0022-202X/85/8501-0054$02.00/0 THE JOUHNAL OF INVESTIGATIV E DEHMATOLOGY, 85:54- 59, 1985 Vol. 85, No.1 Copyright © 1985 by The Williams & Wilkins Co. Printed in U.S.A. Basement Membrane Proteins, Interstitial Collagens, and Fibronectin in Neurofibroma

RAUL FLEISCHMAJER, M.D., RuPERT TIMPL, PH.D., MARIE DziADEK, PH.D., AND MARK LEBWOHL, M.D. Department of Dermatology, Mount S inai School of Medicine, New York, N ew York, U.S.A., and Max Planck Institut fur Biochemie, Martinsried bei Munchen, F.R.G.

The distribution and nature of extracellular matrix of these components is apparently not dependent on the pres­ proteins in neurofibroma tissue was studied by indirect ence of neuronal cells. immunofluorescence, immunoelectron microscopy, im­ Excessive production of extracellular material around periph _ munoblotting, and rotary shadowing. The most striking era! nerves is observed in neurofibromatosis (also knov,rn as feature was an extensive network of basement mem­ von Recklinghausen's disease), one of the most common genetic branes localized mainly around Schwann cells and small human disorders, which is inherited as an autosomal domina11t blood vessels. The major components, collagen IV, lam­ trait. The clinical appearance includes multiple pigmented skin inin, and nidogen, were mainly deposited in the lamina patches ("cafe au lait" spots), axillary freckling, iris Lisch densa. Some laminin and nidogen could be extracted nodules, congenital pseudoarthrosis, and cervicothoracic ky. with 0.5 M NaCl and were shown by electrophoresis to phoscoliosis [12]. Neurofibromas are the most frequently ob. have the characteristic chain and fragment patterns served skin tumors, although Schwannomas and fibromas have described previously for these proteins isolated from the also been described (13] . A gross histologic analysis of neur0 . mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Frag­ fibromas shows abundant reticulin fibers and nerve structures ments of collagen IV and collagen VI were solubilized [13,14]. Schwann cells are t he most prominent cell type with by limited proteolytic digestion and identified after ro­ fibroblasts showing a more scattered distribution [15]. Electron tary shadowing. The more remote interstitial regions of microscopy has shown abundant basement membranes sur­ the tumor contained cross-striated collagen fibrils which rounding Schwann cells and numerous collagen fibrils between were composed of collagen III (diaJileter, 20-30 nm) or these cells [15,16]. collagen I (diameter, 40-50 nm). Collagen fibrils thicker The histologic and cell culture data indicate that the devel­ than 80 nm were not found. The interstitial regions also opment of benign peripheral nerve tumors could involve the contained collagen VI as a fine filamentous network uncontrolled synthesis and deposition of extracellular matrix near cells and between collagen fibrils. Deposits of fi­ proteins. Immunofluorescence analyses have shown increased bronectin were rather small and showed a scattered staining for collagen IV [17] and laminin (18] in neurofibromas. distribution. The data indicate that Schwann cells con­ In the present study we examine the distribution of interstitial tribute considerably to matrix production in neurofi­ collagens I, III, and VI, fibronectin, and several baseme11t broma which may therefore be a suitable model for membrane proteins (laminin, collagen IV, nidogen) in the t\.1- studying basement membranes of neuroectodermal ori­ mor both at the light microscopic and the ultrastructural level. gin. The presence of some of these components is confirmed by biochemical analysis.

