ARTICLE |
Correspondence to: Birte Steiniger, Inst. of Anatomy and Cell Biology, Robert-Koch-Str. 6, D-35033 Marburg, Germany. E-mail: steinigb@mailer.uni-marburg.de
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The precise arrangement of B- and T-lymphocytes in the different compartments of the human splenic white pulp is still largely unknown. We therefore performed a 3D reconstruction of 150 serial sections of a representative adult human spleen alternately stained for CD3 and CD20. The results indicate that the T-cell regions of human spleens may be interrupted by B-cell follicles. Therefore, there is no continuous periarteriolar lymphatic T-cell sheath (PALS) around white pulp arterioles. An arteriole may be surrounded by T-lymphocytes at one level, then run across a follicle without any T-cells around, and finally re-enter a T-cell region. T- and B-cell compartments are intricately interdigitated in the human splenic white pulp. CD4+ T-lymphocytes and the typical fibroblasts of the T-cell region may extend as a thin shell at the follicular surface within the marginal zone. On the other hand, IgD++ B-cells continue from the follicular outer marginal zone along the surface of the T-cell region. Our findings indicate that the microanatomy of the splenic white pulp differs between humans and rodents. This may have consequences for the immigration of recirculating lymphocytes and for initial interactions among antigen-specific T- and B-lymphocytes. (J Histochem Cytochem 51:655663, 2003)
Key Words: human spleen, white pulp, T-cell regions, follicles, marginal zone, CD3, CD20, CCL21
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN CONTRAST TO mice and rats, the compartments of the splenic white pulp are ill-defined in humans. The location of human T-cell regions with respect to the so-called "central arterioles" is especially controversial. In addition, the arrangement of B-cell compartments, especially the location of the marginal zone (MZ), is insufficiently described in humans. Because MZ B-cells are believed to decisively contribute to the sepsis-protective effect of the spleen (
The nomenclature for some parts of the splenic white pulp vasculature was coined in rats (
We have previously shown that the splenic white pulp of humans differs from that of rats with respect to several microanatomic (especially microvascular) features (-actin and myosin, MAdCAM-1, VCAM-1, and VAP-1, as well as thrombomodulin, cytokeratins 8 and 18, and Thy-1 (
To more precisely define the T- and B-cell regions of the human splenic white pulp and their relationship to arterioles, we have performed a 3D reconstruction of 150 serial sections of a representative adult human spleen alternately stained for CD3 and CD20. The specimen was carefully selected from a panel of 73 human spleens primarily derived from traffic accident victims of different ages, which had been thoroughly characterized for the distribution of B- and T-lymphocytes, macrophages, and fibroblasts (
The central question to be answered by our investigation concerned the relationship between white pulp arterioles and follicles. We had frequently observed rather large arterioles without any accompanying T-cells within follicles, and even within germinal centers of the human splenic white pulp. Therefore, we wished to clarify whether these intrafollicular vessels represented genuine "central arterioles" that had lost their T-cell sheaths or whether they should be regarded as side-branches of central arterioles. Together with our previous findings (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Processing of Samples
A representative spleen was chosen from a pool of 73 organs that had been thoroughly investigated by immunohistology (
Immunohistology
Demonstration of CD3 and CD20 in Serial Sections.
A series of 150 sections was prepared on silanized slides and alternately stained for CD3 (polyclonal, DAKO; Hamburg, Germany, no. A 0452) and CD20 (L26, DAKO no. M 0755). In brief, after deparaffinization, formol pigment was removed with picric acid and the sections were preincubated with 0.05% protease type XIV (Sigma, Deisenhofen, Germany; no. P-5147) in TBS for 15 min at RT. After washing, sections were immersed in 0.15% H2O2 in PBS for 30 min at RT to block endogenous peroxidase. After an additional wash, anti-CD3 was applied to the sections at 1:80 and L26 at 1:150 in PBS with 1% BSA, 0.1%NaN3 (PBS/BSA/NaN3), and 3 µg/ml avidin for 60 min at RT. For detection of anti-CD3, a secondary biotinylated goat anti-rabbit IgG (Vector Labs no. BA-1000; via Alexis, Grünberg, Germany) was used at 1:200. For L26, a biotinylated goat anti-mouse IgG (Vector Labs no. PK-6102) was used at the same dilution. The secondary reagents were diluted in PBS with 5% normal rat serum and 20 µg/ml biotin and applied for 30 min at RT. Finally, avidinbiotinylated peroxidase complexes (ABC; Vectastain Elite ABC Kit, Vector Labs no. PK 6102) were prepared according to the manufacturer's recommendation and reacted with the sections for 30 min at RT. After washing in TBS, the peroxidase complexes were revealed by diaminobenzidine and the sections were lightly counterstained with Mayer's hemalum. The first sections of the series served as controls and were incubated with omission of the primary antibody. Normal serum of the respective species in place of primary antibodies did not produce background staining.
