J. Anat. (1996) 189, pp. 335-340, with 5 figures Printed in Great Britain 335

Immunohistochemical and ultrastructural analysis of an extracellular matrix bound antigen preferentially associated with mucosal postcapillary venules

J. SPENCER', T. HUSSELL2, Y. MUSTAFA3 AND M. E. PERRY3 Department of Histopathology, UMDS, St Thomas' Campus, I Department of Respiratory Medicine, Imperial College of Science, Technology and Medicine, St Mary's Hospital Medical School, and I Division of Anatomy and Cell Biology, UMDS, Guy's Campus, London, UK

(Accepted 23 May 1996)

ABSTRACT Human lymphoma derived monoclonal antibody (anti 'mucosal vessel associated antigen' [MVAA]) binds to high endothelial venules (HEV) in gut-associated lymphoid tissue, but shows only weak reactivity with HEV in peripheral lymphoid tissues. We have used immunohistochemistry and immunoelectron microscopy to define the precise ultrastructural distribution of the molecule, and to determine whether there is any association between this molecule and cellular traffic. We have observed that MVAA is a component of basement membrane which is only expressed by a subset of vessels. Although it is restricted to vessels which support traffic within lymphoid tissue, we did not observe any association between the distribution of MVAA and extravasating . MVAA is expressed in the fetal intestine in association with a subset of larger vessels. It is therefore not necessarily induced as a consequence of antigenic challenge. It is likely that MVAA has a structural role related to its restricted microanatomical distribution; possibly the maintenance of integrity of vessel walls which are continuously disrupted by the extravasation of lymphocytes.

Key words: Basement membrane; high endothelial venules; appendix; .

the lymphoma derived autoantibody using immuno- INTRODUCTION electron microscopy (Perry et al. 1991). In an earlier study of lymphoma immunoglobulin HEV are the major site of leucocyte entry into (Ig) specificity we produced a human monoclonal lymphoid organs (Gowans & Knight, 1964; Springer, autoantibody which bound to high endothelial venules 1990; Perry et al. 1992). Biases in lymphocyte traffic (HEV) in Peyer's patches and the appendix but not to between mucosal and peripheral lymphoid tissues palatine or peripheral nodes (Hussell et al. based on expression of endothelial ligands for lym- 1993). Staining of the smooth muscle component of phocyte homing receptors have been described (Kraal some medium sized veins and arteries in various et al. 1983). However, leucocytes must pass through organs was also reported. Although the staining all layers of the vessel wall before entering the appeared to be associated either with basement surrounding lymphoid tissue, and components of the membrane or pericytes, the precise microanatomical extracellular matrix have been reported to mediate the location of the mucosal vessel associated antigen adhesion and migration of lymphocytes in vitro (MVAA) recognised by the monoclonal lymphoma Ig (Shimizu & Shaw, 1991; Chan & Aruffo, 1993). (anti-MVAA), and hence the difference between the MVAA is the only molecule described to date which intestinal and extraintestinal HEV, was not clear at is expressed more abundantly in mucosal than the light microscopic level. Therefore, in this study, we peripheral human HEV. We have looked for any aimed to determine the precise location of the MVAA association between MVAA and extravasating molecule in the and appendix, recognised by lymphocytes at the ultrastructural level. Correspondence to Dr J. Spencer, Department of Histopathology, UMDS, St Thomas' Campus, Lambeth Palace Road, London SE1 7EH, UK. 336 J. Spencer and others

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Fig. 1. Double stained cryostat section of human appendix. Immunoperoxidase is used to detect MVAA (brown) and immuno-alkaline phosphatase with PAL-E to detect endothelium (blue); (a) shows that MVAA is predominantly associated with HEV (arrows); (b) illustrates Antigen associated with mucosal postcapillary venules 337

