Clin. exp. Immunol. (1978) 31, 251-259.
The lymphoid follicles of the human palatine tonsil R. C. CURRAN & E. L. JONES Department of Pathology, Medical School, University of Birmingham, Birmingham
(Received 24 October 1977)
SUMMARY
The distribution of immunoglobulin (Ig) in the lymphoid follicles of the human palatine tonsil has been re-examined by applying the unlabelled antibody peroxidase-anti-peroxidase (PAP) sequence and a two-stage fluorescein isothiocyanate (FITC) technique to trypsinized paraffin sections and cryostat sections. Large numbers of cells containing Ig were demonstrated in the follicle centres, y, p, 3 and a heavy chains being present. Cells containing K and A chains were found in all follicle centres, a finding incompatible with the monoclonality of the centres. Extracellular Ig (y, a, It and 3 heavy chains, Kc and A light chains) was also present in the follicle centres. The lymphocytes of the mantle (zone C) appeared to have y and 3 chains (and K and ) chains) on their surfaces, and were considered to be immature B cells, thereby providing support for our earlier hypothesis that these cells constituted a pool of B memory cells. Trypsinized paraffin sections proved superior to cryostat sections for the demonstration of intracellular Ig, but the reverse was true for surface Ig. Extracellular Ig was equally evident in both types of section. The metalophil method of Marshall (1948) revealed dendritic histiocytes in the follicle centre and lymphocyte mantle (zones B and C). The follicles were supplied by one or more prominent arterioles, around which the dendritic cells were located, an arrangement which may function as a 'traffic area' for trapping circulating B cells on the antigen-bearing dendritic cells.
INTRODUCTION The tonsil is apparently unique amongst the peripheral lymphoid organs in man in possessing large numbers of secondary lymphoid follicles which, at least in specimens removed surgically, invariably have active germinal centres. It is therefore a very suitable tissue for the study of these structures and the many unresolved questions which remain concerning their function. In a previous report (Curran & Jones, 1977) we described the distribution of immunoglobulin, based on the use of paraffin sections and the peroxidase-anti-peroxidase complex (PAP) technique. Following the discovery that trypsinization of paraffin sections 'unmasks' antigens and greatly increases their response in immunohistochemical techniques (Curran & Gregory, 1977), we have re-examined the distribution of the immunoglobulins in the follicles, using trypsinized paraffin sections and also cryostat sections of the same tissues. The opportunity was also taken to determine the structure and distribution of dendritic histiocytes in the lymphoid follicle, by means of Marshall's metalophil method (Marshall, 1948), in an attempt to correlate these with the immunological function of the follicle. MATERIALS AND METHODS Paraffin sections. Eighteen tonsils obtained at operation were sliced and fixed for 24 hr at 20'C in 4%. formaldehyde prepared from paraformaldehyde and containing 0 05 or 0-1 M sucrose. Six other tonsils were fixed by perfusing them for 48-72 hr at 4VC with the same solution by means of a peristaltic pump. From the fixed tissues, paraffin sections (4-6 pm thick) were prepared for histological and immunohistochemical purposes, and thicker (10-20 pm) sections for the Marshall method
Correspondence: Professor R. C. Curran, Department of Pathology, Medical School, University ofBirmingham, Birmingham B15 2TJ.
0099-9104/78/0200-0251$02.00 (C1978 Blackwell Scientific Publications
251
R. C. Curran & E. L. Jones
252
(Marshall, 1948). As well as untreated sections, paraffin sections incubated at 370C in 0-1%4 (pH 7-8) trypsin solution for periods ranging from 1 to 30 min were also subjected to the immunohistochemical procedure (Curran & Gregory, 1977). Cryostat sections. Fourteen tonsils were sliced and quenched in liquid nitrogen for the preparation of cryostat (6-8 jum thick) sections. Some of the cryostat sections were used for the immunohistochemical sequence without fixation, whilst others were fixed prior to this step for 5 min at 20'C in ether-ethanol (40:60) (Leduc, Scott & Avrameas, 1969). Histological methods. Paraffin sections were stained with haematoxylin and eosin by the Gordon & Sweets (1936) method for reticulin, and by the metalophil method for reticuloendothelial cells (Marshall, 1948). Immunohistochemicalprocedures. Ig was demonstrated by the unlabelled antibody PAP method and by a two-stage technique using fluorescein isothiocyanate (FlTC). In the PAP sequence, rabbit antiserum to human heavy or light chains is applied to the tissue section, followed by swine antiserum to rabbit Ig and a third stage consisting of the PAP complex. The swine antiserum of the second stage attaches to the rabbit Ig of the first stage (bound to the antigen in the tissue section) and also to the rabbit Ig in the PAP complex (third stage). The sites of attachment of the PAP complex are revealed by means of a 3,3-diaminobenzidine tetrahydrochloride-peroxide (DAB) solution. In the FITC sequence, the first stage is the same, but in place of the second and third stages, swine anti-rabbit Ig labelled with FITC is applied. Tests were made for y, 0t, ,u, and a heavy chains and for K and A light chains. Occasionally antisera for p and c5 heavy chains were applied simultaneously in the first stage, to determine whether both heavy chains were present on the same cell surfaces (see Curran & Jones, 1977, 1978).