Extracellular matrices of the endoneurium and perineurium MATERIALS AND METHODS are typical structures of peripheral nerves. The endoneurium Tissue membranes which surround Schwann cells consists of basement Postsurgical specimens of neurofibroma tissue (3 tumors) were ob. and nerve axons, and also interstitial regions which contain tained under local anesthesia from patients with neurofibromatosis. collagen fibrils [1,2]. Major components of these matrices are collagen I, III, IV, and V [3], fibronectin and laminin (4,5] Antibodies which have been shown by immunofluorescence staining to be Affinity-purified rabbit antibodies aga inst bovine collagens I and III located in different anatomical regions. Formation of basement and against the aminopropeptides of procollagens I and III were tho%e membranes is dependent on close cell contacts and apparently previously described [19] . Antibodies against collagen VI were raised precedes the deposition of collagen fibrils [6, 7]. against a pepsin fragment obtained from human placenta [20 ], and Normal Schwann cells are able to synthesize collagens I, III, those against human plasma fibronectin were prepared as previously and V [8], collagen IV [9], and Iaminin (4]. In the absence of described (21] . Purified antigens from mouse Engelbreth-Holm-Swar:m nerve axons, synt hesis is decreased or deposition of an intact (EHS) sarsoma were used to prepare antibodies against laminin [22], [23], the 7S-domam of collagen IV [24]. All anttbodies basement membrane is impaired [4,9]. Fibroblasts are thought mdogen and were purified by immunoadsorption, and were specific for each partie. to produce fibronectin and collagen fibrils of large diameter ular antigen examined in the present study. Antibodies raised again%t which are deposited in the perineurium [4,5,8]. Schwannoma antigens of nonhuman origin showed sufficient cross-reactivity with cell lines produce collagen I and an unknown procollagen human tissues to be useful for immunofluorescence [25 ] and immun0 . [10,11) and also fibronectin and laminin [5,11). The synthesis ferritin staining [26]. Antibodies were raised against reduced human laminin from the skin of a patient with hyalinosis cutis et mucosa (27] and used for immunoblotting. Manusc ript received August 1, 1984; accepted for publication Janu· ary 8, 1985. Indirect I mmunofluorescen.ce Microscopy Supported in part by NIH Grants AM 073 76, NATO Grant 1960, and the Deutsche Forschungsge meinschaft (project Ti 95/6-1). Frozen sections, about 6-8 !Lm thick were defatted in ethyl alco_ Reprint requests to: Raul Fleischmajer, M.D. , Department of Der­ hol:ether (1:1) or 70% ethyl alcohol. The sections were digested for 3Q matology, Mount Sinai School of Medicine, Ooe Gustave L. Levy Place, min with hyaluronidase (2 mg/ ml) in phosphate-buffered saline pH 7.2 New York , New York 10029. (PBS). This additional step resulted in brighter fluorescence. Sections Abbreviation s: were treated for 30 min with the first antibody (30- 50 !Lg/ ml), washed EHS: Engelbreth-Holm -Swarm (sarcomfl) with PBS, and treated for 30 min with goat antirabbit gamma globulin PBS: phosphate-buffered saline, pH 7. 2. antibodies conjugated with fluorescein isothiocyanate (Behring-Werke,

54 July 1985 BASEMENT MEMBRANE, COLLAGEN IN NEUROFIBROMA 55 Marburg, West Germany). All specimens were washed in PBS, mounted in PBS:glycerol (1:9, v/v), and examined with a Nikon microscope equipped for epit1uorescence overhead immunofluorescence. Photo­ graphs were taken on Kodak 'Tri X Panchromatic film [28].

