Immunobiol., vol. 164, pp. 136-143 (1983)

1 Dept. of Histology, 2 Dept. of Immunopathology, and 3 Dept. of Electron Microscopy, Medical Faculty, Vrije Universiteit, Amsterdam, The Netherlands

Characterization of Non-Lymphoid Cells in Peyer's Patches of the Rat

T. SMINIA\ M. M. WILDERS2, E. M. lANSE1

, and E. C. M. HOEFSMIT3

Received September 9, 1982 . Accepted in Revised Form December 1, 1982

Abstract

The aim of the present study was to correlate in vitro and in situ observations on non­lymphoid cells in Peyer's patches (PP) of the rat. By carrying out enzyme cytochemical reactions (acid phosphatase, APh, and non-specific esterase, NSE) and immunocytochemistry (Ia antigen staining) on cell suspensions and cryostat sections of PP, two classes of non­lymphoid cells could be distinguished. These were (1) strongly APh- and NSE-positive cells without or with a slight Ia membrane staining and (2) strongly la-positive cells with a weak APh and NSE activity. The first cell class comprised the classical macrophages which, except for tingible body macrophages, were glass-adherent. The second cell class was non-adherent and comprised dendritic (interdigitating) cells.

The role of this la-positive non-adherent cell population was discussed, and a hypothesis was presented on the relation between mononuclear blood cells, veiled cells, and interdigitat­ing cells in PP.

Introduction

It has been shown that Peyer's patch (PP) cells cannot be induced to produce antibody against sheep red blood cells or mount a cytotoxic response against allogeneic cells in vitro without the addition of mac­rophages from another source (7). Adherent cells derived from PP were less able to support antigen-induced proliferation of T cells (3) than adherent cells derived from spleen. These observations strongly suggest that non­lymphoid adherent cells in PP are less active in antigen-presentation than such cells obtained from other peripheral lymphoid tissues. In contrast, more recent studies have shown that PP-adherent cells are capable to present antigen to T cells (14, 19).

Recently, we have studied the in situ localization of non-lymphoid cell types involved in antigen-handling in PP (20). The results of this study indicate that PP do contain in addition to macrophages interdigitating cells (IDC), which are related to antigen-presenting in T cell areas of lymphoid organs (4, 5, 8, 25), as well as follicular dendritic cells (FDC), the anti­gen(immune-complex)-retaining cells typical of follicles (5, 8, 25). These data imply that the non-lymphoid cell types (macrophages, IDe, FDC)

Non-Lymphoid Cells in Peyer's Patches . 137

involved in the initiation, maintenance, and regulation of the immune response are present in PP. Thus there seems to be some discrepancy between the results of the in vitro and the in situ investigations. In order to get a better insight into the cell types used in the in vitro studies and to correlate the in vitro with the in situ observations, we have investigated the non-lymphoid cells in both cryostat sections and suspensions of PP, using enzyme and immunocytochemistry.

Materials and Methods

Animals

Young adult male Wistar rats, obtained from the Central Institute for the Breeding of Laboratory Animals, T.N.O., Zeist, The Netherlands, were used.

Histology

Pieces of the small intestine, including PP, were frozen in liquid nitrogen and stored at -20°C. Cryostat sections (about 8 Ilm thick) were air-dried for at least 30 min, fixed for 10 min in pure acetone, washed 3 times in phosphate-buffered saline (PBS), and used for immuno- and enzyme cytochemical studies.

Isolation of cells

PP were carefully cut from the small intestine and rinsed in cold Earles' solution. Subse­quently, the patches were cut into small pieces and incubated in Earles' medium containing collagenase (1 mg/ml; Worthington CLS III) and magnetically stirred at 37"C for 15 min. After this enzymatic treatment, the tissues were pressed through a nylon filter and washed 3 X

in Earles' to which 0.5 % bovine serum albumine (BSA) was added. Then the cells were resuspended in RPMI medium + 5 % foetal calf serum (FCS) and concentrated at about 107

cells/ml. Drops of this cell suspension and of dilutions of it (104, 105, and 106 cells/ml) were

transferred into plastic culture dishes (diameter 35 mm, height 10 mm) on the bottom of which glass coverslips (22 X 22 mm) had been placed. The cultures were then incubated at 37°C for 2 h. Subsequently, each culture coverslip containing adherent cells was rinsed with Earles' (5 X) in order to remove the non-adherent cells. Then the remaining adherent cells were fixed in pure acetone (for immunocytochemistry) or in Baker's formol (for enzyme cytochemistry) for 10 min at 4°C, rinsed in tap water, and air-dried. The coverslips were split into two halves and sticked, cell side upwards, onto microscope slides. Cytospin preparations were made of the initial cell suspension and of the non-adherent cell population. The preparations were used for immuno- and enzyme cytochemistry.

