Immunology 1977 33 381

Immunofluorescence studies with a specific antiserum to the microfibrillar protein of elastic fibres LOCATION IN ELASTIC AND NON-ELASTIC CONNECTIVE TISSUES

M. A. KEWLEY, F. S. STEVEN & G. WILLIAMS Departments of Medical Biochemistry and Pathology, Stopford Building, University of Manchester, Manchester

Received 13 December 1976; accepted for publication 13 January 1977

1966). During maturation of the elastic fibres, the MFP content decreases progressively with a corresponding increase in elastin (Greenlee et al., 1966; Fahrenbach, Sandberg & Cleary, 1966). As the MFP is laid down before the appearance of elastin, it is thought to be a necessary initiator for elastogenesis (Ross & Bornstein, 1969). A monospecific antibody has been prepared towards the microfibrillar protein of bovine ligamentum nuchae. The antiserum was shown to cross-react with the MFP of embryonic chick aorta elastic fibres using immunoperoxidase techniques at electron microscopy level and the MFP was found to be regularly organized along the surface of the elastic fibres (Kewley, Steven & Williams, 1977a). This may be considered as evidence in support of the hypothesis that MFP plays a role in the spatial organization of elastin monomers which subsequently become polymerized into fibrils which later coalesce to form mature elastin fibres (Kewley et al., 1977b). We have now employed the immunofluorescence technique to locate MFP, or an immunologically similar protein (which for simplicity we will refer to as MFP) in the basement membranes of bovine spleen, mouse liver, kidney, spleen and heart muscle, and human kidney. The results demonstrate a wide distribution of MFP in connective tissues containing either elastic fibres or collagen fibrils. We suggest

Summary. The microfibrillar protein (MFP) of foetal bovine ligamentum nuchae elastic fibres was prepared and used to produce a monospecific antiserum. Indirect immunofluorescence studies employing the specific antiserum demonstrated its selectivity for MFP associated with elastic fibres. The antiserum was demonstrated to react with vascular elastic tissue, perivascular connective tissue and reticular basement membranes in a variety of tissues. This evidence suggests that the MFP (or an immunologically related protein) is not confined to elastic fibres but is widely distributed in connective tissue being associated with both elastin and collagen fibres. INTRODUCTION Foetal elastic fibres have been shown to consist of two structurally distinct components, an internal core of elastin surrounded by a mass of microfibrillar protein (MFP). Fibre formation is characterized by the deposition of MFP in the form of fine fibrils prior to the appearance of the denser but closely associated elastin (Greenlee, Ross & Hartman, Correspondence: Dr F. S. Steven, Department of Medical Biochemistry, Stopford Building, University of Manchester, Manchester M13 9PT.

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that the MFP plays a role in the organization of both developing elastic fibres and collagen fibrils in vivo. MATERIALS AND METHODS Preparation of elastic fibres, MFP and specific antiserum Purified elastic fibres and MFP were prepared from foetal bovine ligamentum nuchae by a modification of the method of Ross & Bornstein (1969). MFP was used to produce a specific antiserum by the method of Shivers & James (1967). This procedure involved the immunization of a rabbit with MFP followed by gel diffusion in Ouchterlony plates with the antiserum against MFP. This produced one major immunoprecipitin band and two subsidiary bands. The major precipitin band was cut out of the gel and introduced into a second rabbit resulting in the formation of a specific antiserum which reacted against MFP with a single immunoprecipitin band and had no crossreactivity with serum proteins (Kewley et al. 1977a). FITC-labelled goat anti-rabbit serum was obtained from Wellcome Reagents Ltd.

Indirect immunofluorescence Cryostat sections of fresh adult bovine spleen,

mouse liver, spleen, kidney and heart muscle and human kidney biopsies were fixed for 2 min in cold acetone. The antiserum, specific for MFP, was diluted 1: 15 in phosphate-buffered saline (PBS), pH 7 6 and applied to the sections for 30 min. Sections were then washed in PBS (pH 7 6) for 40 min and

Figure 2. Adult bovine spleen. A negative control employing indirect immunofluorescence of MFP-absorbed antiserum applied to adult bovine spleen. Blood vessels exhibit very faint fluorescence, demonstrating the insignificant amount of autofluorescence of the tissue. (Magnification x 300.)

incubated at room temperature with 1:10 FITClabelled goat anti-rabbit serum. This was followed by washing in two changes of PBS (pH 7 6) for 40 min before mounting in buffered glycerol and examination with a Vickers Photoplan u.v. Microscope. Control sections were treated with either (a) normal rabbit serum, or (b) rabbit anti-MFP absorbed with MFP, instead of the complete antiserum.