Indirect Immunoelectron Microscopy Immunoelectron microscopy was carried out as previously described (26]. The concentration of antibodies ranged from 0.1-0.3 mg per ml. Following a 20-min fixation with 1% glutaraldehyde, the tissue speci­ mens were exposed for 24 h to the specific antibodies described above, followed by careful washing. This was followed by a second 24-h exposure to goat antirabbit IgG labeled with ferritin and careful wash­ ing. After regular glu taraldehyde and osmium fixation the specimens were dehydrated, embedded in Epon, sectioned into 60-80 nm -thick sections, and stained with uranyl acetate-lead citrate and phospho­ tu ngstic ac id. In control experiments, specific antibodies were replaced FIG 2. Immunofluorescence staining of neurofibroma for collagen I by rabbit lgG from nonimmunized an imals. (a), the aminopropeptide of coll agen III (b), collagen VI (c), and fibronectin (d). Note the uniform staining up to the epidermis (E). x Biochemical Analyses 160. A larger specimen (20 g) of tumor tissue was extracted with 0.5 M NaCl 0.05 M Tris-HCl, pH 7.4, in the prese nce of protease inhibitors fo llo.;ed by 4 M guanidine [23 ]. Both extracts were used for the high density of Schwann cells. The stained regions appeared as immunob lot detection of nidogen following a modified procedure (29]. circular areas or wavy bundles occupying a larger area than Laminin was purified from the NaCl extract by an established proce­ usually seen in staining peripheral nerves [4,5). The upper dure [30]. A portion of the tissue remaining after NaCl extraction was portion of the dermis which was devoid of tumor cells showed subjected to limited digestion with bacterial co llagenase and DEAE cellulose chromatog r ap ~ y for obtainin g collage n IV fragments [31 ]. the more limited staining for normal skin, around blood vessels, Another portion was d1gested w1th pepsm m 0.5 M a-:etic acid for skin appendages, and at the epidermal-dermal junction (Fig 1). solubilizing coll agen IV and VI fragm ents [32]. Both could be precipi­ The staining patterns for the interstitial components were tated from the digest by NaCl (1. 2- 1.7 M). They were examined by rather uniform throughout the whole tissue sections including rota ry shadowing electron m1croscopy as prev10usly described [31,32 ]. the tumor and the uninvolved skin. Differences were noted in A NaCI extract and purified laminin [30] were prepared from the mouse staining intensities with different antibodies, being strongest EHS sarcoma. for collagens III and VI, moderate for collagen I, and weakest for fibronectin (Fig 2). The latter pattern is unusual since RESULTS stronger reactions are observed on normal human skin (28]. Distribution of ExtraceLlular Matrix Proteins Revealed by Immunofluorescence Ultrastructural Localizations Neurofibromas are usually well circumscribed but not encap­ The precise ultrastructural localization of extracellular ma­ sulated tumors t hat affect the dermis and sometimes the sub­ trix components was analyzed by the immunoferritin tech­ cutaneous tissue [33]. We have used immunofluorescence stain­ nique. This technique has been successfully used for localizing ing for an overview of the distribution of various basement basement membrane proteins [34-36], collagens I and III membrane proteins (Fig 1) and of typical components of the [26,37], collagen VI [20], and fibronectin (21] in various tissues dermis (Fig 2). Staining for the basement membrane proteins including human skin. Routine electron microscopy of neuro­ (collagen IV; larninin_. and ni?oge.n) revealed restricted patterns fibromas demonstrated abundant Schwann cells with many which differed only m stammg mtens1ty. The strongest reac­ cytoplasmic projections around nerve axons, all surrounded by tions were always observed in the tumor regions containing a a distinct basement membrane (see control in Fig 3b). In addition, the tumor was composed of regions containing cross­ striated collagen fibrils (range of diameters, 20-55 nm) em­ bedded in an amorphous matrix often containing a network of microfilaments. These regions occasionally showed also base­ ment membrane structures with no apparent contact to a cell. Basement membranes around Schwann cells could be clearly labeled for laminin, collagen IV, and nidogen (Figs 3, 4). Stain­ ing was most prominent in the lamina