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.     

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.     

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 study of calretinin in normal skin and cutaneous adnexal proliferations

Calretinin is a calcium-binding protein member of the EF-hand family. The presence of calretinin has been demonstrated in certain stages of the cellular cycle in a wide variety of normal and neoplastic tissues. The main aims of our study were (1) to investigate what structures of the normal skin and cutaneous adnexal proliferations express immunoreactivity for calretinin and (2) to determine the value of immunohistochemical expression for calretinin as a marker for follicular, sebaceous, apocrine, and eccrine differentiation in cutaneous adnexal proliferations. We studied 139 biopsy specimens, including 10 cases of normal skin of different locations and 129 benign and malignant cutaneous adnexal proliferations. In normal skin, we found that calretinin is expressed in the innermost cell layer of the outer root sheath in anagen hair follicle, in both the duct and sebolemma of the sebaceous gland, in the secretory portion of eccrine glands, and in mast cells of the stroma. In cutaneous adnexal proliferations, we found strong immunoreactivity for calretinin in tricholemmal cysts, tricholemmomas/inverted follicular keratoses, tumors of follicular infundibulum, and in some basal cell carcinomas. Focal positivity was also seen in trichoadenomas, trichoblastomas/trichoepitheliomas, pilomatricomas, proliferating tricholemmal tumors, pilar sheath acanthomas, trichofolliculomas, follicular hybrid cysts, cutaneous mixed tumors, steatocystomas, sebaceous hyperplasias, and sebaceomas. These results demonstrate that immunohistochemical study for calretinin may be helpful to identify the innermost cell layer of the outer root sheath in anagen hair follicle and the cutaneous adnexal proliferations showing differentiation toward this structure. Calretinin immunoreactivity supports eccrine differentiation in some sweat gland neoplasms, and it is also useful in identifying neoplasms with ductal sebaceous differentiation.     

Immunohistochemical demonstration of proteoglycans in the skin of patients with systemic sclerosis

Skin proteoglycan was demonstrated by an immunofluorescent technique using an antibody against bovine cartilage proteoglycan, after the cross-reactivity of human proteoglycan with the antiserum had been confirmed. Normal skin exhibited specific fluorescence mainly in the blood vessels as well as in the subepidermal area. The clinically uninvolved skin of systemic sclerosis (SS) revealed no features different from those of normal skin. However, the vascular proteoglycan deposition of early systemic sclerosis was later replaced by deposition between the collagen fibres, which appeared to progress centrifugally in parallel to the increase in the skin sclerosis, suggesting a vascular initiation of the skin lesion. Sclerotic skin was characterized by random deposition between the collagen fibres. Immunoelectron microscopic studies suggested that the random proteoglycan deposition reflected uncontrolled local accumulation of proteoglycan in the interfibrillar matrix around irregularly arranged collagen fibrils.     

Amyloidogenesis in organ-limited cutaneous amyloidosis: an antigenic identity between epidermal keratin and skin amyloid

Epidermal keratin was extracted and antibody against this protein was produced in rabbits. Various forms of organ-limited cutaneous amyloidosis (lichenoid, macular, and nodular amyloidosis, and basal cell epithelioma) and primary systemic amyloidosis were immunohistochemically examined to test the identity between epidermal keratin and skin amyloid. Amyloids in lichenoid and macular amyloidoses, and in basal cell epithelioma had an identical antigenicity with epidermal keratin, whereas amyloids in nodular amyloidosis and systemic amyloidosis did not have this identity. In addition, amyloid in lichen amyloidosis contained disulfide bonds as in keratin. Connective tissue components including filaments of fibroblasts and vascular endothelial cells did not react with this antikeratin antibody. It was concluded that at least some of the amyloid substance in organ-limited cutaneous amyloidosis is derived from degenerated epidermal keratinocytes through filamentous degeneration or apoptosis.     

Immunohistochemical demonstration of lysozyme in cutaneous histiocytic infiltrates

Intracellular lysozyme (Muramidase) in cells of the monocyte-histiocyte-macrophage series is demonstrated by an immunoperoxidase technique in paraffin-embedded cutaneous sections. Lesions comprising mixed inflammatory and granulomatous infiltrates and tumours of presumed histiocytic origin were studied. The method provides a simple, reliable and reproducible means of identifying histiocytic cutaneous infiltrates in routinely processed tissue.     

Immunoperoxidase and enzyme-histochemical demonstration of human skin tryptase in cutaneous mast cells in normal and mastocytoma skin

Summary Trypsin-like proteinase isolated from human skin was localized in cutaneous mast cells using immunoperoxidase and enzyme-histochemical techniques. Skin biopsy specimens were taken from four mastocytoma and four healthy patients. Immunoperoxidase staining was performed with protein A-sepharose purified rabbit polyclonal antibody raised against human skin tryptase and using aminoethylcarbazole as chromogen. The positively stained cells in the dermis were granular in character. Using peptide 4-methoxy-2-naphthylamide substrates (Bz-Arg-MNA, Z-Lys-Arg-MNA, Z-Gly-Arg-MNA, Z-Pro-Arg-MNA and Z-Gly-Pro-Arg-MNA) and Fast Garnet GBC as chromogen the red azo dye was found to precipitate in the cytoplasmic granules of the cutaneous mast cells. The enzymatic reaction was totally inhibited by diisopropyl fluorophosphate, leupeptin, and benzemidine. No marked inhibition was seen with soybean trypsin inhibitor and alpha-1-antitrypsin. The best substrate was Z-Gly-Pro-Arg-MNA giving the strongest red azo dye when incubation time was 15,30 or 60 min. These results show the localization of human skin tryptase in dermal mast cells and the usefullnes of Z-Gly-Pro-Arg-MNA as a suitable substrate tested for enzyme-histochemical localization of mast cells in healthy or mastocytoma skin.     

