Virchows Arch (Cell Pathol) (1982) 40:181-189

HrchowsA~dvB 9 Springer-Verlag 1982

An Immunohistochemical Study of Cells with Surface and Cytoplasmic Immunoglobulins in situ in Peyer's Patches and Lamina Propria of Rat Small Intestine T. Sminia and Benita E.C. Plesch Department of Histology, Medical Faculty, Free University Amsterdam, P.O. Box 7161, NL-1007 MC Amsterdam, The Netherlands

Summary. The distribution of cells with surface and cytoplasmic immunoglobulins was studied in Peyer's patches (PP) and intestine of rats, using both frozen and paraffin sections, with a two-step peroxidase technique. Anti IgM, IgG, IgA and IgE sera were used. Surface staining was found within PP with all antisera used. Although the villi contained predominantly IgA plasma cells (PC), IgG PC and a few IgM and IgE PC were also found. Within PP, however, no IgA PC were found but IgM and IgG PC were present in all stages of development, mainly in the dome. PC of all types, but mostly IgA cells, were present in and around high endothelial venules (HEV). The results suggest that IgM and IgG PC precursors can develop to PC within PP whereas IgA precursors do not. PC appear to home to the gut preferentially via HEV. Key words: Peyer's patches - Rat - Immunohistochemistry Peroxidase technique

Plasma cells

Introduction It is well known that the small intestine contains large numbers of cells which synthesize and secrete IgA (Allen and Porter 1977). Precursors of IgA secreting cells are present in Peyer's patches (PP) (Craig and Cebra 1971; Rudzik et al. 1975a, b) and it has been shown that mesenteric lymph node (MLN) cells (Parrot and Ferguson 1974; McWilliams et al. 1975; Hall et al. 1977) and IgA blasts in thoracic duct lymph (Pierce and Gowans 1975; Hall et al. 1977) home to the lamina propria (LP) and there secrete IgA. Recently it has been demonstrated directly that PP cells preferentially migrate to MLN for maturation to IgA PC precursors capable of seeding the LP of the small intestine (Roux et al. 1981). Thus a great deal is known about IgA PC development and the results imply that development occurs outside the gut. Send offprint requests to T. Sminia at the above address

0340-6075/82/0040/0181/$ 01.80

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T h e i n t e s t i n e also c o n t a i n s I g M a n d I g G P C (e.g. V a e r m a n a n d H e r e m a n s 1969; W a k s m a n et al. 1973; A l l e n a n d P o r t e r 1977), b u t little is k n o w n a b o u t e i t h e r t h e o r i g i n o r the m a t u r a t i o n site o f t h e s e cells. T h e p u r p o s e o f the p r e s e n t s t u d y was to i n v e s t i g a t e t h e d i s t r i b u t i o n o f cells w i t h s u r f a c e i m m u n o g l o b u l i n s (sIg) a n d c y t o p l a s m i c i m m u n o g l o b u l i n s (cIg) in situ in o r d e r to see if t h e r e was m o r p h o l o g i c a l c o r r o b o r a t i o n o f t h e traffic studies o n I g A P C m a t u r a t i o n a n d to g a i n i n s i g h t i n t o the d e v e l o p m e n t o f t h e o t h e r t y p e s o f P C in P P a n d the s m a l l intestine.