densa. Labeling for collagen IV was weaker than that for laminin and nidogen. A few ferritin deposits were also found in regions remote from basement membranes (Fig 3a, 5a), indicating the deposition of laminin and nidogen in a nonlaminar form. All the staining appeared specific since controls with nonimmune IgG showed only a few scattered ferritin particles (Fig 3b). Small blood vessels with multilayered basement membranes showed strong staining of each lamina densa with laminin (Fig 5) and nidogen. No or only weak staining was found for collagen IV. Large areas of loosely arranged, thin collagen fibrils (diame­ ter, 25- 30 nm)were also present in the tumor which could be labeled wit~ antibodies against the aminopropeptide of procol­ lagen III (Ftg 6b ). Other areas of the tumor consisted of thicker FIG 1. lmmuoofluoresce nce staining of basement membrane pro­ coll agen fibrils (diameter, 40-55 nm) which could be distinctly teins in neurofibroma. The tumor ts located in the mid and lower labeled for collagen I (Fig 6a). Only a few of these fibrils reacted dermi s. Laminin is shown in an overview (a, X 160). Staining for with antibodies against the aminopropeptide of procollagen I collagen IV (b, :>< 360) and nidoge n (c, X 500) are shown at border (not shown). Labehng for collagen VI was mainly observed in regions of the tvmor. E = epidermis; D = uninvolved portion of the areas contammg a network of fine filaments which were about dermis. 5 nm thick. These filaments were located near basement mem- 85, No. 1 56 FLEISCHMAJER ET AL Vol. raised against reduced human laminin (Fig 9), while only weak reactions were observed for the 400,000 chain. The NaCl extract also contained nidogen, as determined by radioimmunoassay, which was not purified. lmmunoblots of this extract showed a' prominent band with M, = 100,000 as found in a s imilar NaCI extract of the mouse tumor (Fig 9). The higher-molecular­ weight forms of 150,000 and 130,000 were, however, not de­ tected in the neurofibroma extract. Two more forms with M~ = 80,000 and 50,000 could be ident ified in a guanidine extract resembling t hose nidogen fragments purified previously from the mouse EHS tumor [23 ). Collagen IV fragments were prepared from neurofibroma after limited digestion with bacterial collagenase. Rotary shad­ showed that the purified fragments consisted mainly of by immunoferritin staining in a owing F IG 3. Locali zation of laminin triple helical strands connected by a Schwann cell s (a) and comparison 78-collagen and dimeric, neurofibroma region containing for collagen IV treated with nonimmune IgG (b). Note the heavy and globule (Fig lOa,c) which are characteristic with a control fragments continuous staining of t he lamina densa in (a) (arrows) and lack of [31] . Limited pepsin digestion was used for preparing staining in the control (b) (arrows). A few regions show also staining of collagens IV and VI. A semipurified preparation of both of the lamina rara close to the plasma membrane (arrowhead) and of fragments together was examined by rotary shadowing and structures not being basement membranes (x). S = Schwann cell; C = cytoplasmic projections. Bars = 100 nm.

FIG 5. lmmunoferritin localization of laminin in the multilayered basement membrane (BM) around a small blood vessel (a). Note the (a) and nidoge n FIG 4. Ultrastructural locali zation of collage n IV heavy staining of the lamina densa and frequent projections of t he ll s. Arrows (b) to the lamina densa around neurofibroma Schwann ce stain into adjacent regions including the lamina rara. Control veii'lsel s in (a) than m denote clusters of ferritin particles being less continuou stained with nonimmune IgG (b) . E = endothelial cell ; L = veii'lsel (b) . S = Schwann cell ; C =cytoplasmic projections. Bars= 100 nm. lumen. Bars = 100 nm. branes, particularly between basement membranes around small blood vessels, and also in close contact with collagen fibrils (Fig ?a). The fibronectin staining (Fig ?b) revealed ferritin particles as small clusters (diameter, 40- 80 nm), often in close contact with small collagen fibrils, and basement membranes. Biochemical Studies The nature and macromolecular organization of some of the extracellular matrix components identified by immunohistol­ ogy were also studied by immunoblotting or rotary shadowing electron microscopy. Sufficient