Eosinophilia-associated muscle disorders: an immunohistological study with tissue localisation of major basic protein in distinct clinicopathological forms.

AIMS: (a) To evaluate tissue eosinophil density, location of eosinophil cytotoxic products, histopathological muscle changes and inflammatory cell types in different eosinophilia-associated myopathies that are clinicopathologically heterogeneous. (b) To determine the immunohistological range of tissue eosinophil density in non-eosinophilic inflammatory myopathies. METHODS: Muscle biopsy specimens from seven patients with blood and/or tissue eosinophilia and clinicolaboratory myopathic signs (five chronic course myopathies, one subacute onset fasciitis/myositis, one acute myositis), and from 18 non-eosinophilic inflammatory myopathies, underwent routine staining, inflammatory infiltrate immunophenotyping, immunostaining for eosinophil major basic protein (MBP) and transmission electron microscopy examination. Eosinophil and total inflammatory cell counts were statistically analysed. RESULTS: Histological examination showed occasional or no infiltrating eosinophils in all cases. MBP staining showed that tissue eosinophil density and percentages in eosinophilia-associated myopathies were significantly higher than in idiopathic myositides. Extracellular MBP diffusion, the hallmark of eosinophil cytotoxicity, was recurrent on sarcolemma and endothelium. Electron microscopy showed eosinophils close to sarcolemma, abundant mast cells, and capillary endothelial swelling. Immunostaining detected a higher mean eosinophil density in idiopathic myositides than previously assessed histologically. CONCLUSIONS: MBP immunohistology on skeletal muscle, previously performed only for acute eosinophilic polymyositis, suggests that eosinophil-mediated injury of muscle cells may occur in a wider spectrum of less aggressive eosinophilia-associated myopathies than previously thought. As conventional histology is likely to underestimate this leucocyte subset, MBP staining may be a useful tool in the analysis of tissue infiltration of eosinophils as a possible treatment target.

Lymphatic vessels in human eyelids: an immunohistological study in dermatochalasis and chalazion.

We investigated lymphatic morphology and expression of endothelin (ET-1) axis molecules in human eyelids affected by an inflammatory state (chalazion) and an age-related degenerative condition (dermatochalasis). Lymphatics were immunohistologically detected by D2-40/LYVE-1 staining. Absorbing lymphatic vessels were localized in papillary dermis and around skin appendages with distinctive morphology. In chalazion, D2-40 reactive flattened lymphatic profiles were compressed by inflammatory infiltrate; in dermatochalasis, large fully opened lymphatics were observed, with a significantly wider total area (lymphatic lumina/200x field; p < 0.05). The lymphatic density (number/200x field) in the two groups was within the same range. Lymphatic dilation is possibly dependent on reduction and fragmentation of the dermal elastic network as well as of oxytalanic fibers in the papillary dermis of dermatochalasis, as shown by Weigert's reaction. Multifunctional peptide ET-1, involved in vasomotion, inflammation and connective proliferation, was faintly and discontinuously localized on lymphatics, as was its type A receptor. In contrast, the consistent expression of type B receptor indicates that lymphatic endothelium is a physiological target for ET-1, whose effects are modulated by multiple pathophysiological conditions. Thus, vasoactive factors play a role in the physiology of richly vascularized eyelids, and therefore, morphofunctional characterization of lymphatic vessels may be useful in suggesting treatment options.

Endothelin-1 and endothelin-converting enzyme-1 in human granulomatous pathology of eyelid: an immunohistochemical and in situ hybridization study in chalazia.

Endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in several functions of eye pathophysiology, such as regulation of intraocular tension and retinal reactive vasoconstriction. As ET-1 pro-inflammatory and fibrosing activity is emerging in different fields of pathology, we investigated the expression of ET-1 and endothelin-converting enzyme-1 (ECE-1) in chalazia, granulomatous lesions of the eyelid. ET-1 and ECE-1 were analyzed by immunohistochemistry (IHC) in twenty surgically removed chalazia, with regard to expression in eyelid structures and inflammatory infiltrate. Phenotype of ET-1 expressing inflammatory cells was established by double immunofluorescence. The cellular localization of prepro-ET-1 (pp-ET-1) mRNA and ECE-1 mRNA was studied by nonisotopic in situ hybridization (ISH). Neutrophils (PMNs), macrophages and T-lymphocytes were scattered in stroma, around alveoli and grouped in lipogranulomas. PMNs, macrophages, basal epithelium of meibomian adenomers and central ducts immunostained for ET-1. ECE-1 protein was found in meibomian adenomers, conjunctival epithelium, tarsal mucous glands and in inflammatory cells. Hybridization signals for pp-ET-1 mRNA and ECE-1 mRNA were recognized in healthy and degenerating meibomian ducts, adenomers, inflammatory cells, as well as in vessel walls. ECE-1 mRNA was also present in conjunctival epithelium and Henle's crypts. Our findings suggest that the multifunctional peptide ET-1 may have a role in molecular genesis of tissue damage in chalazia. ET-1 cytokine activity is likely to support the migration of inflammatory cells and the setting of lipogranulomas; ET-1 stimulation might contribute to proliferation of fibroblasts and collagen synthesis. ET-1 upregulation on meibomian adenomers and ducts may further enhance granulomas formation by stimulating lipid release.