Enzyme cytochemistry

For the demonstration of acid phosphatase, the preparations were incubated in a medium containing naphthol-AS-BI phosphate (Sigma, U.S.A.) as substrate (16). Non-specific esterase was demonstrated using a-naphthyl butyrate (Sigma, U.S.A.) as substrate (17).

Immunocytochemistry

Ia (immune-region associated) antigens were determined with a two-step immunoperoxid­ase technique. The cryostat sections, the adherent cell, and the cytospin preparations were, after rinsing in PBS, incubated for 1 h in mouse anti-rat Ia immunoglobulin (Monoclonal, Sera Lab, U.K.; dilution 1 :800 or 1 :1600 in PBS with 0.2 % BSA) and after washing in PBS (3 times) covered with rabbit-anti-mouse IgG peroxidase conjugate (Miles, Yeda, Israel;

138 . T. SMINIA, M. M. WILDERS, E. M. JANSE, and E. C. M. HOEFSMIT

dilution 1 :200). Slides were again rinsed 3 X in PBS and stained for peroxidase activity with 3,3'-diaminobenzidine-tetra HCl (0.5 mg/ml) in Tris-HCI, pH 7.6, containing om % H 20 2•

After rinsing in tap water, the slides were briefly counterstained with haematoxylin, rinsed in tap water, dehydrated and mounted in Depex or aquamount.

Controls were incubated in normal mouse serum instead of the monoclonal Ia serum. No label was seen. Endogenous peroxidase activity was blocked by incubation of the slides in methanol-H20 2 (24).

Some preparations were used for simultaneous detection of Ia antigens and acid phosphat­ase. The acid phosphatase reaction was performed after the immunocytochemical procedure.

Results

In situ

Cryostat sections of PP stained for acid phosphatase (APh) actlVIty showed that the majority of the APh-positive cells was present in the subepithelial area and in the germinal center of the follicular area (Fig. 1). The APh-positive cells in the subepithelial compartment were relatively small and irregularly shaped; the intensity of the enzyme reaction varied strongly among individual cells. In the germinal center, on the other hand, most APh-positive cells were intensely stained; they were larger and more rounded than those in the subepithelial compartment. In the interfollicular area, only a small number of weakly APh-positive cells was seen. These cells were large and possessed cytoplasmic extensions between the sur­rounding lymphocytes. The enzyme reaction product was present in small granules in the perinuclear region of these cells.

The results of the non-specific esterase reaction on cryostat sections of PP were comparable to those of the APh reaction.

Immunocytochemistry using Ia antiserum showed that the weakly APh­positive cells in the IF A were strongly la-positive (Fig. 2). The APh­positive cells within the subepithelial area and the germinal center were, in contrast, not or only slightly stained with the monoclonal antiserum against la. In addition, la-positive round or irregularly shaped cells without detectable APh activity were found in the subepithelial compartment and occasionally between the epithelium cells. Double staining (APh and Ia) confirmed the results of single staining on alternating sections.

In vitro

The cell population isolated from PP contained only few non-lymphoid cells (less than 10 %), in addition to numerous lymphocytes. Most of these non-lymphoid cells were APh- and NSE-positive. The two APh and NSE­positive cell types seen in cryostat sections (small irregularly shaped cells and larger, more rounded cells) could also be distinguished in the total cell suspension of PP (Fig. 3). The cell suspension further contained large cells with cytoplasmic processes. These cells had an intensive Ia membrane staining (Fig. 4) and were only weakly APh- and NSE-positive.

Non-Lymphoid Cells in Peyer's Patches . 139

Fig. 1. Cryostat section of Peyer's patch (PP) showing the morphological differences between subepithelial macrophages (small, irregularly shaped; arrows) and germinal center mac­rophages (tingible body macrophages; large and round; double arrows). Acid phosphatase reaction, X 400.

Fig. 2. Cryostat section showing part of the interfollicular area of PP with strongly la-positive, dendritic cells (arrows). Immunocytochemistry with anti-Ia serum, X 700.

Figs. 3 and 4. Cytocentrifuge preparations of the total PP suspension. Strongly acid­phosphatase-positive tingible body macrophage (arrow) and a weakly acid-phosphatase­positive large non-lymphoid cell (double arrows). (Fig. 3, X 900). The large non-lymphoid cell (arrow) is strongly la-positive (Fig. 4, X 900); L, Lymphocytes.

Fig. 5. Strongly acid-phosphatase-positive adherent macrophage, X 600.