RESULTS The lymphoid follicles ofthe tonsil have a 'polarity', with zone A at the bottom, zone B in the middle and zone C at the top, beneath the crypt epithelium. Zones A and B comprise the germinal centre and zone C is the mantle of small lymphocytes (Curran & Jones, 1977).
e,~~~~~~~~~~~~~~~~~~k
FIG. 1. Lymphoid follicle showing an arteriole (arrow) entering the germinal centre. Many tingible body macrophages (M) are visible; C = lymphocytes of zone C. Haematoxylin, van Gieson. (Magnification x 175.)
Lymphoid follicles of the tonsil
FIG. 2. Germinal centre of lymphoid follicle, showing paraffin section, PAP method. (Magnification x 330.)
many cells
containing
253
K
chains (black). Trypsinized
FIG. 3. Same germinal centre as in Fig. 2, showing many cells containing A chains (black). Trypsinized paraffin section, PAP method. (Magnification x 330.) H
254
R. C. Curran & E. L. Jones
In a well-orientated section, one or more arterioles (Fig. 1) are seen to enter the base of each follicle. There is a plexus of narrow capillaries in the follicle centre, in zone B particularly, and another plexus of wider calibre capillaries beneath and within the epithelium lining the crypt. Post-capillary venules, with their distinctive tall endothelial cells, lie alongside and occasionally beneath the follicle. The distribution of heavy and light chains in paraffin and cryostat sections is identical in the FITC and PAP preparations. The two methods appear to be equally sensitive, although a weak positive reaction is sometimes easier to pick up initially in an FITC preparation. There are several differences, however, in the results given by the paraffin and cryostat sections. Trypsinized paraffin sections demonstrate Ig within the cells very effectively, the reaction product being present in high concentration and is welllocalized in the cell cytoplasm. In cryostat sections, on the other hand, the Ig is located peripherally and often apparently in a zone around the cell, as a kind of 'halo'; and where there are many positively reacting cells, this sometimes produces a diffuse 'zone' of reaction. Cryostat sections are more satisfactory than paraffin sections for demonstrating Ig on cell surfaces (see below). Extracellular Ig is equally evident in both paraffin and cryostat sections.
Zone A This is the basal part of the follicle centre. Mitotic figures are numerous and there are many large macrophages with ingested chromatin fragments (Fig. 1). These tingible body macrophages are not
A
A
In- X A-
R4 .rr ...
6
4,4" ~
-
s s.
~ ,
U -t '
FIG. 4. Lymphoid follicle. Many plasma cells in and beneath the crypt epithelium (E) contain heavy chains (black). Similar cells are present in the follicle centre, mainly in zone B (B). Strands of extracellular Ig are present in this zone but none in zone A (A). Trypsinized paraffin section, PAP method. (Magnification x 240.) -
Lymphoid follicles of the tonsil
255
metalophilic. Occasionally a few cells are seen which contain Ig (y, a, u or 3 heavy chains; K or ) light chains). There is little or no extracellular Ig. Zone B A considerable number of cells are present containing y, a, 3 and ut heavy chains. Cells containing y heavy chains are the most numerous, and those containing a chains outnumber those containing p or 3 chains. K and A light chains are detectable in all follicle centres (Figs 2 and 3). Considerable concentrations of extracellular Ig are also present, apparently lying between the cells of the zone (Figs 4 and 5): the concentrations of) and ,u heavy chains in this zone are much greater than that of a heavy chain, and very little 3 chain is found. K and A light chains are both present. The Ig does not seem to be associated with the scanty reticulin fibrils present, nor can it be related to the dendritic histiocytes, which are present in considerable numbers in metalophil preparations (Fig. 6a). No link can be detected between the dendritic cells and the reticulin fibrils. Zone C This zone consists largely of a mantle of small lymphocytes, with compact deeply basophilic nuclei. Metalophil dendritic histiocytes (Fig. 6a and b) are also present. Up to 80 or 90 pm in length, these are
FIG. 5. Lymphoid follicle and crypt epithelium (E). Plasma cells containing p heavy chains are present beneath the epithelium and in the follicle centre. # heavy chains are also present around the lymphocytes of zone C (C), but not around the lymphocytes lateral to the follicle (arrow). Extracellular Ig is also present in zone B of the follicle centre (B) but not in zone A (A). Trypsinized paraffin section, PAP method. (Magnification x 240.)
A!
J..A
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.;.