Lymphatic vessels in human sural nerve: immunohistochemical detection by D2-40.

Lymphatics were detected in the epineurium of the human sural nerve by D-240 immunostaining and confirmed by ultrastructural examination.

The pattern of fibrillin deposition correlates with microfibril-associated glycoprotein 1 (MAGP-1) expression in cultured blood and lymphatic endothelial cells.

Fibrillins constitute the major structural components of 10-12nm microfibrils of the extracellular matrix of several elastic and non elastic tissues and of initial lymphatic vessel anchoring filaments. Microfibril-Associated Glycoprotein-1 (MAGP-1) binds fibrillin to tropoelastin during elastogenesis. We recently reported that cultured blood endothelial cells deposit fibrillin in a honeycomb pattern, whereas lymphatic endothelial cells form an irregular web. The aim of this immunohistochemical study was to verify whether the deposition pattern of fibrillin is related to the expression of MAGP-1 in confluent and postconfluent cultures of bovine aortic (AEC), pulmonary artery (PAEC) and lymphatic endothelial cells (LEC). In AEC and PAEC, MAGP-1 and fibrillin co-localized and their deposition increased with time in culture. In AEC, both proteins formed a honeycomb pattern. In LEC, MAGP-1 deposition was still negligible when fibrillin formed an irregular web covering the entire surface. PAEC, which in vivo are exposed to physiological conditions intermediate between AEC and LEC, had an intermediate pattern of deposition of fibrillin and MAGP-1. Assuming that early elastogenesis is an intrinsic functional need for the aorta, but not for the thoracic duct, we propose that delayed appearance of MAGP-1 in LEC may correlate with their irregular fibrillin deposition. Different fibrillin scaffolds could in turn account for the specificity of elastic fibers in compliance with the specific functional requirements of the tissue.

Specific adhesion molecules bind anchoring filaments and endothelial cells in human skin initial lymphatics.

Anchoring filaments are a characteristic feature of initial lymphatic vessels. They connect the abluminal membrane of endothelial cells to the surrounding elastic fibers. The main molecular component of anchoring filaments is fibrillin. Initial lymphatic vessels of human skin were stained with monoclonal antibodies to fibrillin, integrins alpha 2 beta 1, alpha 3 beta 1 and alpha v beta 3, vinculin, talin, beta-actin and focal adhesion kinase (FAK). A double-labeling immunofluorescence method was used to simultaneously stain fibrillin and alpha 3 beta 1 integrin or FAK. Close contiguities between integrins and anchoring filaments were observed. These results suggest that the anchoring filaments connect the extracellular matrix and the endothelial cell cytoskeleton through the transmembrane integrin and FAK molecule. The results also demonstrate the presence of focal adhesions in the wall of initial lymphatic vessels. These connections possibly enable transmission of chemical and/or mechanical stimuli from the extracellular matrix to the endothelial cells. Here, they are transformed in cytoskeleton rearrangements and intracellular signaling events, some of which may contribute to the initial formation of lymph.

Lymphatic vessels of the human heart: precollectors and collecting vessels. A morpho-structural study.

Only topographic and distributional data are available on the lymphatic outflow vessels of the human heart. Here we describe their structural and ultrastructural features. Fragments of the atria, ventricles and fat surrounding the major coronary branches were obtained from hearts of dilated cardiomyopathy patients. Serial semithin sections were observed under light microscopy and used for tridimensional reconstructions. Ultrathin sections were observed by transmission electron microscopy. Precollectors, the initial lymphatic outflow routes of the heart, are small valved vessels with irregular, discontinuous musculature. They originate in the subepicardial region from a network of epicardial, and from scattered myocardial absorbing lymphatic vessels and drain into the collecting vessels accompanying the major coronary branches. Collecting vessels are larger but structurally similar to precollectors. Wall musculature is independent of the size of the vessel. Their ultrastructure is the same as that of precollectors. Endothelial cells have many Weibel-Palade bodies, cytoplasmic filaments and focal adhesions. The basement membrane is discontinuous and anchoring filaments are frequent and conspicuous. The subendothelial layer contains much elastin. Human heart collecting vessels and precollectors may only be distinguished by their size. The scarcity of musculature suggests that lymph progression in this district is mainly ensured by cardiac revolutions. Their ultrastructural features are determined by adaptation to dynamic forces. The architecture of these vessels (random, disorderly, discontinuous, lacking any exact plan) and their large variations in caliber are in line with the ontogenetic hypothesis that peripheral lymphatic vessels originate from the coalescence of mesenchymal lacunae.

