Secondary Lymphoid Follicles Research Articles

Cellular and sub-cellular localisation of PrP in the lymphoreticular system of mice and sheep.

Using immunocytochemistry or immunogold electron microscopy, abnormal PrP accumulation was found in lymphoreticular tissues of Suffolk sheep naturally exposed to scrapie and in the spleens of ME7 infected C57 BL mice at 70 days after infection and at the terminal stage of disease at 170 days. Clinically diseased scrapie affected sheep show widespread PrP accumulation within tingible body macrophages (TBMs) and follicular dendritic cells (FDCs) of secondary lymphoid follicles. Serial tonsillar biopsies taken from 171 ARQ/ARQ sheep at 4 months of age did not contain abnormal PrP accumulations but 80% of biopsies were positive by 14 months. In contrast, whole body necropsies of sheep not previously biopsied failed to detect PrP in the tonsil of sheep at 4, 8, 12 or 16 months of age. These findings suggest that the biopsy procedure of susceptible sheep but not resistant sheep may induce tonsillar infection. In spleen of mice both at 70 and 170 dpi, accumulations of PrP were found within lysosomes of TBMs and also at the plasma-lemma of FDCs. In the light zone of follicles of terminally diseased mice, all FDC dendrites were arranged in the form of highly reactive or hyperplastic labrynthine glomerular complexes. PrP was consistently seen between FDC dendrites in association with abundant electron dense antigen-antibody complexes. At 70 days after challenge, labrynthine complexes were rare and invariably labelled for PrP. However, sparse PrP labelling was also seen on simple FDC dendrites at this stage. These observations suggests that scrapie infected FDCs continually release PrP from the cell surface where it accumulates in excess in association with trapped immune complexes and dendritic extension. It is likely that TBMs acquire lysosomal PrP following phagocytosis of effete FDC processes or from the extracellular space. We suggest that the normal function of PrP may involve cell process extension or immune complex trapping.

BCA-1 is highly expressed in Helicobacter pylori-induced mucosa-associated lymphoid tissue and gastric lymphoma.

Infection with Helicobacter pylori (Hp) induces the formation of lymphoid tissue in the stomach and the occasional development of primary gastric B-cell lymphomas. We have studied the expression of 2 chemokines that attract B lymphocytes, BCA-1 and SLC, in gastric tissue samples obtained from patients with chronic gastritis induced by Hp infection or nonsteroidal anti-inflammatory drugs, as well as from patients with Hp-associated low-grade and high-grade gastric lymphomas. High-level expression of BCA-1 and its receptor, CXCR5, was observed in all mucosal lymphoid aggregates and in the mantle zone of all secondary lymphoid follicles in Hp-induced gastric mucosa-associated lymphoid tissue (MALT). Follicular dendritic cells and B lymphocytes are possible sources of BCA-1, which is not expressed by T lymphocytes, macrophages, or CD1a(+) dendritic cells. Strong expression of BCA-1 and CXCR5 was also detected in the transformed B cells of gastric MALT lymphomas. By contrast, SLC was confined almost exclusively to endothelial cells in and outside the lymphoid tissue. Only scant, occasional SLC expression was observed in the marginal zone of MALT follicles. Our findings indicate that BCA-1, which functions as a homing chemokine in normal lymphoid tissue, is induced in chronic Hp gastritis and is involved in the formation of lymphoid follicles and gastric lymphomas of the MALT type.

Unraveling the Mystery of the Lymphoid Follicle

Unraveling the Mystery of the Lymphoid Follicle

Differential Expression of Ca 2+-Binding Proteins on Follicular Dendritic Cells in Non-Neoplastic and Neoplastic Lymphoid Follicles