.1
Lymphoidfollicles of the tonsil
257
more slender than their counterparts in zone B, but more highly dendritic. Occasional cells of similar shape are also found immediately beneath the crypt epithelium, the basal cells of which are notably metalophylic (Fig. 6a). Very few Ig-containing cells are found in zone C, but ,u heavy chains are detectable in a low concentration around a high proportion of the lymphocytes (Fig. 5). 6 heavy chains are present at the same site, but around smaller numbers of lymphocytes. When antisera for Yi and 6 heavy chains are both applied, the reaction is stronger but the distribution of positive cells appears to be much the same as in the preparation foru chains. K and A light chains are also detectable in association with the lymphocyte surfaces. These reactions are assumed to be caused by surface (receptor) immunoglobulin (SIg). Individual SIg-positive lymphocytes are also found in the vicinity of the follicle, but most of the lymphocytes outside zone C are negative (Fig. 5). Zone C often extends up to the crypt epithelium, and SIgpositive lymphocytes lie within the epithelium. The reactions for SIg are best seen in cryostat sections, and although the reaction for Iu heavy chains can be demonstrated in trypsinized paraffin sections, the reaction for 6 heavy chains cannot, as a rule.
DISCUSSION A number of significant findings concerning the lymphoid follicles of the tonsil emerge from this study: the characterization of the mantle of lymphocytes (zone C) as immature B cells; the resemblance of the blood supply of the follicles to that of the Malpighian bodies of the spleen; the distribution of dendritic reticular cells in and around the follicles; the presence of extracellular Ig in the germinal centre (zone B); and the lack of monoclonality of the germinal centre. The lymphocytes of the mantle (zone C) appeared to be immature B lymphocytes, as judged by the presence on the surface of 4t and 6 heavy chains, and K and A light chains. This is in accord with our earlier hypothesis concerning the function of the lymphoid follicles of the tonsil (Curran & Jones, 1977). This proposed that T lymphocytes sensitized at the epithelial surface interact with a subpopulation of B lymphocytes in the follicle centre, which then divide either to become immunoblasts and plasma cells in zone B, or migrate upwards to constitute a pool of B memory cells in the lymphocyte cap (zone C). These memory cells, on contact with the appropriate antigen in the epithelium, undergo a secondary response to form Ig; and presumably they are the source of the plasma cells in the subepithelial area. The results obtained in the present study with the PAP method suggest that some Ig-negative lymphocytes were probably also present in zone C, but from the very high proportion of positive lymphocytes in the FITC preparations, the numbers of the former must be relatively small. SIg-positive and SIg-negative lymphocytes were also present within the epithelium. The SIg-negative cells may be T cells, concerned with recognition of antigens entering via the crypt. Little information is available regarding the vascular supply of the lymphoid follicle, and although special techniques would be required to determine this accurately, the frequency with which a small artery or arteriole was seen to enter the basal region of a follicle was strongly reminiscent of the blood supply of the Malpighian corpuscles of the spleen, each of which is supplied by a penicillar branch of the central artery of the white pulp. White, French & Stark (1970) demonstrated that dendritic cells are located along the branches of the central artery and that germinal centre formation in the chicken spleen results from a process of progressive capture and aggregation of lymphocytes at the surface of the cytoplasmic extensions of antigen-bearing dendritic processes of these cells. In the lymphoid follicle in the tonsil there may be a similar 'traffic area' centred on an arteriole, in which a circulating population of B lymphocytes becomes trapped on the perikaryon of the dendritic cells. FIG. 6. (a) Lymphoid follicle, showing metalophylic dendritic histiocytes in zones B and C (B and C). Those in zone B are rounder and less dendritic than those in zone C. The macrophages of zone A (A) are generally poorly
metalophil and not dendritic (arrow). The basal cells of the crypt epithelium (E) are also metalophil. Paraffin section, Marshall's metalophil method. (Magnification x 175.) (b) High-power view of part of Fig. 6a, showing some of the dendritic histiocytes in zone C in more detail. Numerous isolated dendrites are visible, an appearance due to the large size of the cells compared with the thickness of the section. 10 jum paraffin section, Marshall's method. (Magnification x 440.)