Materials and Methods Young adult male Wistar rats (200-300 g) were obtained from the C.P.B., Zeist, The Netherlands. They were kept under routine animal house conditions and sacrificed by asphyxiation with 100% CO 2. Pieces of small intestine, including PP, were frozen in liquid nitrogen and stored at - 2 0 ~ C or fixed in a sublimate-formaldehyde solution (6 g HgC12, 5 ml glacial acetic acid, 10 ml 40% formaldehyde solution, 85 ml aq. dest., Bosman et al. 1977). Serial sections, about 8 gm, were mounted on alternating slides, so that different stains could be compared, air dried for 30 min 3 h, fixed for 10 min in pure acetone, dried overnight and used for immunohistochemical demonstration of Ig. Fixed material was routinely dehydrated and embedded in paraffin (melting points 60~ C or 43 ~ C). Serial sections, 6 lam, were stained with methyl green pyronin or haematoxylin-eosin. Paraffin sections were also used for immunohistochemistry after routine removal of paraffin and rehydration but without any further treatment. Before incubation in various antisera, slides were washed in 3 changes of 0.01 M phosphatebuffered saline (PBS) pH 7.4, for 30 min. All procedures were carried out at room temperature. During incubation, slides were placed horizontally and gently shaken. The following antisera were used: goat anti-rat IgM, sheep anti-rat IgG, goat anti-rat IgA, or goat anti-rat IgE (Nordic, Tilburg, The Netherlands) in dilutions between 1:200 and 1:800 in PBS with 0.2% bovine serum albumin (PBS/BSA; dilutions tested for each serum batch). After incubation for 1 h in these sera, slides were washed 3 x in PBS (30 min) and further incubated with rabbit anti-goat IgG peroxidase conjugate (PO; Miles Yeda, Israel or Nordic) or rabbit anti-sheep IgG/PO (Nordic) in a dilution of 1:200 in PBS/BSA for 30 rain. Sections were again rinsed 3 x in PBS and stained for peroxidase activity with 3,Y-diaminobenzidine-tetra HC1 (Sigma, St. Louis, MO, USA) in 0.5 mg/ml Tris-HC1, pH 7.6, containing 0.01% H 2 0 z. After rinsing in tap water, sections were briefly counterstained with haematoxylin, rinsed in tap water, dehydrated and mounted in Entellan (Merck, Darmstadt, FRG). Control slides were incubated with PBS/BSA or pre-immune serum, or incubated in all media except conjugate, and examined for non-specific staining. Endogenous peroxidase activity was sometimes blocked as a control by incubating sections before incubation with antisera, in phenylhydrazine (Straus 1972) or methanol-H20 2 (Streefkerk 1972). Some of the paraffin sections were used for simultaneous detection of cells covtaining different Ig's. All combinations of anti-Ig's were used. For the demonstration of the peroxidase tabel in the first reaction the red chromogen 3-amino-9-ethylcarbazole (Sigma; Graham et al. 1965) was used. Before further incubation slides were washed in 0.1 M glycine-HC1 buffer pH 3.0. After further incubations with antiserum and conjugate the second reaction was visualised with the blue chromogen 4-chloro-l-naphthol (Aldrich, Beerse, Belgium; Nakane 1968). Sections, sometimes lightly counterstained with haematoxylin, were mounted in aquamount.

Results

1. Morphology. P e y e r ' s p a t c h e s are a g g r e g a t e s o f l y m p h o i d tissue in t h e m u c o s a l l a y e r o f t h e i n t e s t i n e (Fig. 1). T h e y c o n s i s t o f l y m p h a t i c n o d u l e s s e p a r a t e d by i n t e r f o l l i c u l a r a r e a ' s ( I F A ; e.g. F a u l k et al. 1971; A b e a n d I t o 1977). T h e m u c o s a l l a y e r a b o v e the i n t e r f o l l i c u l a r a r e a has villi a n d crypts, w h e r e a s t h a t

sIg and clg Cells in Peyer's Patches

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Fig. 1. Cryostat section of PP showing the distribution of sIgM cells. C corona; D dome; GC germinal center; IFA interfolliculararea; M muscularis mucosae; V villi, x200; Inset: Enlargment of a few sIgM cells. Note the thin (black stained) ring around these lymphocytes, x 1,000 above the follicles is flattened. The epithelium in this latter region has the morphological features of lympho-epithelium. The follicles have a conspicuous germinal centre (GC) surrounded by a corona of densely packed small lymphocytes, which separates the follicle from the surrounding interfollicular area. The germinal centre comprises two areas, a light and a dark one. These areas are very striking. The dark area, which is located at the base of the follicle against the muscularis mucosae, contains many blast cells with basophilic cytoplasm. Mitotic figures are frequently seen. The blast cells are intermingled with tingible body macrophages. In the light zone, on the other hand, numerous reticulum cells, large lymphocytes, plasma cells and tingible body macrophages are present. Between the follicles and the lympho-epithelium a dome area can be distinguished (e.g. Abe and Ito 1977; Waksman et al. 1973). This subepithelial zone is populated by several cell types including medium and large lymphocytes, plasma cells and macrophages. Moreover, blood vessels are present in this area. The interfollicular areas comprise numerous small lymphocytes and scattered plasma cells. Furthermore, this area is characterized by high endothelial venules, which are mainly located in the upper half of this area. The villi contain numerous plasma cells in addition to fibroblasts and granulocytes. 2. Immunohistochemistry. In frozen sections surface Ig is clearly visible as a

thin ring around the positive lymphocytes, cytoplasmic Ig appears as a thick ring. In paraffin sections although surface Ig can be seen vaguely when low melting point (43 ~ C) paraffin is used this is not very reproducible. However, in paraffin sections cytoplasmic Ig's can be seen clearly and the morphology

