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.     

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.     

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.     

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.     

Ultrastructural localization of amyloid P component in primary localized cutaneous amyloidosis

Amyloid P component (AP) was specifically localized to dermal amyloid deposits in the papular and nodular variants of primary localized cutaneous amyloidosis by an immunoperoxidase technique using an antibody to serum amyloid P component (SAP). Specific staining with anti-SAP of elastic fibre microfibrils which has previously been observed in normal skin, was also present and was noted in close proximity 10 deposits of amyloid material. AP associated with normal elastic fibre microfibrils may be involved in the deposition of amyloid fibrils in vivo.     

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.     

Dermatitis herpetiformis bodies. Ultrastructural study on the skin of patients using direct preembedding immunogold labeling

Skin samples from three adult patients with dermatitis herpetiformis (DH) and granular IgA deposits in the papillary tips were studied using ultrastructural immunogold technique. IgA positive, so-called DH bodies were identified as amorphous clumps--most probably immunocomplex aggregates--scattered throughout the upper papillary dermis. Dermatitis herpetiformis bodies were seen underneath the basement membrane, sometimes along microfibrillar bundles, as well as adjacent to the papillary collagen fibers and within the surface (microfibrillar) region of elastic tissue. Some DH bodies, however, were not related to any fibrillar components. The collagen and elastic fibers, microfibrillar bundles, anchoring fibrils, and elastic microfibrils themselves were unlabeled. Dermatitis herpetiformis bodies were not found in normal human skin. The results of our ultrastructural study indicate that DH bodies either are bound to a nonfibrillar component of dermal connective tissue or represent deposits of immune complexes trapped in DH skin.     

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.     

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.     

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.     

Depletion of cutaneous peptidergic innervation in HIV-associated xerosis

Severe xerosis occurs in approximately 20% of human immunodeficiency virus seropositive patients. Changes in cutaneous innervation have been found in various inflammatory skin diseases and in xerotic skin in familial amyloid. We have therefore carried out a quantitative examination of the cutaneous peptidergic innervation in human immunodeficiency virus-associated xerosis. Immunohistochemistry and image analysis quantitation were used to compare total cutaneous innervation (protein gene product 9.5), calcitonin gene-related peptide, substance P, and vasoactive intestinal peptide peptidergic fibers, at two sites in the skin of human immunodeficiency virus-associated xerosis patients (upper arm, n = 12; upper leg, n = 11) and site-matched seronegative controls (upper arm, n = 10; upper leg, n = 10). Measurement of lengths of fibers of each type was carried out for each subject in the epidermis and papillary dermis, and around the sweat glands. Immunostained mast cells in these areas were counted. Epidermal integrity and maturation were assessed by immunostaining for involucrin. There were significant (Mann-Whitney U test; p < 0.02) decreases in total lengths of protein gene product 9.5 fibers in both epidermis/papillary dermis and sweat gland fields; of calcitonin gene-related peptide innervation in the epidermis/papillary dermis; and of substance P innervation of the sweat glands. There were no differences in the distribution of mast cells, or in the epidermal expression of involucrin. Depletion of the calcitonin gene-related peptide innervation may affect the nutrient blood supply of the upper dermis, and the integrity and function of basal epidermis and Langerhans cells. Diminished substance P innervation of the sweat glands may affect their secretory activity. Both of these changes may be implicated in the development of xerosis.     

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.     

Anchoring fibrils and type VII collagen are absent from skin in severe recessive dystrophic epidermolysis bullosa

Skin of patients with severe generalized recessive dystrophic epidermolysis bullosa (SGRDEB) was studied by immunoelectron microscopy and immunoblotting with antibodies to type VII collagen, a major structural component of anchoring fibrils. In normal skin, the protein was localized to the dermoepidermal junction zone below the basement membrane and was extractable from the papillary dermis after artificial epidermolysis. In SGRDEB skin, neither immunoreactive material below the basement membrane nor identifiable anchoring fibrils could be recognized and neither the tissue form nor the specific proteolytic fragments of type VII collagen were found in extracts of SGRDEB skin. Very low amounts of type VII collagen alpha-chains could be detected in cultures of SGRDEB-fibroblasts, whereas normal fibroblasts synthesized more of this collagen. These results suggest that a genetic defect in the correct synthesis, secretion, or in the molecular assembly of type VII collagen may underlie SGRDEB.     