258
R. C. Curran C E. L. Jones
It is now thought by some that dendritic cells function by collecting Ig-related substances secreted by T lymphocytes as a specific response to an antigen, the Ig on the dendritic processes then being made available to B lymphocytes. The location of the dendritic histiocytes in the present study accords well with this concept, with a large population of immature B lymphocytes and Ig-secreting cells in close approximation to the dendritic cells. However, the role of the dendritic histiocytes in zone C, where they are surrounded by a uniform population of immature B cells, is less clear. Germinal centres are bone marrow-dependent structures and consist largely of B cells, although thymus cells are known to accelerate the formation of germinal centres (Kotani et al., 1974), and scanty T cells can be demonstrated in primary follicles (Gutman & Weissman, 1972) and at the 'cortical' pole of active germinal centres (Weissman et al., 1977). It is thought that germinal centres are not involved in the initiation of antibody production and that antibody-producing cells are found within them only in vigorous secondary responses. Certainly the large numbers of Ig-containing cells which we found within zone B of each germinal centre is consistent with these views. The source and function of the abundant 'extracellular' Ig, which was also present in this zone, were obscure, however, the material seeming to lie between the various types of cell. Fibrillary materials seem to have a tendency to give 'non-specific' reactions for Ig, but the reactions for extracellular Ig did not seem to be linked in any way with the scanty reticulin fibrils present in the follicle centre. It has often been suggested that the cell population of germinal centres is monoclonal and that each centre may be monospecific with regard to the antigen stimulating its development (Sordat et al., 1970). A single clone of B lymphocytes is committed to the expression of a particular Ig sequence of VH and VL, which together form a particular antibody combining site and determine the binding specificity of the Ig molecule. Monoclonality can be demonstrated by showing that all the cells are restricted to the production of a single combining site, e.g. by use of an anti-idiotype reagent. This has been achieved for multiple myeloma and Waldenstrom's macroglobulinaemia (Wernet, Feizi & Kunkel, 1972; Rudders, Yakulis & Heller, 1973), by producing anti-idiotype reagents from the monoclonal Ig and using them to identify the malignant clone. The method is not readily applicable, however, and light chain typing, based on the restriction within a clone to the production of Ig light chains of either K or A type, is the more usual method of detecting monoclonality: the finding of a single light chain type being strong presumptive evidence of monoclonality. Our finding of K and A light chains in every follicle centre therefore provides strong evidence that the population of Ig-secreting cells of the germinal centres of the lymphoid follicles of the tonsil is not monoclonal. We are grateful to Professor P. G. H. Gell for much helpful discussion during the preparation of the manuscript. Our thanks are also due to Mr J. Gregory and Miss Philomena Byrne for technical assistance and to Mrs D. Harding for typing the manuscript. Generous financial support was provided by the Endowment Fund Medical Research Committee of the Central Birmingham Health District.
REFERENCES thymus cells in the formation of germinal centres. Acta CURRAN, R.C. & GREGORY, J. (1977) The unmasking of anat. 90, 585. antigens in paraffin sections of tissue by trypsin. ExLEDuc, E.H., SCOTT, G.B. & AvRAMEAs, S. (1969) Ultraperientia, 33, 1400. structural localization of intracellular immune globulins in CURRAN, R.C. & JONES, E.L. (1977) Immunoglobulinplasma cells and lymphoblasts by enzyme-labelled containing cells in human tonsils as demonstrated by antibodies. J. Histochem. Cytochem. 17, 211. immunohistochemistry. Clin. exp. Immunol. 28, 103. CURRAN R.C. & JONES, E.L. (1978) Hodgkin's disease: an MARSHALL, A.H.E. (1948) A method for the demonstration of reticuloendothelial cells in paraffin sections. I. Path. immunohistochemical and histological study. J. Path. (In Bact. 60, 515. Press.) GORDON, H. & SWEETS, H.H. (1936) A simple method for RUDDERS, R.A., YAKULIS, V. & HELLER, P. (1973) Double myeloma: production of both IgG type lambda and IgA the silver impregnation of reticulum. Am. I. Path. 12, type lambda myeloma proteins by a single plasma cell line. 545. Am. J. med. 55, 215. GUTMAN, G.A. & WEISSMAN, I.L. (1972) Lymphoid tissue architecture. Experimental analysis of the origin SORDAT, B., SORDAT, M., HESS, M.W., STONER, R.D. & COTTIER, H. (1970) Specific antibody within lymphoid and distribution of T cells and B cells. Immunology, 23, germinal center cells of mice after primary immunization 465. with horseradish peroxidase: a light and electron microKOTANI, M., NAWA, Y., Fujii, HI., FUKUMOTO, T., MIYAscopic study. 7. exp. Med. 131, 77. MOTO, M. & YAMASHITA, A. (1974) Involvement of
Lymphoid follicles ofthe tonsil WEISSMAN, I.L., GUTMAN, G.A., FRIEDBERG, S.H. & JERABEK, L. (1977) Lymphoid tissue architecture. III. Germinal centres, T cells and thymus-dependent vs thymus-independent antigens. Immune Reactivity of Lymphocytes; Development, Expression and Control (eds M. Feldman and A. Globerson). Plenum Press, New York. Adv. exp. biol. Med. 66. WERNET, P.T., FEIZI, T. & KUNKEL, H.G. (1972) Idiotype
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determinants of immunoglobulin detected on the surface of human lymphocytes by cytotoxicity assays. J. exp. Med. 136, 650. WHITE, R.G., FRENCH, V.I. & STARK, J.M. (1970) A study of the localization of a protein antigen in the chicken spleen and its relation to the formation of germinal centres. ]. med. Microbiol. 3, 65.