Epidermal origin of the amyloid in localized cutaneous amyloidosis

A case of localized cutaneous amyloidosis which developed after a lichen planus-like skin reaction is reported. The amyloid consisted of amyloid fibrils enveloped by heparan sulphate granules. These amyloid fibrils reacted to anti-human keratin antibody, indicating an epidermal origin for the fibrils.     

Unusual skin manifestation of cutaneous amyloidosis

A 76-year-old man with a 20-year history of extensive cutaneous amyloidosis is reported. He had asymptomatic symmetric brownish reticulated pigmented patches with well-demarcated borders on his thighs, lower legs, dorsal feet and both arms. The trunk and popliteal fossae were not affected. A skin biopsy specimen showed abundant amyloid deposits in the papillary dermis and reticular dermis. Despite the extensive cutaneous involvement and large amount of amyloid in the deep dermis, no evidence of systemic amyloidosis could be found. Various manifestations of cutaneous amyloidosis are reviewed. We report this case to remind dermatologists of the protean presentations of cutaneous amyloidosis.     

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.     

Immunohistochemical demonstration of breast-derived and/or carcinoma-associated glycoproteins in normal skin appendages and their tumors

Sixty-six benign and malignant skin appendage tumors were studied for the expression and localization of the glycoproteins identified by the monoclonal antibodies (MoAbs) GCDFP-15, CU18, B72.3, and VU-1D9. Formalin-fixed, paraffin-embedded tissue was processed by the avidin-biotin complex method. In normal eccrine and apocrine sweat glands, GCDFP-15, CU18, B72.3, and VU-1D9 staining was localized differently (intracellular, membranous, or intraluminal), whereas eccrine glands showed no B72.3 staining. There were various patterns of positive staining of tumors arising from sweat glands, but no immunoreactivity for B72.3 was found in eccrine-derived tumors. CU18 and VU-1D9 labeled mature sebocytes in a vacuolar fashion and stained sebaceous carcinomas. VU-1D9 labeled membranes of the secondary germ cells in early anagen of a hair follicle bulb as well as the basaloid cells of trichoepitheliomas and basal cell carcinomas. These MoAbs appear to be valuable markers for the study of normal skin appendages and their tumors.     

A monoclonal anti-keratin antibody reactive with amyloid deposit of primary cutaneous amyloidosis

Specimens from cutaneous amyloidoses (lichen amyloidosis and macular amyloidosis) were stained immunohistochemically with monoclonal anti-keratin antibodies. One monoclonal antibody raised against hair keratin (HKN-6) reacted with the amyloids of both primary amyloidoses. Another monoclonal antibody, HKN-2, did not decorate the amyloid deposit. HKN-6 did not stain the interfollicular epidermis, but HKN-2 did. The possible explanations of these findings are 1) amyloid deposits contain keratin protein modulated to react with HKN-6; 2) amyloid deposits contain a protein unrelated to keratin protein, but reactive to HKN-6.     

Nodular primary cutaneous amyloidosis. Isolation and characterization of amyloid fibrils

A case of nodular cutaneous amyloidosis and Sj?gren's syndrome occurred in a 63-year-old woman. Nodules had been seen for the past ten years and Sj?gren's syndrome had accompanied amyloidosis for the last three years. The concomitant occurrence of nodular cutaneous amyloidosis and Sj?gren's syndrome may not be by chance, since four of 12 cases of nodular cutaneous amyloidosis that have been reported to date in Japan were in patients with both amyloidosis and Sj?gren's syndrome. The amyloid deposits in the tissue were stained with anti-lambda light-chain amyloid antibody. Amyloid fibrils were purified from the skin lesions in this patient and were characterized biochemically, immunologically, and ultrastructurally. The results indicated that the amyloid fibrils consisted of 29,000-, 20,000-, and 17,000- dalton peptides, the 29,000-dalton peptide of which was shown to react with the lambda light chain of immunoglobulin by immunoblot study.     

