament aggregates (KIFA) coated with IgM anti-KIF autoantibodies, are present in normal human skin and occur in increased quantities in certain skin diseases. Keratin bodies are normally rapidly removed, but in primary localized cutaneous amyloidosis (PLCA) they are converted by an unknown mechanism to amyloid. Amyloid P component (AP), a glycoprotein identical to, and derived from, the normal plasma protein serum amyloid P component (SAP), is present in all forms of amyloid including PLCA. We investigated the interaction between SAP, keratin bodies, and KIFA. Immunofluorescence staining of normal skin using fluoresceinated anti-SAP and rhodamine-conjugated anti-IgM, or AE-1/AE-3 anti-keratin antibodies followed by Texas Red-conjugated anti-mouse immunoglobulin, showed that 52% +/- 4 (mean +/- sem, n = 6) of keratin bodies bound anti-SAP. Similar findings were present in a biopsy from a patient with lichen planus. Isolated KIFA, prepared by 8M urea extraction of normal human epidermis or cultured keratinocytes, were preincubated with normal human serum as a source of SAP and then stained with fluoresceinated anti-SAP. Bright fluorescence seen when the incubation medium contained Ca++ was absent in the presence of ethylenediamine tetraacetic acid. Specific Ca++-dependent binding of SAP to KIFA was confirmed using immunoblotting. Binding of SAP to KIFA did not prevent their degradation following exposure to trypsin or alpha-chymotrypsin. Similarly, partial enzymatic digestion of KIFA did not abrogate their ability to bind SAP. Our findings, that SAP is associated with keratin bodies in skin and exhibits Ca++-dependent binding to KIFA in vitro, identify keratin filaments as a newly recognized ligand for SAP.     

IgA immunoreactive deposits collocal with fibrillin immunoreactive fibers in dermatitis herpetiformis skin

The IgA immunoreactive granules or fibrils, characteristically found in dermal papillae of patients with dermatitis herpetiformis, were previously reported to be associated with microfibrillar bundles. Recently, fibrillin, a component of such 8-12 nm microfibrils, was identified. In normal skin, the fibrillin immunoreactive microfibrils are present at the periphery of elastic fibers and are also present without concomitant amorphous elastin in the dermal papillae close to the lamina densa. The localization of the IgA immunoreactive material in the dermal papillae of 17 patients with dermatitis herpetiformis was compared with the distribution of the fibrillin immunoreactive fiber network. Immunofluorescence methods using FITC- and TRITC-labelled antibodies, an avidin-biotin-peroxidase complex technique, and standard elastin staining procedures, were used in several sequential and double staining procedures. In 13 specimens, in which the IgA reactivity was granular, most of the granules were located at the sites of fibrillin-reactive structures. As it could not be excluded that the collocality was coincidental, it could not be ascertained whether the IgA granules were in fact related to the fibrillin immunoreactive fibers in these specimens. However, in 4 specimens with both granular and fibrillar IgA immunoreactive deposits, these were clearly related to and located at the sites of fibrillin-reactive fibrils in the dermal papillae. The results confirm earlier reports of an association of IgA reactive deposits with microfibrillar bundles in dermatitis herpetiformis skin, though the possibility of their binding to other extracellular matrix component(s) has not been ruled out. The findings suggest that fibrillin may be the structural component (or one of them) to which IgA reactive deposits bind in the skin of patients with dermatitis herpetiformis.     

Dermal elastic fibres in the inherited hypermobile disorders

BACKGROUND: Elastic fibres in the inherited hypermobile disorders are probably abnormal on the inherited grounds. The abnormality may contribute for diagnosis and investigation of connective tissue biology. OBJECTIVE: The abnormality will be specific for every disorders and disclosed in the age-dependent change when exposure-dependent change was excluded. MATERIALS AND METHODS: Skin specimens from Ehlers-Danlos syndrome and hypermobile syndrome, Marfan syndrome, Osteogenesis imperfecta type I, homocysteinuria and normal controls are studied by routine electron microscopy. Age-dependent ultrastructural change of broad elastic fibres was evaluated in reticular dermis. RESULTS: Age-dependent change was categorized in infantile, adolescent, adult and senile pattern. Infantile pattern showed normal ultrastructure. Degeneration was first found in adolescent pattern by disarrayed microfibrils and degenerate matrix. Degeneration proceeded in adult pattern and ended in senile pattern. Typical abnormality developed in adult pattern. Ehlers-Danlos syndrome and hypermobile syndrome showed no specific abnormality but the degeneration began earlier and was severer than the control. Marfan syndrome showed defects of microfibrils and matrix surface. Moth-eaten figure was characteristic. Osteogenesis imperfecta showed abnormal matrix and revealed homogenous bulges of matrix. Homocysteinuria was specified by numerous microfibrils on the matrix surface in infantile pattern. CONCLUSION: Abnormality of elastic fibre was recognized in the reticular dermis of elbow. Ehlers-Danlos syndrome and hypermobile syndrome showed no specific abnormality but the degeneration was more intensive than the control. Abnormality of Marfan syndrome was degeneration of elastic microfibrils and matrix surface, Osteogenesis imperfecta was characterized by excess amount of matrix. Homocysteinuria revealed numerous microfibrils. Ultrastructural abnormality provided grounds for studies on histopathology and biology of elastic fibre.