Lymphocytes begin to populate human fetal in- sections in human monoclonal autoantibody obtained testine between 14 and 16wk of gestation in the as supernatant from a mouse myeloma/human absence of exogenous immunological stimuli. Primi- lymphoma heterohybridoma cell line (anti-MVAA) at tive organised Peyer's patches are apparent at around 4 'C. Bound anti-MVAA was detected using a murine 19 wk of gestation (Spencer et al. 1986). In this study anti-idiotypic antibody specific for anti-MVAA. we analyse the expression ofMVAA in the intestine of Murine anti-idiotypic antibody was detected using 11 to 19-wk-old human fetuses to determine whether rabbit antimouse Ig conjugated to horse radish MVAA is associated with the movement of lympho- peroxidase and DAB substrate. The anti-idiotypic cytes into the developing gut, and whether the MVAA antibody cross reacted with a small population of expression and distribution at different anatomical lymphocytes ( < 1 %), but this did not interfere with sites may be due to variation in local antigenic the interpretation of results (Hussell et al. 1993). stimulation. For double immunoenzyme histochemistry, frozen sections were first stained to detect MVAA as described above and following a period of washing in MATERIALS AND METHODS running tap water, the sections were incubated in Tissues PAL-E. Binding of PAL-E was then detected using The following normal human tissues were received biotinylated rabbit antimouse Ig, followed by avidin fresh in the laboratory: 8 specimens of fetal ileum conjugated to alkaline phosphatase and fast blue between the ages of 11 and 19 wk, 4 specimens of substrate. No counterstain was used with double appendix which were obtained from right hemi- stained preparations. All secondary antibody reagents colectomy for colonic carcinoma and 1 normal were purchased from Dako Ltd and chemicals from nasopharangeal tonsil (adenoid) which was obtained Sigma UK Ltd, unless otherwise stated. from routine adenoidectomy for noctumal apnoea. Controls for both single and double immunohisto- For frozen sections, samples of all specimens were chemistry were included. For single immunohisto- snap frozen by dropping tissues in vials into liquid chemistry the primary antibody was omitted. For nitrogen. We find that this method gives good double immunohistochemistry, each of the primary preservation of morphology for immunohistohemical antibodies was omitted in turn on separate sections. analysis of frozen sections, provided the tissue is fresh. Tissues were then stored at -70 °C until required. Immunoelectron microscopy For pre-embedding immunoelectron microscopy, dis- sected pieces of 1 normal adenoid and 1 appendix Cryostat sections (30 jim) were cut and incubated in a were fixed for 1 h in freshly prepared 4% para- wet chamber at 4 'C for 24 h with anti-MVAA, formaldehyde and 0.1 % glutaraldehyde. They were followed by a 24 h incubation at 4 'C in the murine then rinsed in Tris-buffer, pH 7.6, immersed in 0.6 M anti-idiotypic antibody specific for anti-MVAA, fol- sucrose for 12 h, snap frozen in liquid nitrogen and lowed by a 2 h incubation in a wet chamber at room stored at -70 °C until required. temperature with goat antimouse IgG conjugated to 5 nm gold particles (Amersham Life Science), diluted in Tris buffer (pH 7.6) normal human Immunohistochemistry for light microscopy containing serum. After washing in serum/Tris buffer and Acetone-fixed 8 gm cryostat sections were stained postfixation with 2% glutaraldehyde, the sections using monoclonal antibody PAL-E, which recognises were silver-enhanced using an IntenSE M silver all endothelium, though arterial endothelium is enhancement kit (Amersham Life Science), osmicated only weakly stained (Eurodiagnostica, Apeldoorn, for 30 min in 2 % aqueous osmium tetroxide, rinsed in Holland: Schlingemann et al. 1985). Primary antibody distilled water, dehydrated in graded alcohols, imme- was followed by rabbit antimouse Ig conjugated to rsed in the link reagent propylene oxide and infiltrated horseradish peroxidase and diaminobenzidene subs- with a mixture of equal quantities of propylene oxide trate. MVAA was detected by incubating tissue and resin, before flat embedding in TAAB-resin.

MVAA surrounding the endothelium. Background staining due to endogenous peroxidase, which was also observed in the controls, is indicated by arrowheads. Fig. 2. Serial cryostat sections of human fetal intestine (16 wk gestation) stained using immunoperoxidase (brown) to detect (a) MVAA and (b) all endothelium. MVAA is only detected around a few of the larger vessels (arrow). Weak staining directly associated with some areas of epithelium (arrowheads) is nonspecific. x 80. 338 J. Spencer and others

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Three control sections were prepared which included the omission either of the anti-MVAA or the anti- idiotypic antibody, or both. The processed 30 gm thick sections were examined by light microscopy and areas selected from which ultrathin sections were cut, stained and viewed using a Hitachi 7000 transmission electron microscope (Perry et al. 1991).