amounts of laminin were puri­ fied from a 0.5 M NaCI extract of the tumor to be analyzed by FIG 6. Immunoferritin staining for coll agen I (a) and the aminOPro­ electrophoresis. The material migrated as a single band of high peptide of procollagen III (b) in an interstitial region of neurofibro\na. molecular mass which was split into two chains with M, = Note the heavy staining along cross-striated collagen fibrils very often amorphous rr>giQ n s. 200,000 and 400,000 after reduction (Fig 8). The 200,000 chain in periodic patterns, and absence of stain in the showed a distinct reaction in immunoblots using antibodies Bars = 100 nm. July 1985 BASEMENT MEMBRANE, COLLAGEN IN NEUROFIBROMA 57

FIG 7. Ult rastructura l ident ification of coll agen VI (a) and fi bronectin (b) by immunoferritin staining of neurofi ­ broma. Note clusters of stain for collagen VI in regions containing fi lamentous materia l (arrows) a nd lack of staining of cross-striated collagen fibrils in a peri ­ odic fashi on. F ibronectin staining oc­ curred in fer ritin clusters occasionally > ; along a n i solated basement. membrane (BM, arrows) and in m tersttba l regwns '. ~....u .. • ~- (arrowheads). Bars= 100 nm. : . ..;:_.b it. ,

Basement membranes normally consist of two layers, a lam­ +ME -ME ina rara (Iucida) close to the cells a nd a more remote lamina densa. Immunoferri tin staining localized all t hree basement membrane p roteins primarily to the lamina densa around Schwann cells a nd t he mult ilayers of lamina densa at the periphery of small blood vessels. Ferritin staining also extended into t he lamina rara, part icularly for laminin. A s imilar locali­ zation of collagen I V has been r eported fo r epidermal (39] and re nal (34,36] basement membranes, while data fo r laminin are di verse and include restrictions to the lamina rara [35,36,40], the lamina densa (41], or a localization in both (42). A unifo rm distribution of laminin was fo und in Reichert's membrane (43] which i s a n extremely t hick b asement membrane. This could N M N M indicate diffe rences in the molecular architecture of basement FIG 8. Polyacryla mide gel electrophoresis o f l aminin purified from membranes in di fferent a natomical regions. neurofibroma (N) and compari son wit h m ouse Iaminin (M ). Samples T he strong immunoferritin staining of the lamina densa in we re a na lyzed prior to (-ME) and after (+ME) reduction wit h 2% 2- our study demonstrates an unlimited penetration of these struc­ mercaptoetha no!. tures by t he fe rritin conjugate. This was a s urprising fi nding since other basement membranes, particularly glomerular base­ rmally barriers fo r the passage of such Nd ment membranes are no large proteins [44]. 1t ' -1.00 Nidogen, a recently described ubiquitous basement mem­ brane protein [23], was not previously examined in ultrastruc­ 150- I"'! - 200 distribution in the lamina densa with 13 0- tural studies. The close co _; laminin was not unexpected since large fo rms of nidogen (M, 100- = 100,000) show strong interactions with l ~ minin [45]. La~ in in 80- extracted from the neurofibroma co nsisted of constituent chains having M , = 200,000 and small amounts of a M, = 50- 400,000 chain. Laminin synthesized b y rat Schwann cells [4] or Schwannoma cells [5] essentially lac k t he 400,000 chain, which could indicate t he increased d egradation of this c hain from these sources, and could explain the low amounts of this G N M N M chain in neurofibroma laminin. High proteolytic activity could e a bsence of higher-molecular-weight FIG 9. hnJ1lunoblot detection of nidogen components (N d) and of also be responsible for th laminin chains (L) extracted from n eurofibroma with N aCl (N ) or forms of nidogen in tumor extracts. guanidine (0). They were compared wi th si1nila r materi al obtained The presence of collagen I V in neurofibroma was confirmed from m ouse E HS sarcoma (M ). Ntdogen was used in nonpurified f orm by solubilizing fragments of t he p rotein and identifying t hem and was not reduced. Laminin was purifie d and reduced prior to by rotary s hadowing. These fragments included the cross­ electrophoresis. Affinity-purifie d a ntibodies against mouse nidoge n linking domains of the protein (tetramers connected by t he 7S [23 ] and against human lammm [27] were used at a concent ration of collage n d omain, dimers connected by t