140 . T. SMINIA, M. M. WILDERS, E. M. lANSE, and E. C. M. HOEFSMIT

The adherent cell population comprised almost exclusively non-lym­phoid cells; most lymphocytes were found in the remaining non-adherent cell population. It was remarkable that almost all adherent cells were small, irregularly shaped, and strongly APh- and NSE-positive cells (Fig. 5). The larger more rounded APh- and NSE-positive cells present in the total cell suspension were hardly seen in the preparations of adherent cells; they were found in the cytospin preparations of the non-adherent cell population. The same was the case for the la-positive cells; the strongly la-positive cells were not seen in the adherent cell population. Most of the adherent cells were la-negative, a small fraction (less than 10 %) showed a slight la membrane staining. These latter cells were mostly small in dimension. The non-adherent cell population comprised, in addition to numerous lympho­cytes, the strongly la-positive cells and the large rounded, strongly APh­and NSE-positive cells; this latter cell population was la-negative.

Discussion

It is evident from both the present observations and the literature (10,12) that in PP at least two populations of macro phages are present, viz. 1) germinal center macrophages with the morphological features of tingible body macrophages (TBM; large rounded cells containing pycnotic lympho­cytes in various stages of digestion), and 2) subepithelial macrophages. The latter cell population is morphologically and enzyme cytochemically heterogeneous (d. 12). Obviously, the main function of both populations of cells is to remove foreign material and effete autologous products by phagocytosis. Most likely, they do not play an important role in the presentation of antigenic material to lymphoid cells as they lack surface la antigens which are of functional significance in initiating an immune response (2, 11, 15).

In addition to these two populations of classical macrophages, strongly la-positive dendritic cells were found in sections and cell suspensions of PP; these cells, which had only a slight activity of the lysosomal enzymes APh and NSE, were not glass-adherent. These results suggest that the in vitro observations that adherent cells of PP are less active in stimulating lym­phoid cells (3, 6) may be due to a lack of la-positive (dendritic) cells in the adherent cell population. Most likely the active antigen-presenting cells are present in the non-adherent fraction. It must be stressed that the ability of dendritic cells to adhere to glass is species-dependent: dendritic cells isolated from lymphoid tissues of mice are glass adherent (21, 22, 23), whereas rat dendritic cells are non-adherent (9, 27).

It has been shown that the la-positive non-lymphoid cell population in PP does comprise interdigitating cells and veiled cells (20, 26). The relation with respect to function and origin of these cell types in peripheral

Non-Lymphoid Cells in Peyer's Patches . 141

LE 5153 I BL

IFA

~6AL .---__ ----1 J '----......,

PC~ Fig. 6. Schematic drawing of the possible relation between monocytes, veiled cells and interdigitating cells in the Peyer's patch (PP) - mesenteric lymph node (MLN) complex. AL, afferent lymph; BL, basal lamina; BV, blood vessel; IFA, interfollicular area (T cell area); LE, lymphoepithelium; PC, paracortex MLN; SEA, subepithelial area. 1. monocyte; 2. monocyte­like macrophage; 3. veiled cell within the epithelium; 4. veiled cell; 5. interdigitating cell in PP; 6. veiled cell in afferent lymph; 7. interdigitating cell in MLN.

lymphoid organs is still unclear. la-positive interdigitating (dendritic) cells in the medulla of the thymus are of bone marrow origin (1). It has been suggested that veiled cells are derived from non-lymphoid mononuclear blood cells, probably monocytes or a subpopulation of monocytes; these veiled cells apparently home into the T -dependent areas of lymphoid organs and mature into IDe (6).

Recently, it has been shown that intestinal lymph contains bone-marrow­derived strongly la-positive dendritic (veiled) cells, which are potent stimulators of the primary mixed leukocyte response (18). It has been suggested strongly that these cells home into the mesenteric lymph nodes, as following mesenteric lymphadenectomy strongly la-positive dendritic cells appear in the thoracic duct lymph (13). Taken together, these observa­tions lead to the suggestion that bone-marrow-derived non-lymphoid mononuclear cells enter PP and transform into veiled cells, which in turn may either remain mobile and leave as dendritic (veiled) cells the PP and home into the paracortical area of mesenteric lymph nodes as mature IDe or mature locally to IDe and settle into the IFA of the PP (Fig. 6). Further studies using 3H-thymidine-Iabelled non-lymphoid mononuclear blood cells are in progress to test this hypothesis.

Acknowledgements

The authors are indebted to MARTINE ROEST for typing the manuscript.

142 . T. SMINIA, M. M. WILDERS, E. M. JANSE, and E. C. M. HOEFSMIT

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Dr. T. SMINIA, Department of Histology, Medical Faculty, Vrije Universiteit, P.O. Box 7161, NL-1007 Amsterdam, The Netherlands