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Northern hybridization of total RNA isolated from adult human sciatic nerve demonstrated a readily detectable hybridization signal for glucose transporter 1 (GLUT 1) mRNA. Western blot analysis demonstrated that GLUT 1 proteins extracted from adult human and from mature rat sciatic nerves had different electrophoretical mobilities. The migration positions of human and rat GLUT 1 proteins corresponded to 60-70 kDa and 55-60 kDa, respectively, as estimated by markers with known molecular masses. Indirect immunofluorescence staining localized GLUT 1 to the perineurium in the adult human sciatic nerve. Only a few endoneurial capillaries of human adult nerve stained positively for GLUT 1, which was in contrast to rat peripheral nerve where most endoneurial capillaries were positive for GLUT 1. In developing human nerves, the staining pattern for GLUT 1 was markedly different from that of the adult nerves: at 14 weeks, the perineurial cells were entirely negative for GLUT 1. Between 22 and 26 weeks of gestation, the staining reaction for GLUT 1 in the perineurium became markedly more prominent, and by 35 weeks the intense perineurial staining resembled that observed in the adult human nerves. In contrast to adult nerves, both endo- and epineurial blood vessels stained intensely for GLUT 1 in the fetal samples. Thus, the immunoreactivity for GLUT 1 in the perineurium seems to increase concomitant with the maturation of barrier properties of perineurium, whereas the transient expression of GLUT 1 in the vasculature of developing nerve may have a specific role in the proliferating endothelial cells.

The expression of beta 4 integrins in adult and fetal human skin as well as in cultured keratinocytes was studied by immunodetection with monoclonal antibodies, and compared with that of beta 1 integrins. The distribution of the beta 1 and beta 4 integrin epitopes was entirely different in the adult epidermis. As reported previously by us (J Clin Invest 84:1916, 1989), the beta 1 epitopes were present most prominently at lateral cell-cell contact points, whereas staining with the beta 4 antibody demonstrated a linear staining pattern polarizing to the basal surface juxtaposed to the dermal-epidermal basement membrane. In contrast, in fetal skin, the staining patterns both with beta 1 and beta 4 antibodies were similar and demonstrated the presence of epitopes surrounding the entire cell surface of both basal and suprabasal keratinocytes. Distinct polarization of beta 4 integrin epitopes was noted in cultured keratinocytes maintained in low-calcium (0.15 mM) medium, and the expression of these integrins was also noted in human papilloma virus-transformed keratinocytes. Switch of the cultures to high-calcium (1.2 mM) medium resulted in redistribution of the epitopes to a pattern suggesting their location at under-surface of the cells adjacent to the substratum. This Ca(++)-induced redistribution of beta 4 integrin epitopes could be counteracted by 10(-7) M vitamin D3. Finally, incubation with anti-beta 4 integrin antibody reduced the capacity of keratinocytes to attach to plastic substratum. The results provide further evidence for the role of beta 4 integrins in cell-matrix interactions.

Strikingly selective expression patterns of beta 1, alpha 2, alpha 3, and alpha 5 integrin subunits were revealed in endoneurium, perineurium, and epineurium of fetal and adult human peripheral nerve by immunostaining with specific antibodies. The alpha 2 subunit was expressed only on Schwann cells both in fetal and adult nerve, whereas the alpha 3 epitopes were expressed exclusively in the adult tissue and were primarily present on perineurial cells. The alpha 5 epitopes were expressed only on the innermost cell layer of perineurium of fetal and adult nerve. The tumor cells within schwannomas and cutaneous neurofibromas expressed both alpha 2 and alpha 3 subunits, indicating that Schwann cells have the potential to express also the alpha 3 subunit in vivo. Cell cultures established from human fetal nerve and neurofibromas revealed expression of the alpha 2 and alpha 5 epitopes on Schwann cells, perineurial cells, and fibroblasts, whereas only Schwann cells contained the alpha 3 epitopes which were occasionally concentrated on the adjacent Schwann cells at cell-cell contacts. Our findings emphasize that nerve connective tissue cells change their profiles for expression of extracellular matrix receptors under conditions which have different regulatory control signals exerted by, for example, axons, humoral factors, or the extracellular matrix of the peripheral nerve. This plasticity may play an important role during nerve development and in neoplastic processes affecting the connective tissue compartments of peripheral nerve.