Demonstration of CCL21 (SLC) and Smooth Muscle -actin.
Rabbit anti-human CCL21 (Exodus-2, SLC) affinity-purified antibodies were obtained from Preprotech (No. 500-P109) via TEBU (Frankfurt, Germany). The antibodies were diluted 1:1000 in PBS/BSA/NaN3 and applied for 20 hr at 4C to paraffin sections that had been autoclaved for 20 min in H2O/EDTA, pH 8.0, for antigen retrieval. MAb asm-1 against smooth muscle
-actin was obtained from Progen (Heidelberg, Germany; no. 61001) and used at 1:200. Before incubation with this antibody, paraffin sections were autoclaved in citrate buffer, pH 6.0. Demonstration of bound antibody was as described above, using anti-rabbit or anti-mouse ABC. Omission of primary antibody and application of normal rabbit or mouse serum were used as controls.
Image Acquisition and 3D Reconstruction
The right angle cut at one corner of the sections was used for overall orientation. A representative area close to this landmark, containing three follicles, was chosen and photographed with a digital video camera using a x4 lens with an x1.25 optovar. The pictures were superimposed with the help of the Openlab program (Version 1.7.8; Improvision, Heidelberg, Germany) and the sectioned arterioles were used for orientation. By means of this program, the sections could only be moved in x- and y-directions without rotation. T- and B-cell regions and vessels were delineated and imported into two new files. These were used for 3D reconstruction with 3D-Doctor (Version 2.0 for demonstration; Able Software, Lexington, KY). Areas of interest were delineated and visualized by "complex surface rendering" and "full rendering" to view surface structures of compartments. For transparent pictures demonstrating branching of the arterioles inside the respective compartment, "surface-rendering" and "transparent object-setting" were chosen.
Four informative levels were defined and represented in a different color for direct comparison with the immunostained sections. For improved visualization, 3D images of the computer-generated reconstructions were drawn by hand.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arrangement of Arterioles in Relation to B- and T-cell Regions of the White Pulp
Two and a half follicles, designated I to III, were reconstructed from serial sections stained for CD20 using the L26 antibody (Fig 1A and Fig 1B). For direct comparison with the immunohistological staining results, sections are highlighted in yellow or green color at four different levels (Fig 1 and Fig 2). Surface rendering includes the most peripheral immunoreactive lymphocytes of the white pulp. Thus, Ia in Fig 1A and Fig 1B indicates a T-cell region covered by a thin continuous layer of L26-positive B-cells, which continue into the oMZ of the adjacent follicles. The lower part of this T-cell region is still observed as a hollow area at level 2 in Fig 1D and Fig 1F. A similar phenomenon occurs in the vicinity of follicle III (Fig 1D, Fig 1F, Fig 2D, Fig 2F, Fig 3G, and Fig 3H). L26 reacts with all B-cells, and therefore, germinal center, mantle zone, and MZ are not distinguished after immunostaining (Fig 3C and Fig 3E). However, these internal regions do show up in sections stained for CD3 because of the nuclear counterstaining in the CD3- mantle zones and the CD3+ T-cells in the germinal centers (Fig 3D and Fig 3F).
|
|
|
Only arterioles in the white pulp, i.e., vessels with smooth muscle cells in their walls, were included for reconstruction. Two larger arterioles, named 1 and 2, and their branches are represented in blue color in Fig 1 and Fig 2. They form the innermost structures of the reconstructed white pulp area. The specimen was orientated such that these vessels enter from above and branch into finer arterioles towards the bottom of the reconstruction. Successive branches of the arterioles are designated in numerical order. The blood flow in the arterioles is from level 1 to level 4. To permit a better integration of 2D and 3D information and to prevent superpositioning of the vessels, the reconstructed part of the white pulp is also depicted after slight rotation. In the "normal" position, the lower margin of the immunostained areas in Fig 3 represents the anterior aspect of the white pulp in Fig 1A, Fig 1C, Fig 2A, and Fig 2C). From this position the specimen is rotated 15° to the right and 25° anteriorly, as indicated in Fig 1B, Fig 1D, Fig 2B, and Fig 2D.