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Surface (o) and cytoplasmic (.) Ig's in Peyer's patches of the rat

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IgE 0

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Fig. 2. Schematic drawing of PP showing the distribution and relative numbers of slg and cIg cells. C corona; D dome; GC germinal center; IFA interfollicular area; IMC immune complexes; V villi of the cells and nuclei is excellent. No difference in cIg distribution is seen using the different types of paraffin, except for cytoplasmic IgE which could only be demonstrated in low melting point paraffin. Double staining of cIg's provided no extra evidence in itself but confirmed the results of single staining. The results of immunohistochemistry are summarised in Fig. 2. Villi. In the LP of villi cells bearing sIg are seldom seen but very many cIg cells are present, most of which are mature PC. About 75% of these are IgA PC, 20% IgG PC, 5% IgM PC and < 1 % IgE PC. In the villi mast cells which were positive for IgE were quite frequently observed (cf. Mayrhofer et al. 1976). The stain is less intense than in IgE PC. D o m e a n d c o r o n a contain many sIgM and sIgG bearing cells evenly distributed

over these areas (Fig. 1); sIgA cells are scattered in groups, giving a patchy appearance and sIgE cells are dotted over the same areas. It is difficult to estimate numbers in the thick frozen sections but an estimate of the percentage of sIg cells gives 40% sIgM, 40% sIgG, 15% sIgA and 5% sIgE. It is possible that some cells bear both sIgM and sIgG. Although the distribution of sIg cells is similar in dome and corona, the dome contains far more macrophages

Figs. 3 and 4. Paraffin sections of PP stained for IgM (Fig. 3) and IgA (Fig. 4). The black dots (arrows) are cells containing Ig. Note the differences in distribution of clgM and clgA cells. C corona; D dome; GC germinal center (d dark area; I light area in which immune complexes are clearly visible, double arrows); IFA interfollicular area with high endothelial venule (HEV); V villi, • lnsets: Enlargment of part of the GC with IgM containing cells (Fig. 3) and of part of the IFA with IgA blast cells (Fig. 4), x 1,000

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Fig. 5. Part of the IFA at the base of the villi (E, gut epithelium). Note the clgA blast cells

(arrows) in and around the high endothelial venule (HEV). Paraffin section, z 500

Fig. 6. Numerous immature and mature IgA plasma cells (arrows) are present in the lamina propria of the villi, x 1,000

and T-cells (Sell et al. 1981 in rabbit; own observations in rat) which appear negative with anti Ig stains. Throughout the dome large numbers of cIgM and rather fewer cIgG cells are seen; most of these are immature and mature PC. A few cIgM and cIgG cells are found in the corona, cIgE cells are found in both dome and corona but in low numbers, cIgA cells are only very seldom present. Germinal Centre. In the light part of the GC heavy intercellular staining occurred. This was present in frozen sections stained with all antisera but was heaviest with anti IgM and IgG and only very light with anti IgE; in paraffin sections it was only seen after anti IgM staining (Fig. 3). This is probably due to staining of naturally occurring immune complexes trapped by (follicular) dendritic reticular cells (Nossal and Ada 1971). In the GC only very few cells stained with sIgM but many had cIgM. These were mainly large blasts and immature PC (Fig. 3). Many cells were sIgG positive, and blasts and immature PC with cIgG were also present. Large sIgA cells were present in the GC but only very occasional cIgA cells. No surface staining was observed with anti IgE in the G C but a few cIgE cells were always present. More cIg cells are present in the upper part than the lower part of the GC. At the base of the GC, immediately adjacent to the muscle layer a few PC of all types were observed, in particular IgA PC (Fig. 4).

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Interfollicular Area. In and around HEV in the IFA sIg and cIg cells of all isotypes were observed, in particular many immature and mature IgA PC. Groups of PC, predominantly IgA, were also present at the base of the IFA and between HEV and the LP (Figs. 5 and 6). Discussion