We studied the Ca(2+)-capture ability of follicular dendritic cells (FDCs) in tonsillar secondary lymphoid follicles (LFs) and the expression of six Ca(2+)-binding proteins (CBPs), caldesmon, S-100 protein, calcineurin, calbindin-D, calmodulin, and annexin VI in LFs of various lymphoid tissues and caldesmon and S-100 protein in neoplastic follicles of follicular lymphomas. First, Ca(2+)-capture cytochemistry revealed extensive Ca(2+) capture in the nuclei and cytoplasm of FDCs, but little or none in follicular lymphocytes. All six CBPs were localized immunohistochemically in the LFs and were always present in the basal light zone. Immunoelectron microscopic staining of FDCs was classified into two patterns: caldesmon was distributed in the peripheral cytoplasm like a belt; S-100 protein, calcineurin, calbindin-D, and calmodulin were distributed diffusely in the cytosol. Annexin VI was, however, negative on FDCs. Immunocytochemistry also demonstrated CBP-positive FDCs within FDC-associated clusters isolated from germinal centers. In situ hybridization revealed diffuse calmodulin mRNA expression throughout the secondary LFs. These data indicate that the CBPs examined may regulate Ca(2+) in the different subcellular sites of FDCs, and the roles of CBPs may be heterogeneous. We also investigated the distribution of caldesmon and S-100 protein in follicular lymphomas on paraffin-embedded tissue sections. FDCs within grades I and II neoplastic follicles clearly expressed caldesmon, but not S-100 protein, except a part of grade II neoplastic follicles. FDCs within grade III follicles showed no caldesmon, but frequently expressed S-100 protein. These results demonstrate that the caldesmon and S-100 protein staining patterns of grade I follicular lymphomas are different from those of grade III follicular lymphomas and suggest that FDC networks in grade I neoplastic follicles may be similar to those in the light zone within non-neoplastic follicles, FDC networks in grade III neoplastic follicles may be similar to those in dark and basal light zones within non-neoplastic follicles, and grade II follicles may be intermediate between grade I and grade III follicles.

Bcl-6 and Bcl-2 protein expression in diffuse large B-cell lymphoma and follicular lymphoma: Correlation with 3q27 and 18q21 chromosomal abnormalities

The bcl-2 gene on chromosome 18 at q21 and the bcl-6 gene on chromosome 3 at q27 are both highly regulated during B-cell differentiation and show an inverse relationship of expression in the normal secondary lymphoid follicle. The objective of this study was to investigate the relationship between bcl-2 and bcl-6 protein expression and the relationship between protein expression and the corresponding chromosomal alterations in malignant lymphomas, including those associated with the germinal center. Expression of bcl-2 and bcl-6 proteins was studied in 55 cases of diffuse large B-cell lymphoma (DLBCL) and 21 cases of follicular lymphoma (FL), and the results correlated with the presence of t(14;18) and 3q27 abnormalities in a subset of 52 cases with cytogenetic analysis. These cases were selected to represent a spectrum of nodal and extranodal lymphomas, including those with and without a t(14;18). It was shown that the neoplastic cells in 71% of DLBCLs and 100% of FLs expressed bcl-6 protein. Expression of bcl-6 was seen more frequently in diffuse large B-cell lymphomas with large noncleaved morphology compared with immunoblastic morphology (82% v 27%, P = .0015), but failed to correlate with 3q27 abnormalities. Thirty-eight percent of cases with 3q27 abnormalities were bcl-6 protein negative, whereas 85% of cases without a 3q27 abnormalities were bcl-6 protein positive. Expression of bcl-2 protein was shown in 51% DLBCLs (nodal v extranodal, 71% v 30%, P = .012). bcl-2 protein was expressed in 89% of FLs with t(14;18), in contrast to 25% of FLs without t(14;18) ( P = .016). In DLBCL and FL with t(14;18), the most common pattern of expression was bcl-2 + bcl-6 + . In lymphomas without t(14;18), there was not an inverse relationship between bcl-2 and bcl-6 protein expression. In conclusion, these data suggest that mechanisms other than gene rearrangements can deregulate bcl-2 and bcl-6 expression in lymphomas, and there does not appear to be an inverse relationship between these two proteins as seen in the normal germinal center.

Development of follicular dendritic cells: a study using short-term bone marrow cell grafting in SCID mice.