Slight distortions of the single sections during the cutting process, the fact that only every second section was represented, and the inability of rotating the sections with the Openlab program led to an irregular outline of the vessels in Fig 1C, Fig 1D, Fig 2C, and Fig 2D. In addition, the four highlighted section levels were difficult to visualize after transparent rendering. To overcome these restrictions, an additional optimized version of the reconstruction was drawn by hand (Fig 1E, Fig 1F, Fig 2E, and Fig 2F).
The reconstruction revealed the following course of white pulp arterioles. The feeding arterioles 1 and 2 are connected by an anastomosis at level 1. Arteriole 1 gives off two branches within follicle I, designated 1.1 and 1.2 in Fig 1 Fig 2 Fig 3. Arteriole 2 splits into three branches, 2.1, 2.2, and 2.3, at the upper pole of follicle II. Arterioles 2.1 and 2.2 divide into three further branches at the border between white and red pulp, while arteriole 2.3 gives off branch 2.3.1 within follicle II and then continues in direction to follicle III. Therefore, there is no continuous single "central arteriole," but arterioles regularly branch into vessels of higher order, especially in white pulp follicles.
The location of the arterioles with respect to B- and T-cell regions is also evident from Fig 1 Fig 2 Fig 3. At levels 1 and 2 (Fig 1F, Fig 2F, and Fig 3A3D), the vessels are covered by mixtures of B- and T-cells. Between both levels the vessels have, however, passed a T-cell-rich region, which is still partially visible at level 2 (Fig 1F, Fig 3C, and Fig 3D). Mixtures of T- and B-cells also occur around arterioles of higher order, especially at the surface of follicles, e.g., around arterioles 2.1, 2.1.1 and 2.1.2 (Fig 3C and Fig 3D). The course of arteriole 2.3 is most informative. This vessel is still located within the T-cell region at its origin above level 2. It then passes into a small region of mixed cellularity and enters follicle II, where it is accompanied only by B-cells, as observed at level 3 (Fig 1F, Fig 2A2F, Fig 3E, and Fig 3F). At this level, arteriole 2.3 runs through the mantle zone of follicle II (Fig 3 F). It then leaves follicle II and again enters a T-cell region close to follicle III at level 4, where it occupies an eccentric position (Fig 2E, Fig 2F, Fig 3G, and Fig 3H). Because there is no other arteriole of this size associated with the white pulp at both levels 3 and 4, this finding indicates that "central arterioles" may run through a T-cell region, then leave this region, cross a follicle, and finally re-enter the next T-cell region. In other words, T-cell regions may be interrupted by follicles and then reappear again, so that a continuous PALS does not exist. In addition, most arterioles do not occupy a central position in the T-cell region or in the follicle.
Superficial Interdigitation of B- and T-cell Regions and Distribution of CCL21
The 3D reconstruction also demonstrated an intricate interdigitation of B- and T-cells at the surface of the respective regions. Previous investigations (-actin in the MZ and also in the entire T-cell region (
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The precise localization of T- and B-cell regions in human spleens can be revealed only by 3D imaging. Because the diameter of a follicle is on the order of about 1 mm, there is presently no alternative to reconstruction of immunostained serial sections. Immunostaining for light microscopy gives superior morphological information and permanent specimens. This method, however, requires that primary 2D non-digital information must be transformed into digital data for further processing. Therefore, certain limitations cannot be avoided, which are discussed below.
Our reconstruction of two and a half human splenic follicles associated with two larger T-cell regions reveals fundamental differences to splenic white pulp morphology described in rodents: Human T-cell regions do not represent continuous PALS but instead are elliptical elongated structures, which are interrupted by follicles. However, a thin extension of the outer T-cell region, composed of typical fibroblasts and CD3+CD4+ T-cells, surrounds the follicles within the MZ. The fact that these fibroblasts probably contain CCL21, which is a chemokine with a preferential effect on naive CD4+ T-lymphocytes (-actin and CCL21 needs to be shown by double staining. However, this is difficult because of the different methods of antigen retrieval necessary for the two primary reagents used. Up to now, we have not found phenotypical differences between CD4+ MZ T-cells and the CD4+ T-cells in the T-cell areas.
The shape of the T-cell accumulations in the follicular MZ represented in Fig 2 is somewhat astonishing. The T-cells do not appear to form a closed shell around follicle I, but instead appear as ring-like structures visible only around the equator of the follicle. This phenomenon is most likely caused by omission of every second section and by tangential and even horizontal sectioning through the T-cell shell in the upper and lower parts of the follicle. At such low angles, band-like accumulations of T-cells can no longer be distinguished because the cells are increasingly dispersed in the plane of cutting. Rings of T-cells are also present in the MZ of follicle II above level 3, but their number is somewhat reduced in comparison to follicle I. Therefore, in Fig 2 MZ T-cells are only visualized where they are found most strongly accumulated. In the upper part of follicle III, MZ T-cells were almost indistinguishable from the red pulp. If follicle III is larger than follicles I and II, this may be also due to primarily tangential sectioning in the upper part of this follicle.