Vasa vasorum of superficial collecting lymphatics of human thigh.

Collecting lymphatics were obtained from human thigh fat for light microscopy and tridimensional reconstruction at time of operation for varicose veins. No patient had lymphedema and routine sections showed no inflammation or notable pathologic alteration of the surrounding soft tissue. Abundant vasa vasorum was observed around the musculature of superficial collecting lymphatics of human thigh. Within intervalvular portions of the lymphatic collectors where the muscle coat was thicker and more compact, the vasa vasorum penetrated between smooth muscle cells and was in contact with the endothelium. In valvular portions of the collecting lymphatics where the muscle layer was thinner and more fragmented, there were fewer vasa vasorum. Tri-dimensional reconstructions of the collecting lymphatic wall showed two communicating plexi of vasa vasorum--one outside and the other inside the muscle layer. Arteries and veins of similar size did not have such an abundant vasa vasorum. The explanation for this difference may relate to the fact that a relatively low oxygen and nutrient content of lymph is insufficient to nourish the collecting lymphatic. Moreover, diffusion of nutrients from the external plexus is likely also impeded by the thickness and density of the muscle layer. The vasa vasorum deep in the muscular layer and in the subendothelial space probably sustain adequate nutrition and oxygenation to the collecting lymphatic.

The structure of superficial lymphatics in the human thigh: precollectors.

BACKGROUND: Little is known about the morphology of precollectors, the lymphatic vessels connecting the absorbing and the collecting vessels, which are regarded as the initial drainage routes of lymph. The aim of this study was to describe the structural features of human precollectors. METHODS: Samples of fat from around the saphenous veins were obtained from patients undergoing varicotomy, and serial sections were observed under light and transmission electron microscopy. Tridimensional reconstructions were also obtained by computer analysis. RESULTS: Precollectors were characterized by an irregular and discontinuous arrangement of smooth muscle cells in their wall. This arrangement was unrelated to the site of valves. When present, muscular elements were arranged helicoidally, as shown in tridimensional reconstructions. Under transmission electron microscopy, the endothelium of precollectors was similar to that of absorbing lymphatic vessels, irrespective of the presence of smooth muscle cells, and was thin, rich in pinocytotic vesicles, supported by a discontinous basal lamina, and connected by anchoring filaments to the surrounding connective tissue. Myoendothelial contacts were frequent. Valves were similar to those of collecting vessels, except for the presence of numerous zonulae adherentes connecting the characteristic "tip cells" of the free edge. CONCLUSIONS: Human thigh precollectors are characterized by the alternation of portions with a well-developed muscular coat and portions with an absorbing structure. These morphological features suggest that the precollectors contribute to fluid absorption and lymph propulsion. The frequent myoendothelial contacts suggest that smooth muscle contraction is regulated locally.

Subendothelial nerve fibers in bovine mesenteric lymphatics: an ultrastructural and immunohistochemical study.

In the lymphatic vessels of man and most animals the nerve fibers are confined to the adventitia. However, immunohistochemical studies suggest that acetylcholinesterase-positive and monoamine-containing fibers reach as far as the endothelium in bovines. The aim of this study was to verify the presence of subendothelial nerve fibers by transmission electron microscopy (TEM) in bovine mesenteric lymphatics and to determine whether typical sensory neurotransmitters such as Substance P (SP) and calcitonin gene related peptide (CGRP) could be detected in these fibers. TEM revealed numerous unmyelinated nerve fibers in the subendothelial connective environment in close association with endothelial cells. Their axons were devoid of Schwann cell sheath on the endothelial side and contained small clear vesicles and large nerve fibers were demonstrated to be SP and CGRP-immunoreactive with mouse monoclonal antibodies against SP and rabbit polyclonal antibodies against CGRP. It is hypothesized that these fibers act as mechanoceptors capable of detecting intraluminal pressure and vessel wall tension variations and of locally releasing SP and CGRP. Since SP, potentiated by CGRP, is known to be a vasoconstrictor in lymphatics, we propose that the contraction of bovine mesenteric lymphatics may also be neurogenic.