The lymphoid follicles of the human palatine tonsil R. C. CURRAN & E. L. JONES Department of Pathology, Medical School, University of Birmingham, Birmingham
(Received 24 October 1977)
SUMMARY
The distribution of immunoglobulin (Ig) in the lymphoid follicles of the human palatine tonsil has been re-examined by applying the unlabelled antibody peroxidase-anti-peroxidase (PAP) sequence and a two-stage fluorescein isothiocyanate (FITC) technique to trypsinized paraffin sections and cryostat sections. Large numbers of cells containing Ig were demonstrated in the follicle centres, y, p, 3 and a heavy chains being present. Cells containing K and A chains were found in all follicle centres, a finding incompatible with the monoclonality of the centres. Extracellular Ig (y, a, It and 3 heavy chains, Kc and A light chains) was also present in the follicle centres. The lymphocytes of the mantle (zone C) appeared to have y and 3 chains (and K and ) chains) on their surfaces, and were considered to be immature B cells, thereby providing support for our earlier hypothesis that these cells constituted a pool of B memory cells. Trypsinized paraffin sections proved superior to cryostat sections for the demonstration of intracellular Ig, but the reverse was true for surface Ig. Extracellular Ig was equally evident in both types of section. The metalophil method of Marshall (1948) revealed dendritic histiocytes in the follicle centre and lymphocyte mantle (zones B and C). The follicles were supplied by one or more prominent arterioles, around which the dendritic cells were located, an arrangement which may function as a 'traffic area' for trapping circulating B cells on the antigen-bearing dendritic cells.
INTRODUCTION The tonsil is apparently unique amongst the peripheral lymphoid organs in man in possessing large numbers of secondary lymphoid follicles which, at least in specimens removed surgically, invariably have active germinal centres. It is therefore a very suitable tissue for the study of these structures and the many unresolved questions which remain concerning their function. In a previous report (Curran & Jones, 1977) we described the distribution of immunoglobulin, based on the use of paraffin sections and the peroxidase-anti-peroxidase complex (PAP) technique. Following the discovery that trypsinization of paraffin sections 'unmasks' antigens and greatly increases their response in immunohistochemical techniques (Curran & Gregory, 1977), we have re-examined the distribution of the immunoglobulins in the follicles, using trypsinized paraffin sections and also cryostat sections of the same tissues. The opportunity was also taken to determine the structure and distribution of dendritic histiocytes in the lymphoid follicle, by means of Marshall's metalophil method (Marshall, 1948), in an attempt to correlate these with the immunological function of the follicle. MATERIALS AND METHODS Paraffin sections. Eighteen tonsils obtained at operation were sliced and fixed for 24 hr at 20'C in 4%. formaldehyde prepared from paraformaldehyde and containing 0 05 or 0-1 M sucrose. Six other tonsils were fixed by perfusing them for 48-72 hr at 4VC with the same solution by means of a peristaltic pump. From the fixed tissues, paraffin sections (4-6 pm thick) were prepared for histological and immunohistochemical purposes, and thicker (10-20 pm) sections for the Marshall method
Correspondence: Professor R. C. Curran, Department of Pathology, Medical School, University ofBirmingham, Birmingham B15 2TJ.
0099-9104/78/0200-0251$02.00 (C1978 Blackwell Scientific Publications
251
R. C. Curran & E. L. Jones
252
(Marshall, 1948). As well as untreated sections, paraffin sections incubated at 370C in 0-1%4 (pH 7-8) trypsin solution for periods ranging from 1 to 30 min were also subjected to the immunohistochemical procedure (Curran & Gregory, 1977). Cryostat sections. Fourteen tonsils were sliced and quenched in liquid nitrogen for the preparation of cryostat (6-8 jum thick) sections. Some of the cryostat sections were used for the immunohistochemical sequence without fixation, whilst others were fixed prior to this step for 5 min at 20'C in ether-ethanol (40:60) (Leduc, Scott & Avrameas, 1969). Histological methods. Paraffin sections were stained with haematoxylin and eosin by the Gordon & Sweets (1936) method for reticulin, and by the metalophil method for reticuloendothelial cells (Marshall, 1948). Immunohistochemicalprocedures. Ig was demonstrated by the unlabelled antibody PAP method and by a two-stage technique using fluorescein isothiocyanate (FlTC). In the PAP sequence, rabbit antiserum to human heavy or light chains is applied to the tissue section, followed by swine antiserum to rabbit Ig and a third stage consisting of the PAP complex. The swine antiserum of the second stage attaches to the rabbit Ig of the first stage (bound to the antigen in the tissue section) and also to the rabbit Ig in the PAP complex (third stage). The sites of attachment of the PAP complex are revealed by means of a 3,3-diaminobenzidine tetrahydrochloride-peroxide (DAB) solution. In the FITC sequence, the first stage is the same, but in place of the second and third stages, swine anti-rabbit Ig labelled with FITC is applied. Tests were made for y, 0t, ,u, and a heavy chains and for K and A light chains. Occasionally antisera for p and c5 heavy chains were applied simultaneously in the first stage, to determine whether both heavy chains were present on the same cell surfaces (see Curran & Jones, 1977, 1978).