The interdigitation of T- and B-cells at the surface of the follicles and, respectively, of the T-cell regions may provide a means of alleviating cellular interactions among both cell types early after immigration into the white pulp. From previous investigations (Steiniger et al. 2000,
Periarteriolar areas of mixed TB cellularity are also unknown in rodents. We are convinced that this phenomenon is due to the simultaneous presence of T- and B-cells and not to T-cell crossreactivity of the CD20 reagent used. It has been described that CD20 may be present in T-cell neoplasias (
The reconstruction also reveals fundamental differences among rodents and humans with respect to the localization and branching pattern of white pulp arterioles. In humans, the same vessel may run through T-cell regions and then through follicles. Most arterioles branch dichotomously at the follicular surface, or even within a follicle. The arterioles do not occupy a central position in the T-cell areas or follicles. Therefore, the present nomenclature for splenic arterioles is not appropriate to describe the microanatomy of the human splenic white pulp.
Our findings can be generalized, although the extent of the white pulp is clearly variable in humans. Up to now, we have thoroughly investigated 73 spleens for most of the immunologically relevant cell types (
The need to also perform drawings of the 3D computer-generated images indicates that the distortions caused by cutting and mounting of paraffin sections and the incongruencies introduced by omitting every second section could not be remedied by the versions of the computer programs applied for reconstruction. In addition, the fully transparent and 3D impression is perceived only if the image is freely rotated on the computer display. Therefore, we had to resort to hand-made drawings to integrate all information. To exclude misinterpretation of the data, we defined four levels in the reconstructed image, which can be directly compared to the original 2D immunostained sections shown in Fig 3.
Further research should be centered on elucidating the reasons for the peculiar arrangement of T- and B-cell regions in human spleens. In vitro studies of isolated fibroblasts, T-cells, and antigen-presenting cells may clarify how fibroblasts contribute to specific T-cell responses. Microdissection of immunostained B-cells from the surface of the T-cell region and from the follicular oMZ and iMZ may contribute to verifying these cells as recirculating B-cells and MZ B-memory cells, respectively.
![]() |
Acknowledgments |
---|
Immunostaining and 3D reconstruction were performed by L. Rüttinger as part of his M.D. thesis. We thank C. Fiebiger for precise observations and the talented artwork optimizing the 3D reconstructions. B. Herbst and A. Seiler provided excellent technical assistance. Thanks are due to Dr T. Hurek (Max-Planck Institute for Terrestrial Microbiology; Marburg, Germany) for support and discussions during data acquisition for 3D reconstruction and for the permission to use the Openlab program. Dr K. Troidl (Hochschulrechenzentrum of Marburg University) kindly provided assistance with scanning and processing of the photographs. Dr M. Bette and O. Stehling (Institute of Anatomy and Cell Biology, University of Marburg, Germany) also spent much time on adapting the digital images to a format suitable for publication.
Received for publication September 24, 2002; accepted December 18, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amlot PL, Hayes AE (1985) Impaired human antibody response to the thymus-independent antigen, DNP-Ficoll, after splenectomy. Lancet I: 10081011
Buckley PJ (1991) Phenotypic subpopulations of macrophages and dendritic cells in human spleen. Scan Microsc 5:147-158
Buckley PJ, Smith MR, Braverman MF, Dickson SA (1987) Human spleen contains phenotypic subsets of macrophages and dendritic cells that occupy discrete microanatomic locations. Am J Pathol 128:505-520[Abstract]
Dammers PM, Visser A, Popa ER, Nieuwenhuis P, Kroese FGM (2000) Most marginal zone B cells in rat express germline encoded Ig VH genes and are ligand selected. J Immunol 165:6156-6169
Dijkstra CD, Döpp EA, Joling P, Kraal G (1985a) The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies. Immunology 54:589-599[Medline]