Figure 1. Purified elastic fibres. Indirect immunofluorescence employing anti-MFP applied to 6-p cryostat sections of purified foetal bovine elastic fibres. Fluorescence is confined to the surface of the fibres where the microfibrillar protein (MFP) is located. The elastin component of the fibres is unstained even though the central regions of these fibres have been exposed by sectioning. (Magnification x 144.)

Histology In addition to the immunological investigations, representative specimens were taken from the specified tissues, fixed in formalin and prepared for routine histological examination. Sections were stained with (a) haemalum and eosin, (b) elastic Van Gieson (EVG) combination for elastin and collagen (Miller, 1971), (c) periodic acid-Schiff (PAS) for basement membranes (McManus, 1946) and (d) by silver impregnation for reticulin fibres, including basement membranes (Gordon & Sweet, 1936).

Immunofluorescence studies

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Figure 3. Adult bovine spleen. Indirect immunofluorescence employing complete antiserum applied to adult bovine spleen. The elastic tissue of the arteriole intima (I) and the surrounding connective tissue (CT) are highly fluorescent (compare Fig. 2). The reticular basement membranes of the surrounding splenic pulp also fluoresce.

Figure 4. Adult bovine spleen. In stained bovine spleen, the elastic tissue of the R.V.G. central arterioles is outlined. The supporting septum comprises both collagenous (C) and darker elastic fibres (E). (Magnification x 120.)

Figure 5. Adult bovine spleen. Silver impregnation technique applied to bovine spleen. Reticulin stain outlines the basement membranes within lymphoid tissue. Compare this figure with the fluorescent pattern obtained in Fig. 3 and note the correlation between immunofluorescence and distribution of reticulin. (Magnification x 270.)

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Figure 6. Adult bovine spleen. Indirect immunofluorescence applied to adult spleen illustrating the location of MFP on basement membranes and elastic tissue of the blood vessels. (Magnification x 365.)

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Figure 7. Adult bovine spleen. Indirect immunofluorescence applied to adult bovine spleen showing the fluorescence of the reticular basement membranes (on the right of the figure) and the fibrous splenic capsule (S) on the left of the figure. (Magnification x 160.)

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Imnmunofluorescence studies RESULTS Bovine ligamentum nuchae elastic fibres Purified bovine foetal elastic fibres examined after treatment with anti-MFP serum and indirect immunofluorescence showed intense fluorescence confined to the surface of the elastic fibres (Fig. 1). Controls (prepared by methods 1 and 2 described above) exhibited no fluorescence. Bovine spleen Ultraviolet microscopy of (a) adult bovine spleen sections and (b) spleen treated with MFP-absorbed antiserum and FITC anti-rabbit serum exhibited only faint autofluorescence with the arterioles outlined (Fig. 2). By contrast, in spleen treated with complete antiserum (Fig. 3) the arterioles appeared brightly fluorescent. The arterioles possessed two distinct fluorescent regions: (i) their intimal lining and (ii) their peri-arteriolar connective tissue. Selective stains for elastin and reticulin fibres gave positive reactions with the perivascular connective tissue (Figs 4 and 5) and with the splenic capsule. The splenic capsule and the splenic reticular basement membranes also reacted with the indirect fluorescent antiserum to MFP (Figs 6 and 7).

Human kidney Indirect immunofluorescence techniques applied to human kidney biopsy specimens demonstrated MFP in the basement membranes of the tubules and the glomerular capillaries (Fig. 8).

Mouse kidney, liver, spleen and heart muscle As with the bovine spleen and human kidney, MFP was located by indirect immunofluorescence techniques in the same sites as reticulin and basement membranes stained with silver impregnation and PAS.

DISCUSSION Immunoperoxidase studies (Kewley et al., 1 977a) and the immunofluorescence results described in this investigation with the monospecific antibody directed to the MFP of foetal bovine elastic tissue have clearly shown that MFP (or an immunologically similar protein) is not confined to the surface of elastic fibres but is widely distributed in a number of connective tissues. It is associated with collagenous structures in the form of 'reticulin fibres' and basement mem-

Figure 8. Adult human kidney. Indirect immunofluorescence applied to human kidney showing the MFP located on the basement membranes of the tubules and glomerular capillaries. (Magnification x 365.)