RESULTS The lymphoid follicles ofthe tonsil have a 'polarity', with zone A at the bottom, zone B in the middle and zone C at the top, beneath the crypt epithelium. Zones A and B comprise the germinal centre and zone C is the mantle of small lymphocytes (Curran & Jones, 1977).
e,~~~~~~~~~~~~~~~~~~k
FIG. 1. Lymphoid follicle showing an arteriole (arrow) entering the germinal centre. Many tingible body macrophages (M) are visible; C = lymphocytes of zone C. Haematoxylin, van Gieson. (Magnification x 175.)
Lymphoid follicles of the tonsil
FIG. 2. Germinal centre of lymphoid follicle, showing paraffin section, PAP method. (Magnification x 330.)
many cells
containing
253
K
chains (black). Trypsinized
FIG. 3. Same germinal centre as in Fig. 2, showing many cells containing A chains (black). Trypsinized paraffin section, PAP method. (Magnification x 330.) H
254
R. C. Curran & E. L. Jones
In a well-orientated section, one or more arterioles (Fig. 1) are seen to enter the base of each follicle. There is a plexus of narrow capillaries in the follicle centre, in zone B particularly, and another plexus of wider calibre capillaries beneath and within the epithelium lining the crypt. Post-capillary venules, with their distinctive tall endothelial cells, lie alongside and occasionally beneath the follicle. The distribution of heavy and light chains in paraffin and cryostat sections is identical in the FITC and PAP preparations. The two methods appear to be equally sensitive, although a weak positive reaction is sometimes easier to pick up initially in an FITC preparation. There are several differences, however, in the results given by the paraffin and cryostat sections. Trypsinized paraffin sections demonstrate Ig within the cells very effectively, the reaction product being present in high concentration and is welllocalized in the cell cytoplasm. In cryostat sections, on the other hand, the Ig is located peripherally and often apparently in a zone around the cell, as a kind of 'halo'; and where there are many positively reacting cells, this sometimes produces a diffuse 'zone' of reaction. Cryostat sections are more satisfactory than paraffin sections for demonstrating Ig on cell surfaces (see below). Extracellular Ig is equally evident in both paraffin and cryostat sections.
Zone A This is the basal part of the follicle centre. Mitotic figures are numerous and there are many large macrophages with ingested chromatin fragments (Fig. 1). These tingible body macrophages are not
A
A
In- X A-
R4 .rr ...
6
4,4" ~
-
s s.
~ ,
U -t '
FIG. 4. Lymphoid follicle. Many plasma cells in and beneath the crypt epithelium (E) contain heavy chains (black). Similar cells are present in the follicle centre, mainly in zone B (B). Strands of extracellular Ig are present in this zone but none in zone A (A). Trypsinized paraffin section, PAP method. (Magnification x 240.) -
Lymphoid follicles of the tonsil
255
metalophilic. Occasionally a few cells are seen which contain Ig (y, a, u or 3 heavy chains; K or ) light chains). There is little or no extracellular Ig. Zone B A considerable number of cells are present containing y, a, 3 and ut heavy chains. Cells containing y heavy chains are the most numerous, and those containing a chains outnumber those containing p or 3 chains. K and A light chains are detectable in all follicle centres (Figs 2 and 3). Considerable concentrations of extracellular Ig are also present, apparently lying between the cells of the zone (Figs 4 and 5): the concentrations of) and ,u heavy chains in this zone are much greater than that of a heavy chain, and very little 3 chain is found. K and A light chains are both present. The Ig does not seem to be associated with the scanty reticulin fibrils present, nor can it be related to the dendritic histiocytes, which are present in considerable numbers in metalophil preparations (Fig. 6a). No link can be detected between the dendritic cells and the reticulin fibrils. Zone C This zone consists largely of a mantle of small lymphocytes, with compact deeply basophilic nuclei. Metalophil dendritic histiocytes (Fig. 6a and b) are also present. Up to 80 or 90 pm in length, these are
FIG. 5. Lymphoid follicle and crypt epithelium (E). Plasma cells containing p heavy chains are present beneath the epithelium and in the follicle centre. # heavy chains are also present around the lymphocytes of zone C (C), but not around the lymphocytes lateral to the follicle (arrow). Extracellular Ig is also present in zone B of the follicle centre (B) but not in zone A (A). Trypsinized paraffin section, PAP method. (Magnification x 240.)
A!
J..A
.4w . ko
.;.