Dijkstra CD, van Vliet E, Döpp EA, van der Lelij AA, Kraal G (1985b) Marginal zone macrophages identified by a monoclonal antibody: characterization of immuno- and enzyme-histochemical properties and functional capacities. Immunolgy 55:23-30[Medline]
Grogan TM, Jolley CS, Rangel CS (1983) Immunoarchitecture of the human spleen. Lymphology 16:72-82[Medline]
Grogan TM, Rangel CS, Richter LC, Wirt DP, Villar HV (1984) Further delineation of the immuno-architecture of the human spleen. Lymphology 17:61-68[Medline]
GulbransonJudge A, MacLennan ICM (1996) Sequential antigen-specific growth of T cells in the T zones and follicles in response to pigeon cytochrome c. Eur J Immunol 26:1830-1837[Medline]
Gunn MD, Tangemann K, Tam C, Cyster JG, Rosen SD, Williams LT (1998) A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc Natl Acad Sci USA 95:258-263
Hsu S-M (1985) Phenotypic expression of B lymphocytes. III. Marginal zone B cells in the spleen are characterized by the expression of Tac and alkaline phosphatase. J Immunol 135:123-130
Hsu S-M, Cossman J, Jaffe ES (1983) Lymphocyte subsets in normal human lymphoid tissues. Am J Clin Pathol 80:21-30[Medline]
Hultin LE, Hausner MA, Hultin PM, Giorgi JV (1993) CD20 (pan-B-cell) antigen is expressed at a low level on a subpopulation of human T lymphocytes. Cytometry 14:196-204[Medline]
Kraal G (1992) Cells in the marginal zone of the spleen. Int Rev Cytol 132:31-74[Medline]
Liu Y-J, Oldfield S, MacLennan ICM (1988) Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones. Eur J Immunol 18:355-362[Medline]
Liu Y-J, Zhang J, Lane PJL, Chan EY-T, MacLennan ICM (1991) Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur J Immunol 21:2951-2962[Medline]
Nieuwenhuis P, Ford WL (1976) Comparative migration of B- and T-lymphocytes in the rat spleen and lymph nodes. Cell Immunol 23:254-267[Medline]
QuintanillaMartinez L, Preffer F, Rubin D, Ferry JA, Harris NL (1994) CD20+ T-cell lymphoma. Neoplastic transformation of a normal T-cell subset. Am J Clin Pathol 102:483-489[Medline]
Snook T (1950) A comparative study of the vascular arrangements in mammalian spleens. Am J Anat 87:31-77
Snook T (1964) Studies on the perifollicular region of the rat's spleen. Anat Rec 1148:149-159
Steiniger B, Barth P (2000) Microanatomy and function of the spleen. Adv Anat Embryol 151:1-100
Steiniger B, Barth P, Herbst B, Hartnell A, Crocker PR (1997) The species-specific structure of microanatomical compartments in the human spleen: strongly sialoadhesin-positive macrophages occur in the perifollicular zone, but not in the marginal zone. Immunology 92:307-316[Medline]
Steiniger B, Barth P, Hellinger A (2001) The perifollicular and marginal zones of the human splenic white pulp: do fibroblasts guide lymphocyte immigration? Am J Pathol 159:501-512
Storie I, Wilson GA, Granger V, Barnett D, Reilly JT (1995) Circulating CD20dim T-lymphocytes increase with age: evidence for a memory cytotoxic phenotype. Clin Lab Haematol 17:323-328[Medline]
Tanaka H, Takasaki S, Sakata A, Muroya T, Suzuki T, Ishikawa E (1984) Lymphocyte subsets in the white pulp of human spleen in normal and diseased cases. Acta Pathol Jpn 34:251-270[Medline]
Timens W, Boes A, Poppema S (1989) Human marginal zone B cells are not an activated B cell subset: strong expression of CD21 as a putative mediator for rapid B cell activation. Eur J Immunol 19:2163-2166[Medline]
Timens W, Poppema S (1985) Lymphocyte compartments in human spleen: an immunohistologic study in normal spleens and noninvolved spleens in Hodgkin's disease. Am J Pathol 120:443-454[Abstract]
Van Krieken JHJM, te Velde J (1986) Immunohistology of the human spleen: an inventory of the localization of lymphocyte subpopulations. Histopathology 10:285-294[Medline]
Van Krieken JHJM, te Velde J (1988) Normal histology of the human spleen. Am J Surg Pathol 12:777-785[Medline]
Van Krieken JHJM, Te Velde J, Kleiverda K, LeenheersBinnendijk L, Van de Velde CJH (1985) The human spleen; a histological study in splenectomy specimens embedded in methylmethacrylate. Histopathology 9:571-585[Medline]
Van Rooijen N, Claassen E, Kraal G, Dijkstra CD (1989) Cytological basis of immune functions of the spleen. Immunocytochemical characterization of lymphoid and non-lymphoid cells involved in the in situ immune response. Prog Histochem Cytochem 19:1-69
Veerman AJP, van Ewijk W (1975) White pulp compartments in the spleen of rats and mice. A light and electron microscopic study of lymphoid and non-lymphoid cell types in T- and B-areas. Cell Tissue Res 156:417-441[Medline]