尖锐湿疣患者皮损中朗格汉斯细胞和浆细胞样树突细胞的检测

目的 探讨朗格汉斯细胞（LC）和浆细胞样树突细胞（pDC）在尖锐湿疣（CA）感染发病机制中的可能作用。 方法 免疫组化SP法检测23例CA患者皮损中CD1a+LC和CD2AP+pDC、CD123+pDC的表达和分布情况,以其中13例CA组织的边缘正常皮肤为对照,比较CA皮损和边缘正常皮肤中CD1a+LC、CD2AP+pDC、CD123+pDC表达密度和阳性率的差异。 结果 CD1a+LC主要位于表皮基底层上棘层,少数在真皮乳头层。CA皮损中CD1a+LC在表皮层和真皮层的密度及阳性率与边缘正常对照组比较,差异无统计学意义（P > 0.05）。CD2AP+pDC、CD123+pDC主要位于真皮乳头层,CA患者皮损中CD2AP+pDC、CD123+pDC的密度和阳性率均比边缘正常对照组显著增高（P < 0.05）。 结论 人乳头瘤病毒感染影响局部黏膜的pDC数量,LC数量未见变化。     

Loss of hyaluronan in the basement membrane zone of the skin correlates to the degree of stiff hands in diabetic patients

Glycosaminoglycans are important components of all extracellular matrices. One of the glycosaminoglycans is hyaluronan, which is ubiquitously distributed throughout the connective tissue. Hyaluronan is especially abundant in the skin, in which it is of both structural and functional importance. This study describes the localization and distribution of hyaluronan in the skin of healthy individuals and of 23 patients with insulin-dependent diabetes mellitus and various degrees of limited joint mobility. In normal skin, hyaluronan staining was seen in all layers but most prominently in the papillary dermis and the basement membrane zone. In the skin from diabetic patients with normal or only moderately restricted mobility of the hands (limited joint mobility grades 0 and 1), the distribution of hyaluronan was similar to that of normal skin. In the skin of patients with severe restriction in joint mobility (limited joint mobility grade 2) the staining pattern was significantly different with weak hyaluronan staining in the papillary dermis and the basement membrane zone almost devoid of hyaluronan. Moreover, an increased epidermal thickness in the latter patients was evident as well as a pronounced hyaluronan staining compared with normal epidermis.     

Extensive variant of cutaneous amyloidosis: report of a case with electron-microscopic and immunohistochemical studies of the basement membrane zone at sites of amyloid production.

A 60-year-old Japanese female developed widespread lichenoid eruptions with pigmentation, which initially appeared in preceding erythematous skin lesions due to dermatomyositis. Thioflavine T and Dylon stainings, electron microscopy and immunohistochemistry revealed that thick amyloid deposits were present in the papillary dermis particularly beneath the epidermis. Autopsy showed no evidence of systemic amyloidosis. Electron microscopy of the lesional skin disclosed the disturbance of lamina densa formation in the epidermal basement membrane zone (BMZ). There was disruption and dissociation of the lamina densa from the basal cell, and a lamina-densa-like substance was found in the amyloid deposits. Immunofluorescence and immunoelectron microscopy showed that type IV and VII collagens, LDA-1 antigen (a noncollagenous component of the BMZ) and laminin were distributed in irregular thick deposits along the BMZ and were also present within the amyloid itself. These findings indicate that morphological and immunohistochemical abnormalities of the lamina densa may be involved in amyloid production at the interface of the epidermis and dermis, at least in this case.     