RESULTS Distribution of MVAA in lymphoid tissue of the appendix The distribution of MVAA was compared with the distribution of vascular endothelium in the appendix by double immunohistochemical staining with anti- MVAA (brown) and PAL-E (blue) (Fig. 1 a). The PAL-E marked all endothelial cells. All MVAA staining observed was associated with vessels, though MVAA was largely restricted in its distribution to the vessels located in the interfollicular areas. in the germinal centre, vessels in the dome region of Fig. 5. Immunoelectron micrograph ofpart ofa lymphoid follicle in the lymphoid follicle and vessels in the lamina propria human appendix. There is no expression of MVAA within the were HEV avascular trabeculae of fine connective tissue (arrows) in this field. of the appendix all MVAA negative. Most x 10000. were associated with MVAA. Examination of the HEV under a higher magnification showed that MVAA surrounded the endothelium (Fig. 1 b). In fibrils or filaments, and appeared to follow their addition, staining associated with some larger vessels pathway (Fig. 3). However, staining with a more in the muscularis mucosae was also observed and diffuse pattern was also seen. No label was associated again the staining was located around the endo- with the periendothelial striated fibres of type I thelium. collagen or the basal lamina structures where type IV collagen is located. There was no evidence of any association either between MVAA and the pericyte Ontogeny of MVAA expression membrane or MVAA and trafficking lymphocytes. In fetal intestine, expression of MVAA was present in The most abundant MVAA was found in association all specimens studied from 11 wk gestation onwards, with HEV in the appendix, rather than the adenoid but it was only observed around few large vessels with (Fig. 4). Interestingly, no MVAA staining was flat endothelium. Most vessels were MVAA negative observed in areas of connective tissue without blood and there was no association between the sparse vessels (Fig. 5). lymphocyte population or clusters of lymphoid tissue and vessels expressing MVAA (Fig. 2 and unpublished data). DISCUSSION We have shown that MVAA is a component of basement membrane associated with a subset ofblood Ultrastructural analysis of the distribution of MVAA vessels. The restricted distribution of MVAA within MVAA was located extraendothelially in the fibro- lymphoid tissue, a property which is not shared by reticular laminae of the basement membrane material other known basement membrane components in- where the MVAA marker was frequently adjacent to cluding fibronectin, laminin and collagen, suggests

Fig. 3. Immunoelectron micrograph of an HEV in human appendix illustrating the distribution of abundant MVAA marker. The marker (arrows) is located in the connective tissue of the perivascular extracellular matrix, but the endothelial cells are negative (double arrows). x 15000. Fig. 4. Immunoelectron micrograph of an HEV in human adenoid demonstrating high endothelial cells (arrows) migrating lymphocytes in the vessel wall (double arrows) and the sparse amount of MVAA (arrowheads). x 10000. 340 J. Spencer and others that this molecule is novel. Within lymphoid tissue, however, that it may be upregulated by factors MVAA is present predominantly in association with produced in the course of an immune response. The HEV, though some flat endothelium in the inter- molecular structure of MVAA has evaded us to date follicular areas may also be associated with MVAA. due, in part, to the difficulty in working with a human Capillaries in the lymphoid follicles and vessels in the monoclonal antibody of IgM isotype (anti-MVAA) lamina propria are MVAA negative. The distribution which is produced by an unstable hybridoma. Hope- is clearly different, almost reciprocal, to the dis- fully the future production of a murine monoclonal tribution of the basement membrane molecule recog- antibody to this interesting basement membrane nised by monoclonal antibody 4C7 which is prefer- component will enable thorough molecular charac- entially associated with basement membrane of terisation. capillaries in the follicle centre and . In addition, 4C7 is detected in some epithelial basement membranes (Jaspars et al. 1993). Differences in the REFERENCES expression of basement membrane molecules which CHAN PY, ARuFFo A (1993) VLA-4 integrin mediates lymphocyte are associated with vessels with distinct functional migration on the inducible endothelial ligand VCAM-l and the extracellular matrix ligand fibronectin. Journal of Biological properties suggests that the basement membrane Chemistry 263, 24655-24664. molecules themselves have a function related to their GowANs JL, KNIGHT EJ (1964) The route of recirculation of microanatomical distribution. lymphocytes in the rat. Proceedings of the Royal Society of London (Biology) 159, 257-282. HEV, the dominant site ofMVAA, are the principle HUSSELL T, ISAACSON PG, CRABTREE JE, DoGAN A, SPENCER J sites of lymphocyte extravasation into lymphoid (1993) Immunoglobulin specificity of low grade gas- tissues. We therefore considered it possible that trointestinal lymphoma of mucosa-associated lymphoid tissue MVAA may be associated with this role since (MALT)-type. 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