Immunohistochemical demonstration of MAM-3 and MAM-6 antigens in normal human skin appendages and their tumors

Summary The expression of MAM-3 and MAM-6 antigens was immunohistochemically investigated on 110 tumors of human skin appendages. Forty-two samples from tumor-adjacent normal skin appendages were also studied. MAM-3 antigens, as detectable by monoclonal antibodies (MoAbs) 67D11, 115G3, and 115H10 were present in the inner layer cells but not in the outer layer cells of normal eccrine excretory ducts. Sporadic positivity was also found in cytoplasm of apocrine acini with 115G3, while 67D11 and 115H10 were negative. MAM-6 antigens, as detectable by the MoAbs 115D8, 115F5, 139H2, 140C1, and 126E7 were found in the secretory canaliculi of normal eccrine acini and within the apical lumina at the terminal portion of ducts. Apocrine acinar cells mainly exhibited an apical staining, but a focal supranuclear dot-like staining could also be observed. A foamy reaction pattern for MAM-6 was noted in mature sebocytes. However, none of the antigenic epitopes was expressed in normal squamous epithelium or hair follicles. In benign tumors, the staining patterns for both antigens, in general, resembled their distribution in the corresponding normal tissues. However, carcinomas orginating from sweat glands, sebaceous glands, and the pilar apparatus expressed both antigens in a more irregular and heterogeneous pattern. This might preferably be explained by the loss of those mechanisms controlling the antigen expression in mature, functional tissues. Conclusions from these immunohistochemical studies with regard to the histogenesis mainly of the malignant skin appendage tumors should be drawn with caution.     

Advanced glycation end product-modified beta2-microglobulin is a component of amyloid fibrils of primary localized cutaneous nodular amyloidosis

Primary localized cutaneous nodular amyloidosis is a rare form of cutaneous amyloidosis. Amyloid fibrils in primary localized cutaneous nodular amyloidosis have been reported to be originated from immunoglobulin light chains. Immunohistochemical studies on the lesional skins of four patients with primary localized cutaneous nodular amyloidosis demonstrated that amyloid deposits of all cases showed a positive reaction with the antibodies for beta2-microglobulin and advanced glycation end products as well as immunoglobulin light chain (kappa or lambda). No beta2-microglobulin and advanced glycation end product immunoreactivity was found in the amyloid deposits of other primary localized cutaneous amyloidosis (lichen amyloidosis and macular amyloidosis). Double immunofluorescence study of the lesional skin of primary localized cutaneous nodular amyloidosis showed that anti-kappa light chain, anti-beta2-microglobulin and anti-advanced glycation end product antibodies mostly reacted with the same area of amyloid deposit. Amyloid proteins were sequentially extracted with distilled water from one case of primary localized cutaneous nodular amyloidosis and recovered in the five water-soluble fractions (fractions I-V). Immunoblot assay of amyloid fibril proteins demonstrated that immunoreactive polypeptides with anti-kappa light chain antibody (29 kDa) and with anti-beta2-microglobulin antibody (12 kDa) were detected in fractions I-V, whereas immunoreactive polypeptide with anti-advanced glycation end product antibody (12 kDa) was detected exclusively in fractions III-V but not in fractions I and II. Two-dimensional polyacrylamide gel electrophoresis revealed that 12 kDa polypeptide in fractions I and II was electrophoretically identical with authentic beta2-microglobulin and that beta2-microglobulin in fractions III-V was advanced glycation end product-modified beta2-microglobulin with more acidic pI value. These results indicate that beta2-microglobulin is another major component of amyloid fibrils in primary localized cutaneous nodular amyloidosis and that beta2-microglobulin in primary localized cutaneous nodular amyloidosis is partly subjected to the modification of advanced glycation end product.     

Formation of immunoglobulin light chain amyloid oligomers in primary cutaneous nodular amyloidosis

Background Primary cutaneous nodular amyloidosis (PCNA) is thought to be a plasma cell dyscrasia. The amyloid deposits are found in the dermis and subcutis, and they contain clonal immunoglobulin light chains, produced by a local proliferation of plasma cells. New insights into amyloid diseases have revealed that the pathology is due more to the presence of small, misfolded protein species termed oligomers than to the deposition of fibrillar material. Objectives To demonstrate the presence of amyloid oligomers in PCNA and to provide evidence that cutaneous amyloid diseases share a common pathogenic pathway similar to other amyloid diseases. Methods Immunohistochemical staining with conformation‐specific and sequence‐specific antibodies was used to localize different amyloid species of light chain immunoglobulins in a case of PCNA. Additionally, in vitro characterization of immunoglobulin oligomers and fibrils was performed to determine, through toxicity studies in a human keratinocyte cell line, which amyloidogenic form of the immunoglobulin is toxic in PCNA. Results Amyloid oligomers were identified in PCNA. Oligomers were mainly formed by lambda light chain immunoglobulins, and kappa light chain oligomers were detected in lesser amounts. Amyloid species were detected intra‐ and extracellularly. In addition, amyloid oligomers and fibrils, derived from unknown protein sources, were detected. This finding suggests that immunoglobulin amyloids can act as seeds capable of inducing the aggregation of heterogeneous proteins in the skin. Furthermore, cytotoxicity studies demonstrated that immunoglobulin oligomers, but not monomers or fibrils, are toxic to human keratinocytes. Conclusions These data indicate that PCNA has common pathways with other amyloid diseases with respect to protein misfolding and pathogenesis. Immunoglobulin oligomers may prove to be targets for the treatment of PCNA.     

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.