Elastic fibres in normal and sun-damaged skin: an immunohistochemical study

Sun-exposed and sun-protected skin obtained at post mortem from the nape of the neck in 14 subjects was immunostained using antisera to elastin, lysozyme, amyloid P component, and the plasma protease inhibitors alpha-I antitrypsin, alpha-I antichymotrypsin and alpha-2 macroglobulin. Both the normal elastic fibres in sun-protected skin, and elastosis in sun-exposed skin were positively immunostained for elastin, lysozyme and amyloid P component. Collagen fibres were unstained. No immunostaining of normal elastic fibres or elastosis in the skin was obtained with antisera to alpha-I antitrypsin, alpha-I antichymotrypsin or alpha-2 macroglobulin. It was concluded that the elastosis in sun-exposed skin does contain elastic fibres. The absence of immunostaining for plasma protease inhibitors probably indicates that the elastic material is mature, and not newly-formed.     

Elastolysis in lichen ruber planus.

The dermal elastic fiber network was studied in specimens from five patients with lichen ruber planus, using a standard elastin staining procedure (orcein), results being compared with those for the elastin-associated microfibrillar network stained using anti-fibrillin antibodies in an immunofluorescence and an avidin-biotin-peroxidase complex technique. Additional specimens of healed apparently normal skin were taken from two of the patients. Orcein-stained fibers were scarce or absent in the inflammatory zone in all the lesions. In contrast, an extensive fibrillin immunoreactive network was present in the papillary zone in all the specimens, in a pattern similar to that of normal skin. In specimens from healed lesions of lichen ruber planus, dermal orcein-stained fibers were present in the papillary dermis. The findings indicate that the amorphous component of elastic fibers is destroyed during the acute phase of lichen ruber planus. Hypothetically, the elastolysis is caused by elastases released from macrophages known to be present in the lichenoid infiltrate. In contrast, the fibrillin fiber network seems to be less or not at all affected by proteolytic events during the inflammatory phase of lichen ruber planus.     

Enhanced expression of human metalloelastase (MMP-12) in cutaneous granulomas and macrophage migration

Accumulation of inflammatory cells such as macrophages may lead to degeneration of connective tissue matrix in various skin diseases. Macrophage metalloelastase, is a matrix metalloproteinase (MMP-12) capable of degrading elastin as well as various basement membrane components. To investigate the role of human macrophage metalloelastase in skin, we assessed by in situ hybridization and immunohistochemistry 66 specimens representing skin diseases characterized either by changes in elastic fibers or by pronounced infiltrations of extravasating and migrating macrophages. CD68 immunostaining was performed to identify the human macrophage metalloelastase-positive cells and Weigert's Resorcin-Fuchsin staining to reveal the status of elastic fibers. We found abundant expression of human macrophage metalloelastase mRNA in macrophages in areas devoid of normal elastic fibers in granulomatous skin diseases sarcoidosis, necrobiosis lipoidica diabeticorum, and granuloma annulare. Positive cells for human macrophage metalloelastase protein could be detected in the same regions as well as positive immunostaining for urokinase plasminogen activator. Of the other matrix metalloproteinases capable of degrading elastin, 92 kDa gelatinase colocalized with human macrophage metalloelastase, while 72 kDa gelatinase was produced by surrounding fibroblast-like cells. Furthermore, human macrophage metalloelastase was expressed by macrophages in areas with disrupted basement membrane, as assessed by type IV collagen staining, in pityriasis lichenoides and dermatitis herpetiformis. Specimens of anetoderma, acrodermatitis chronica atrophicans and pseudoxanthoma elasticum showed no signal for human macrophage metalloelastase. Matrilysin was not detected in any of the samples investigated. Our study suggests that human macrophage metalloelastase may contribute to elastin degradation occurring in granulomatous skin diseases and may aid macrophage migration through the epidermal and vascular basement membranes in inflammatory disorders.     

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.