.1
Lymphoidfollicles of the tonsil
257
more slender than their counterparts in zone B, but more highly dendritic. Occasional cells of similar shape are also found immediately beneath the crypt epithelium, the basal cells of which are notably metalophylic (Fig. 6a). Very few Ig-containing cells are found in zone C, but ,u heavy chains are detectable in a low concentration around a high proportion of the lymphocytes (Fig. 5). 6 heavy chains are present at the same site, but around smaller numbers of lymphocytes. When antisera for Yi and 6 heavy chains are both applied, the reaction is stronger but the distribution of positive cells appears to be much the same as in the preparation foru chains. K and A light chains are also detectable in association with the lymphocyte surfaces. These reactions are assumed to be caused by surface (receptor) immunoglobulin (SIg). Individual SIg-positive lymphocytes are also found in the vicinity of the follicle, but most of the lymphocytes outside zone C are negative (Fig. 5). Zone C often extends up to the crypt epithelium, and SIgpositive lymphocytes lie within the epithelium. The reactions for SIg are best seen in cryostat sections, and although the reaction for Iu heavy chains can be demonstrated in trypsinized paraffin sections, the reaction for 6 heavy chains cannot, as a rule.
DISCUSSION A number of significant findings concerning the lymphoid follicles of the tonsil emerge from this study: the characterization of the mantle of lymphocytes (zone C) as immature B cells; the resemblance of the blood supply of the follicles to that of the Malpighian bodies of the spleen; the distribution of dendritic reticular cells in and around the follicles; the presence of extracellular Ig in the germinal centre (zone B); and the lack of monoclonality of the germinal centre. The lymphocytes of the mantle (zone C) appeared to be immature B lymphocytes, as judged by the presence on the surface of 4t and 6 heavy chains, and K and A light chains. This is in accord with our earlier hypothesis concerning the function of the lymphoid follicles of the tonsil (Curran & Jones, 1977). This proposed that T lymphocytes sensitized at the epithelial surface interact with a subpopulation of B lymphocytes in the follicle centre, which then divide either to become immunoblasts and plasma cells in zone B, or migrate upwards to constitute a pool of B memory cells in the lymphocyte cap (zone C). These memory cells, on contact with the appropriate antigen in the epithelium, undergo a secondary response to form Ig; and presumably they are the source of the plasma cells in the subepithelial area. The results obtained in the present study with the PAP method suggest that some Ig-negative lymphocytes were probably also present in zone C, but from the very high proportion of positive lymphocytes in the FITC preparations, the numbers of the former must be relatively small. SIg-positive and SIg-negative lymphocytes were also present within the epithelium. The SIg-negative cells may be T cells, concerned with recognition of antigens entering via the crypt. Little information is available regarding the vascular supply of the lymphoid follicle, and although special techniques would be required to determine this accurately, the frequency with which a small artery or arteriole was seen to enter the basal region of a follicle was strongly reminiscent of the blood supply of the Malpighian corpuscles of the spleen, each of which is supplied by a penicillar branch of the central artery of the white pulp. White, French & Stark (1970) demonstrated that dendritic cells are located along the branches of the central artery and that germinal centre formation in the chicken spleen results from a process of progressive capture and aggregation of lymphocytes at the surface of the cytoplasmic extensions of antigen-bearing dendritic processes of these cells. In the lymphoid follicle in the tonsil there may be a similar 'traffic area' centred on an arteriole, in which a circulating population of B lymphocytes becomes trapped on the perikaryon of the dendritic cells. FIG. 6. (a) Lymphoid follicle, showing metalophylic dendritic histiocytes in zones B and C (B and C). Those in zone B are rounder and less dendritic than those in zone C. The macrophages of zone A (A) are generally poorly
metalophil and not dendritic (arrow). The basal cells of the crypt epithelium (E) are also metalophil. Paraffin section, Marshall's metalophil method. (Magnification x 175.) (b) High-power view of part of Fig. 6a, showing some of the dendritic histiocytes in zone C in more detail. Numerous isolated dendrites are visible, an appearance due to the large size of the cells compared with the thickness of the section. 10 jum paraffin section, Marshall's method. (Magnification x 440.)