Full thickness biopsies of affected skin and fascia from one patient with diffuse fasciitis and eosinophilia (DF), two patients with generalized morphea (GM), and five patients with progressive systemic sclerosis (PSS) of recent onset were examined for the expression of transforming growth factor beta 1 (TGF beta 1) and type I procollagen genes by in situ hybridization with human sequence-specific cDNA. An increased number of fibroblasts showing clearly detectable expression of pro alpha 1(I)collagen gene was found in all fibrotic lesions when compared with unaffected skin from the patient with DF and skin from two normal individuals examined in parallel. Expression of the TGF beta 1 gene was noted in a fibroblast subpopulation of the affected tissues from the patients with DF and GM. In contrast, the TGF beta 1 gene was not expressed at a detectable level in affected skin from the five patients with PSS. The results suggest that TGF beta 1 may play a role in the development of skin fibrosis in cases of DF and GM. However, from these studies, we cannot implicate TGF beta 1 in the pathogenesis of skin fibrosis in PSS.

The formation of the connective tissue compartments of human sciatic and tibial nerves was studied with special reference to the maturation of the basement membranes during foetal development (11-35 weeks of gestation). All Schwann cells were surrounded by continuous basement membranes as early as at week 11, while the perineurial cells became covered by basement membranes gradually between weeks 17 and 35, as estimated by electron microscopy. The first laminin subunits detectable within the nerve were the B1, B2 and M chains. These laminin subunits were present in Schwann cell basement membrane zone at week 11, and in the perineurium at week 17 and later. Laminin A and S chains were first detected at 26 weeks in the perineurium, and at a later stage (35 weeks) on Schwann cells. In mature nerves, all these five laminin chains could be demonstrated in both Schwann cell and perineurial cell basement membrane zones, although A, S and B2 chains predominated in the perineurium, and M, B1 and B2 were the predominant chains in Schwann cell basement membranes. Beta 1 and beta 4 integrins were expressed by all Schwann cells in samples from the youngest foetuses (11-17 weeks). At 22-35 weeks, however, only a subpopulation of Schwann cells stained positively for beta 1 and beta 4 integrins. Perineurial cells expressed beta 1 integrins at all ages studied. Staining for beta 4 integrin in perineurium became detectable and intensified concomitant with the formation of structural basement membranes. The results demonstrate that Schwann cells and perineurial cells change their laminin and integrin expression profiles during the maturation of peripheral nerve.

The effects of different glucose concentrations on the expression of extracellular matrix genes were examined in primary cell cultures initiated from sciatic nerves of nondiabetic Sprague-Dawley rats. The cells were incubated in medium containing 5.5, 15, or 25 mM D-glucose, and the expression of type I and IV collagens, laminin, and fibronectin genes was examined at mRNA steady-state levels by Northern hybridizations. Incubation of cell cultures, consisting of Schwann cells, perineurial cells, and fibroblasts, in high glucose concentrations (15 or 25 mM D-glucose) resulted in elevation of pro-α 1(IV), pro-α 2(IV), and pro-α 1(I) collagen chain and fibronectin mRNAs after only 3 days of incubation, whereas laminin B2 chain mRNA levels appeared unaltered. These observations suggest that hyperglycemia may partially contribute to basement membrane thickening in peripheral nerves of diabetic individuals by increasing the expression of genes coding for basement membrane components, e.g., type IV collagen.

Publisher Summary This chapter describes the simultaneous use of in situ hybridization (ISH) to detect cellular mitochondrial RNAs (mRNAs) and peroxidaseantiperoxidase (PAP) method to identify and localize protein epitopes in tissue samples and in cell cultures. In situ hybridization (ISH) technique is used to localize specific cellular RNA or DNA sequences in tissues or in cell cultures by molecular hybridizations with complementary nucleotide probes. As the half-life of functional mRNA molecules within cells is relatively short, varying from minutes to hours, the in situ detection of specific mRNAs allows both the temporal and spatial examination of cellular gene expression. The steady state level of a given antigen within tissue or cell culture samples reflects the balance between its local synthesis and degradation or its deposition from other sources, such as plasma. Thus, the presence of an antigen does not necessarily imply its synthesis by the resident cells.

Cutaneous lesions from three patients with segmental neurofibromatosis were evaluated. Routine histologic studies revealed the presence of rudimentary neural structures within an abundant collagenous matrix. The majority of the cells in all three cases expressed S-100 protein, suggesting their identity as Schwann cells. The stromal component stained positively for fibronectin and type IV collagen; the latter indicated the presence of basement membrane material. Embedded in the tumor mass were glandular epithelial structures that stained with epithelial membrane antigen antibody. Staining for factor VHI-related antigen revealed vascular endothelium and multiple scattered mast cells. In one case strands of cells stained with antibodies to desmin, suggesting muscle cell differentiation. This case may represent a distinct subset of neurofibromas.