In this study the distribution of cells with surface and cytoplasmic Ig was studied in situ. The use of a sensitive two-step immunoperoxidase technique on both frozen sections, in which sIg and cIg can be demonstrated, and paraffin sections, in which cIg can be visualised with excellent preservation of morphology, proved to be very useful, since the techniques give complementary information. The sublimate-formaldehyde fixative used for paraffin sections appears to be superior to the formalin fixatives commonly used by others and obviated extra steps such as enzyme digestion (cf. Mepham et al. 1979). Possibly the precipitating mercury salt makes antigens more accessible than does the cross-linking formalin alone, while apparently preserving cytoplasmic antigens since cIg compared very favorably to cIg in frozen sections. The morphological observations agree in general with those obtained in studies of PP of other rat strains or mammalian species (e.g. Faulk et al. 1970). The PP comprises several compartments, characterised by different structures and cell populations. The question arises as to whether the different compartments have different functions and furthermore whether the whole represents a functional unit in the immune response. The present results showed that within PP surface staining cells of all the isotypes studied (IgM, IgG, IgA and IgE) were present and in the villi cells with cIg of the same isotypes were found, so that the PP cells could be the direct or indirect precursors of the cells populating the LP. The relative numbers of both sIg and cIg cells of the different isotypes varied from other observations but there may be considerable species differences (cf. Allen and Porter 1977; Rudzik et al. 1975a; Haaijman et al. 1977; Sell et al. 1980; Butler et al. 1981). Other authors mostly failed to demonstrate plasma cells within PP. Apart from possible species differences, techniques may also be important, since we could not demonstrate Igs reproducibly using anti Ig (total) antisera, or in one-step techniques nor on cytospin preparations that had not been fixed with alcohol: ether. All stages of development of IgM PC were found within the dome of PP and large cIgM blast cells in the GC. This would suggest that IgM cells are primed within the GC and migrate to the dome where they complete their maturation to PC within PP. Recently, the same suggestion has been made on the basis of results of studies on plaque-forming cells in PP (TlaskalovfiHogenov~t and Stepankovfi 1980). From there they might migrate to other parts of the gut. This would also apply to IgG PC on the same grounds. However, since these cIgG cells are fewer than cIgM cells in the dome, whereas there are more in the villi, development of IgG PC outside PP as well seems likely, cIgE cells show a similar pattern to cIgM and cIgG cells but they are far less in number.

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c I g A cells are c o n s p i c u o u s b y their absence in the follicular a r e a o f PP. This w o u l d agree with the o b s e r v a t i o n s m e n t i o n e d in the i n t r o d u c t i o n which show t h a t I g A P C m a t u r e in M L N . L a r g e n u m b e r s o f I g A PC are seen in a n d a r o u n d H E V , which w o u l d seem to indicate that they r e t u r n to the intestine via this route. The absence o f I g A PC in or a r o u n d o t h e r b l o o d vessels in the gut a n d the l o c a l i z a t i o n o f P C not only between H E V and the L P b u t also between H E V and the m u s c u l a r i s m u c o s a e ( M M ) , a n d a l o n g the M M u n d e r the patches, suggests t h a t H E V m a y be the m a j o r route o f e n t r y in n o r m a l animals, whence they migrate, possible a l o n g the M M to o t h e r p a r t s o f the gut. It has been shown t h a t PP are n o t necessary for I g A PC h o m i n g ( M c D e r m o t t et al. 1980) a n d o f course n o t all a n i m a l s have H E V ; nevertheless they m a y prefer this r o u t e when available. A s m e n t i o n e d above, I g G P C within PP are fewer t h a n I g M PC, whereas their n u m b e r in the L P is m u c h higher. B o t h types, however, are seen a r o u n d H E V , p a r t i c u l a r l y I g G PC. This m a y m e a n that a l t h o u g h p r e c u r s o r s can d e v e l o p to P C within PP they m a y also leave to m a t u r e , possible after a m p l i f i c a t i o n , elsewhere. F o r I g A PC, d e v e l o p m e n t outside PP a p p e a r s to be o b l i g a t o r y . The r e a s o n for this r o u n d a b o u t r o u t e is n o t certain, but p r e c u r s o r s for I g A PC have been s h o w n to m i g r a t e f r o m the gut to o t h e r m u c o s a l surfaces, thus a f f o r d i n g p r o t e c t i o n a g a i n s t gut antigens as p a r t o f a c o m m o n m u c o s a l i m m u n e system ( M c D e r m o t t a n d Bienenstock 1979; Bienenstock a n d Befus 1980). This m a y also a p p l y to I g M , I g G a n d p r o b a b l y IgE cells, b u t to a less m a r k e d degree.

Acknowledgements. The authors wish to thank Marja van de Ende and Marga Janse for their technical assistance, and Martine Roest for typing the manuscript.

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Received December 10, 1981 / Accepted March 15, 1982