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R. C. Curran C E. L. Jones
It is now thought by some that dendritic cells function by collecting Ig-related substances secreted by T lymphocytes as a specific response to an antigen, the Ig on the dendritic processes then being made available to B lymphocytes. The location of the dendritic histiocytes in the present study accords well with this concept, with a large population of immature B lymphocytes and Ig-secreting cells in close approximation to the dendritic cells. However, the role of the dendritic histiocytes in zone C, where they are surrounded by a uniform population of immature B cells, is less clear. Germinal centres are bone marrow-dependent structures and consist largely of B cells, although thymus cells are known to accelerate the formation of germinal centres (Kotani et al., 1974), and scanty T cells can be demonstrated in primary follicles (Gutman & Weissman, 1972) and at the 'cortical' pole of active germinal centres (Weissman et al., 1977). It is thought that germinal centres are not involved in the initiation of antibody production and that antibody-producing cells are found within them only in vigorous secondary responses. Certainly the large numbers of Ig-containing cells which we found within zone B of each germinal centre is consistent with these views. The source and function of the abundant 'extracellular' Ig, which was also present in this zone, were obscure, however, the material seeming to lie between the various types of cell. Fibrillary materials seem to have a tendency to give 'non-specific' reactions for Ig, but the reactions for extracellular Ig did not seem to be linked in any way with the scanty reticulin fibrils present in the follicle centre. It has often been suggested that the cell population of germinal centres is monoclonal and that each centre may be monospecific with regard to the antigen stimulating its development (Sordat et al., 1970). A single clone of B lymphocytes is committed to the expression of a particular Ig sequence of VH and VL, which together form a particular antibody combining site and determine the binding specificity of the Ig molecule. Monoclonality can be demonstrated by showing that all the cells are restricted to the production of a single combining site, e.g. by use of an anti-idiotype reagent. This has been achieved for multiple myeloma and Waldenstrom's macroglobulinaemia (Wernet, Feizi & Kunkel, 1972; Rudders, Yakulis & Heller, 1973), by producing anti-idiotype reagents from the monoclonal Ig and using them to identify the malignant clone. The method is not readily applicable, however, and light chain typing, based on the restriction within a clone to the production of Ig light chains of either K or A type, is the more usual method of detecting monoclonality: the finding of a single light chain type being strong presumptive evidence of monoclonality. Our finding of K and A light chains in every follicle centre therefore provides strong evidence that the population of Ig-secreting cells of the germinal centres of the lymphoid follicles of the tonsil is not monoclonal. We are grateful to Professor P. G. H. Gell for much helpful discussion during the preparation of the manuscript. Our thanks are also due to Mr J. Gregory and Miss Philomena Byrne for technical assistance and to Mrs D. Harding for typing the manuscript. Generous financial support was provided by the Endowment Fund Medical Research Committee of the Central Birmingham Health District.
REFERENCES thymus cells in the formation of germinal centres. Acta CURRAN, R.C. & GREGORY, J. (1977) The unmasking of anat. 90, 585. antigens in paraffin sections of tissue by trypsin. ExLEDuc, E.H., SCOTT, G.B. & AvRAMEAs, S. (1969) Ultraperientia, 33, 1400. structural localization of intracellular immune globulins in CURRAN, R.C. & JONES, E.L. (1977) Immunoglobulinplasma cells and lymphoblasts by enzyme-labelled containing cells in human tonsils as demonstrated by antibodies. J. Histochem. Cytochem. 17, 211. immunohistochemistry. Clin. exp. Immunol. 28, 103. CURRAN R.C. & JONES, E.L. (1978) Hodgkin's disease: an MARSHALL, A.H.E. (1948) A method for the demonstration of reticuloendothelial cells in paraffin sections. I. Path. immunohistochemical and histological study. J. Path. (In Bact. 60, 515. Press.) GORDON, H. & SWEETS, H.H. (1936) A simple method for RUDDERS, R.A., YAKULIS, V. & HELLER, P. (1973) Double myeloma: production of both IgG type lambda and IgA the silver impregnation of reticulum. Am. I. Path. 12, type lambda myeloma proteins by a single plasma cell line. 545. Am. J. med. 55, 215. GUTMAN, G.A. & WEISSMAN, I.L. (1972) Lymphoid tissue architecture. Experimental analysis of the origin SORDAT, B., SORDAT, M., HESS, M.W., STONER, R.D. & COTTIER, H. (1970) Specific antibody within lymphoid and distribution of T cells and B cells. Immunology, 23, germinal center cells of mice after primary immunization 465. with horseradish peroxidase: a light and electron microKOTANI, M., NAWA, Y., Fujii, HI., FUKUMOTO, T., MIYAscopic study. 7. exp. Med. 131, 77. MOTO, M. & YAMASHITA, A. (1974) Involvement of
Lymphoid follicles ofthe tonsil WEISSMAN, I.L., GUTMAN, G.A., FRIEDBERG, S.H. & JERABEK, L. (1977) Lymphoid tissue architecture. III. Germinal centres, T cells and thymus-dependent vs thymus-independent antigens. Immune Reactivity of Lymphocytes; Development, Expression and Control (eds M. Feldman and A. Globerson). Plenum Press, New York. Adv. exp. biol. Med. 66. WERNET, P.T., FEIZI, T. & KUNKEL, H.G. (1972) Idiotype
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determinants of immunoglobulin detected on the surface of human lymphocytes by cytotoxicity assays. J. exp. Med. 136, 650. WHITE, R.G., FRENCH, V.I. & STARK, J.M. (1970) A study of the localization of a protein antigen in the chicken spleen and its relation to the formation of germinal centres. ]. med. Microbiol. 3, 65.