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branes, both of which possess glycoprotein constituents (Leblond, Glegg & Eidinger, 1957; Pras & Glynn, 1973; Kefalides, 1973; Pras, Johnson, Holborow & Glynn, 1974). The glycoprotein nature of MFP has been demonstrated by Ross & Bornstein (1970). The association of morphologically identical microfibrillar structures with elastic fibres and basement membranes is well established by transmission electron microscopy. Microfibrils have been noted at the periphery of the basement membrane and throughout the adventitial tissue in trachea (Rhodin, 1955), lung (Low, 1962), aorta (Karrer, 1961), myocardium (Low, 1962) and glomerular capillaries (Farquhar, 1961). Microfibrils are also numerous in the tunica propria of various organs (Karrer, 1958; 1961). The present study has extended these EM observations by establishing the immunological relationship between the observed basement membrane microfibrils and the extracted MFP of elastic fibres. MFP has already been implicated in the fibrillogenesis of elastic fibres (Ross & Bornstein, 1969) and the present evidence has shown that the MFP (or an immunologically related protein) is widely distributed in connective tissue, whether elastin-containing, or elastin-free as in the spleen and kidney basement membranes. It is our contention that the MFP may play a hitherto unrecognized role in the organization of collagen and in elastin polymerisation in vivo. It is conceivable that interference with MFP (immunologically or otherwise) might contribute to connective tissue disorders.

ACKN OWLEDGMENTS We are grateful to Mr D. Bradley for the histology preparations, Mrs Pat Tarpey for providing the kidney biopsy sections and to Mr G. Humberstone for assistance with the photography. We wish to thank the Arthritis and Rheumatism Council for their financial support, without which this study could not have been carried out. REFEREN CES FAHRENBACH W.H., SANDBERG L.B. & CLEARY E.G. (1966)

Ultrastructural studies on early elastogenesis. Anat. Rec. 155, 563. FARQUHAR M.G., WISSIG S.L. & PALADE G.E. (1961) Glomerular permeability. I. Ferritin transfer across the normal glomerular capillary wall. J. exp. Med. 113, 47. GREENLEE T.K., JR, Ross R. & HARTMAN J.L. (1966) The fine structure of elastic fibres. J. cell. Biol. 30, 59. GORDON H. & SWEET H.H. (1936) A simple method for the silver impregnation of reticulum. Amer. J. Path. 12, 545. KARRER H.E. (1958) The fine structure of connective tissue in the tunica propria of bronchioles. J. Ultrastruct. Res. 2, 96. KARRER H.E. (1961) An electron microscope study of the aorta in young and aging mice. J. Ultrastruct. Res. 5, 1. KEFALIDEs N.A. (1973) Structure and biosynthesis of basement membrane. Int. Rev. conn. Tissue Res. 6, 63. KEWLEY M.A., STEVEN F.S. & WILLIAMS G. (1977a) Preparation of a specific antiserum to the microfibrillar protein of elastic tissue. Immunology, 32, 483. KEWLEY M.A., STEVEN F.S. & WILLIAMS G. (1976b). The presence of fine elastin fibrils within the elastin fibre observed by scanning electron microscopy. J. Anat. 123, 129. LEBLOND C.P., GLEGG R.E. & EIDINGER D. (1957) Presence of carbohydrates with free 1,2-glycol groups in sites stained by the periodic acid-Schiff technique. J. Histochem. Cytochem. 5, 445. Low F.N. (1961) The extracellular portion of the human blood air barrier and its relation to tissue space. Anat. Rec. 139, 105. Low F.N. (1962) Microfibrils, fine filamentous components of the tissue space. Anat. Rec. 142, 131. MCMANUS J.F.A. (1946) Histological demonstration of mucin after periodic acid. Nature (Lond.), 158, 202. MILLER P.J. (1971) An elastin stain. Med. Lab. Tech. 28, 148. PRAS M. & GLYNN L.E. (1973) Isolation of a non-collagenous reticulin component and its primary characterisation. Brit. J. Exp. Path. 54, 449. PRAS M., JOHNSON G.D., HOLBOROW E.J. & GLYNN L.E. (1974) Antigenic properties of a non-collagenous reticulin component of normal connective tissue. Immunology, 27, 469. RHODIN J. & DALHAMN T. (1955) Electron microscopy of collagen and elastin in lamina propria of the tracheal mucosa of rat. Exp. Cell Res. 9, 371. Ross R. & BORNSTEIN P. (1969) The elastic fibre. The separation and partial characterisation of its macromolecular components. J. cell. Biol. 40, 366. Ross R. & BORNSTEIN P. (1970) Studies of the components of the elastic fiber. Chemistry and Molecular Biology of the Intercellular Matrix, (ed. by E.A. Balazs), volume 1, p. 641. Academic Press, New York. SHIVERS C.A. & JAMES J.M. (1967) Specific antibodies produced against antigens of agar-gel precipitates. Immunology, 13, 547.

Immunofluorescence studies with a specific antiserum to the microfibrillar protein of elastic fibres. Location in elastic and non-elastic connective tissues.

Immunology 1977 33 381 Immunofluorescence studies with a specific antiserum to the microfibrillar protein of elastic fibres LOCATION IN ELASTIC AND N...
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