Previous observations have demonstrated that fibronectin is deposited in high abundance in basal cell carcinoma stroma. In this study, the nature of fibronectin and the site of its synthesis were explored in 10 basal cell carcinomas of the nodulo-ulcerative type by immunocytochemistry and in situ hybridization. First, simultaneous localization of epithelial tumor cell islands and fibronectin epitopes was carried out by double immunofluorescence staining with monoclonal anti-cytokeratin antibodies and polyclonal fibronectin antibodies, the latter recognizing both the cellular and plasma types of the protein. Large amounts of fibronectin were deposited in the basal cell carcinoma stroma, with the highest concentration present in the immediate proximity of the epithelial cell islands. Immunofluorescence with a monoclonal anti-fibronectin antibody, which is directed against the ED-domain of cellular fibronectin and does not recognize the plasma type of fibronectin, revealed essentially the same staining pattern as that obtained with the polyclonal anti-fibronectin antibody. This observation suggested that fibronectin in BCC was predominantly of the cellular type. Second, in situ hybridizations, utilizing a human fibronectin specific cDNA, demonstrated that the highest concentration of fibronectin mRNA was found in the most peripheral cell layer of the epithelial tumor islands. The presence of fibronectin mRNAs in the tumor cells of the central regions of the islands, as well as within occasional stromal cells, was also noted. Thus, two lines of evidence suggest that the epithelial tumor cells are predominantly responsible for the synthesis and deposition of fibronectin in basal cell carcinoma. The presence of fibronectin may explain the characteristic biologic behaviour of basal cell carcinomas, including low degree of metastatic potential and local destructive nature of the tumors.

Perineurial cell cultures were established from the sciatic nerves of adult Wistar rats. Highly enriched cultures were studied with respect to the production of extracellular matrix components under conditions free from the influence of Schwann cells, axons, or the extracellular matrix of peripheral nerves. Indirect immunofluorescence staining revealed the presence of collagen type IV epitopes, and electron microscopy demonstrated patches of basement membrane on the perineurial cell surfaces. Collagenous fibrils with a diameter of 15-20 nm were also observed in the intracellular space. SDS-PAGE of radiolabeled medium proteins showed a pattern of bands suggesting the synthesis and secretion of fibronectin, and type I and IV collagens. Northern hybridizations revealed characteristic polymorphic mRNA transcripts corresponding to fibronectin, laminin B2 chain, as well as to the alpha-chain subunits of type I, III, and IV collagens. Furthermore, in situ hybridizations suggested expression of these genes by cultured perineurial cells without apparent heterogeneity within the cell populations. In situ hybridizations of sciatic nerve tissue from 2-wk-old rats also suggested that perineurial cells express alpha 1(I) and alpha 2(IV) collagen, as well as laminin B2 chain genes in vivo. This profile of matrix gene expression is different from that of Schwann cells, which do not synthesize fibronectin, or that of fibroblastic cells, which do not form a cell surface basement membrane. The capability of perineurial cells to express genes for the basement membrane zone and for interstitial collagens further adds to our understanding of the functional role of perineurial cells in developing and healing peripheral nerve, as well as in certain neoplastic lesions of neural origin, such as von Recklinghausen's neurofibromas.

Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.

Diabetes mellitus (DM) is known to be associated with widespread connective tissue changes. However, the connective tissue of peripheral nerves in diabetes has gained little attention. Thickening of the basement membranes of Schwann, perineurial and endothelial cells suggests a perturbation in the metabolism of type IV collagen. We studied the ultrastructure of endoneurial collagen fibrils in the sciatic nerves of spontaneously diabetic BB rats and found that the fibrils in the diabetic rats were significantly thicker than in the age and sex matched non-diabetic BB rats, both in the proximal (52.6 vs. 46.1 nm) and the distal part (52.4 vs. 45.5 nm) of the nerve, respectively. The mechanism for the thickening of endoneurial collagen fibrils remains unknown, but conceivably involves changes in the biochemical composition of the nerve connective tissue matrix: e.g. altered metabolism of type I and/or type III collagen, increased glycosylation of the collagen molecules, or changes in the glycosaminoglycan content of the ground substance.