he g lobular d omain), e numbers on each s ~rl.e indicate the apparent molecular 10-20 11 g/ml. Th indicating t hat excessive deposition in the t umor tissue does masses of the components (M, X 10 · ). not significa nt ly interfere wit h the characteristic self-assembly patterns (31] . Similarly, collagen VI structures could be de­ shown to contain tetramers of collagen VI (Fig lOb) as we ll as tected after solubilization with pepsin, and consisted of dimers, other, unide ntified material. tetramers, and filamentous aggregates. The cross-striated collagen fibrils were shown to be composed DISCUSSION of either collagen III (diameter, 20-30 nm) or collagen I (di ­ In the present study, we demonstrate by immunohistologic ameter around 45). The presence of only relatively t hin fibri ls and biochemical analyses that neurofibroma t issue contains agrees with previous o bservations [46] while t hicker fibrils of basement membrane proteins (collagen IV, laminin, nidogen), 80- 90 nm diameter which h ave also been r eported could not be collagens I, III, and VI, a nd fibronectin. T his composition of observed in our material. Based on qualitative histologic a nal­ the e xtracellular m atrix corresponds to that of p eripheral yses, it appears that neurofibroma tissue contains more collagen nerves [3,38] and the synt hetic products of cultured Schwann III than co llagen I. Biochemical analyses of human femoral cells [4,8,9] and/or Schwannoma cells [5, 11] . Our data t hus nerves demonstrated a ratio of 4:1 fo r collagen I a nd III [38], confi rm and extend previous o bservations (15- 18] t hat abun­ but no data are available for n eurofibromas. dant a mounts of b asement membranes are associated with The immunohistologic data also indicated a relatively low neurofibromas. production and/or deposition of fibronectin. This protein is 58 FLEISCHMAJER ET AL Vol. 85, No. 1

FIG 10. Rotary shadowing images of fragments of collagen IV (a,c) and col­ lagen VI (b) obtained from neurofibroma tissue. Material in (a) was solubilized with co llagenase and purified, and showed 78-collagen particles (arrow) and dimeric strands connected by t he globular domain of collagen IV (arrow­ heads). Materials in (b and c) were sol­ ubi lized with pepsin and not sep a rat~ d. Note in (c) a typical collagen IV tetrarner connected by the 7S-domain (msteri.sk) and in (b) dimers (a r rowheads) and te­ tramers (arrow) of collagen VI.

usually synthesized by fibroblasts of peripheral nerves and 13. Jungueira L Ch, Montes GS, Kaupert D, Shigihara KM, Bolanha ni deposited in t heir perineurium [4,5]. TM, Knsztan RM: Morpholog1cal and histochemical studies on the co llagen in neurinomas, neurofibromas, and fibromas. J The composition of the extracellular matrix of neurofibromas Neuropathol Exp Neural 40:123-133, 1981 suggests t hat t he major contributors to its synthesis are t he 14. McNairy ,DJ._Montgomery H: Cutaneous tumors of von Recklin _ proliferating Schwann ce ll s rather than fibroblasts. The rea­ hausen s d1 sease (neurofibromatosis). Arch Dermatol Syp hil ~ l sons for increased matrix deposit ion are not known. It could 51:384-390, 1945 15. Weber K, Braun-Falco 0: Zur Ultrastruktur der Neurofibromatose be an inherent property of transformed Schwann cell phenotype Hautarzt 23: 116- 122, 1977 · [5,10,11] or r efl ect lack of modulation by nerve axons [6-8]. 16. Waggener JD: Ultrastructure of benign peripheral nerve sheath Neurofibroma tissue can be obtained in sufficiently large tumors. Cancer 19:699- 709, 1966 amounts t hat it offers the possibility for isolating basement 17. Gay R~, Gay S, Jones RE: Histological and immunohistological membrane protein tdenttficatwn of coll agens in basement membranes of Schwann s such as laminin and nidogen frorn a human _ce ll~ of neurofi_bromas. Am J Dermatopathol 5:317-325, 1983 source, and also screening for co mponents that could be unique 18. M1ettmen M, Fo1da.rtJM, Ekbl?m P: Immunohistochemical deJU.­ to peripheral n erve basement membranes. onstratJOn of lammm, the '?aJor ~ l ycoprotei n of basement meJU. ­ branes, as an a1d m the d1agnos1s of soft tissue tumors. Am J We appreciated the s killed technical assistance of Ms. O livia Love­ Clin Pathol 79:306-311, 1983 lace, Ms. H anna Wiedemann, and Ms. Vera van Delden. 19. Nowack _H. , Gay S! 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