VLA integrins in human skin were examined by indirect immunofluorescence utilizing antibodies recognizing the beta 1, alpha 2, alpha 3, or alpha 5 subunits. Staining of fetal, newborn, or adult skin with antibodies to beta 1, alpha 2, or alpha 3 subunits gave essentially similar staining patterns: intense staining was associated with the basal layer of the epidermis, hair follicles, and blood vessel walls. The alpha 5 subunit could be detected only in epidermis and the inner root sheath of hair follicles in fetal skin. In epidermis, the staining reaction for the beta 1 subunit was not only found in sites interfacing with the basement membrane zone, but also around the entire periphery of these cells. We speculate that these receptors might have previously unrecognized functions in cell-cell interactions or that these findings may suggest the presence of previously unrecognized ligands in the intercellular spaces of keratinocytes. Examination of nine nodular basal cell carcinomas revealed a prominent staining reaction with anti-beta 1 and anti-alpha 3 antibodies at the periphery of the tumor islands. In contrast, staining of five squamous cell carcinomas revealed either the absence of integrins or altered and variable expression. Thus, matrix components and their receptors may participate in modulation of growth, development, and organization of human skin.

Simplified and expedient methodologies for examination of cellular gene expression at the mRNA and protein levels, utilizing in situ hybridization and peroxidase-anti-peroxidase immunodetection, were developed. These techniques were first optimized for the detection of extracellular matrix genes expressed by cultured human skin fibroblasts and keratinocytes, the two principal cell types of human skin. In situ hybridizations and Northern transfer analyses with human-sequence-specific cDNAs encoding collagenous and noncollagenous protein sequences demonstrated selective expression of different matrix genes by these two cell types, indicating different biosynthetic capacities of these cells and attesting to the specificity of the hybridizations. The utility of in situ hybridization was also demonstrated in mixed primary cell cultures established from cutaneous neurofibromas consisting of Schwann cells, perineurial cells, and fibroblasts. The methodologies developed here were further utilized for simultaneous detection of fibronectin mRNA and immunoreactive protein in fibroblast cultures. This procedure allowed detection of grains representative of radioactively labeled cDNA-mRNA hybrids and protein epitopes, as visualized by peroxidase-anti-peroxidase immunodetection on the same cells. This methodology, with appropriate modifications, may be applicable to other cell types as well as tissue specimens.

The synthesis of collagen and its deposition into the extracellular matrix were studied in scleroderma and normal skin fibroblast cultures using immunocytochemistry, electron microscopy and electrophoretic analysis. Indirect immunofluorescence localized type I collagen to intracellular granules of normal skin fibroblasts but not to the extracellular matrix. A 48-h ascorbic acid-treatment of scleroderma and normal skin fibroblasts induced the appearance of fibrillar, collagen-containing deposits in the extracellular space as indicated by indirect immunofluorescence and electron microscopy. Interestingly, similar type I collagen-containing fibrils were observed in cultures of scleroderma skin fibroblasts even without preceding ascorbic acid treatment. Electrophoretic analysis revealed that ascorbic acid increases the conversion of procollagen to collagen to same extent both in scleroderma and control cell layers. The accumulation of collagen to the cell layer of scleroderma fibroblasts was increased when compared to normal skin fibroblasts; whereas the relative conversion of procollagen to collagen was identical in scleroderma and normal fibroblasts. The results show that the conversion of procollagen to collagen in the cell layers of both scleroderma and normal skin fibroblasts is dependent on ascorbic acid, due to improved secretion of procollagen molecules in the presence of ascorbic acid.

Cutaneous neurofibromas, characteristic lesions of neurofibromatosis 1, are composed of an abundant extracellular matrix and nerve connective tissue-derived cell types: Schwann cells, perineurial cells, and fibroblasts. In this study, the extracellular matrix gene expression by these cells was examined under culture conditions that allowed them to be metabolically active and readily identifiable by morphologic and immunocytochemical criteria. Northern hybridizations demonstrated expression of genes for type I, III, IV, and VI collagens, as well as for fibronectin, laminin, and elastin. In situ hybridizations revealed that all three cell types expressed pro alpha 1 (I), pro alpha 2 (VI), and laminin B1 chain genes. However, fibroblasts did not contain [35S]cDNA-mRNA hybrids specific for type IV collagen, whereas both Schwann cells and perineurial cells expressed these genes. Perineurial cells and fibroblasts readily expressed the fibronectin gene whereas Schwann cells were essentially devoid of the corresponding mRNA. Perineurial cells also expressed the gene for laminin A chain. The results indicate that the extracellular matrix gene expression profiles of Schwann cells, perineurial cells, and fibroblasts are distinct: all three cell types are capable of expressing some of the genes for extracellular matrix components, such as type I and VI collagens, whereas Schwann cells and perineurial cells may have the primary role in synthesizing basement membrane zone components, type IV collagen and laminin. These observations potentially relate to the mechanisms of growth and development of human neurofibromas. The results attest to the applicability of the methodology utilized here to study other human tumors with mixed cell populations.