To evaluate the cellular origin of follicular dendritic cells (FDC) in lymphoid follicles (LFs), severe combined immunodeficient (SCID) mice (H-2d) were grafted with 5-bromo-2'-deoxyuridine (BrdU)-incorporated bone marrow cells from CB-17 mice (H-2d) and with non-BrdU-incorporated bone marrow cells from C3H mice (H-2k) and Wistar rats (RT1u). This procedure was followed by antigenic stimulation with horseradish peroxidase and related immune complex (mouse peroxidase anti-peroxidase) administration. Secondary LFs in the lymph nodes and spleen of the reconstructed SCID mice were examined morphologically and immunocytochemically. LFs reconstructed with CB-17 mouse bone marrow cells contained FDCs capable of trapping and/or retaining mouse peroxidase anti-peroxidase as immune complexes. Secondary LFs contained BrdU-incorporated germinal center lymphocytes but not non-lymphoid stromal cells. A cell grafting study in SCID mice using bone marrow cells from C3H mice and Wistar rats demonstrated that FDCs in reconstructed LFs exhibited a marker specific for the recipient but not for the donor. These data indicate that functionally active FDCs occur de novo in reconstructed LFs in SCID mice, and do not support the view that FDCs originate from bone marrow cells in short-term reconstructed LFs.

The Role of CD8+ CD40L+ T Cells in the Formation of Germinal Centers in Rheumatoid Synovitis

In rheumatoid synovitis, lymphocytes can be arranged in follicular structures resembling secondary lymphoid follicles. To understand the organizing principles of this ectopic lymphoid tissue, the cellular components contributing to synovial follicles were examined. In 9 of 24 synovial tissue biopsies, lymphoid aggregates were found consisting of CD4+ T cells and CD20+ B cells. In four of the nine patients, the follicular centers were occupied by CD23+ CD21+ cellular networks representing follicular dendritic cells involved in germinal center reactions. In five patients, CD23+ cells were absent from the centers of the aggregates, suggesting that fully developed germinal centers are generated in only a subset of patients. To identify factors involved in the regulation of the synovial microarchitecture, cell populations contributing to the follicles were quantified by digital image analysis of immunostained tissue and by flow cytometry of tissue-derived lymphocytes. Proportions of CD4+, CD20+, and CD68+ cell subsets were surprisingly invariant, irrespective of the presence or absence of CD23+ follicular dendritic cells. Instead, tissue biopsies with CD23+ germinal center-like regions could be distinguished from those with CD23- T cell-B cell aggregates by a fourfold increase in the frequency of tissue-infiltrating CD8+ T cells, a fraction of which expressed CD40 ligand (CD40L). The data suggest a previously unsuspected role of CD8+ lymphocytes in modulating germinal center formation and raise the possibility that CD8+ CD40L+ T cells are involved in aggravating pathologic immune responses in rheumatoid synovitis.

The Leukocyte Semaphorin CD100 Is Expressed in Most T-Cell, but Few B-Cell, Non-Hodgkin's Lymphomas

The 150-kd transmembrane protein CD100 is the first semaphorin protein shown to be expressed in lymphoid tissue. CD100 is present in the interfollicular T cell zones and is also expressed by B cells in the germinal centers of secondary lymphoid follicles, but not in the mantle zones. The CD100 molecule was recently cloned, and CD100 transfectants were shown to induce homotypic aggregation of human B cells and improve their viability in vitro, suggesting that CD100 may play a role in lymphocyte aggregation and germinal center formation. We studied the expression of CD100 in 138 clinical cases representing a range of lymphoproliferative disorders, to determine whether this molecule is expressed in these neoplastic processes. In general, we found CD100 expression to be common in peripheral T-cell non-Hodgkin's lymphomas but rare in B-cell non-Hodgkin's lymphomas. CD100 expression was not detectable in low-grade B-cell non-Hodgkin's lymphomas, including cases of small lymphocytic lymphoma (18 cases), marginal zone lymphoma (10 cases), and mantle cell lymphoma (10 cases), as might be expected for these neoplasms that are not of follicular center cell origin. Surprisingly, we found that the vast majority of follicular lymphomas (37 of 40 cases) as well as diffuse large-cell lymphomas of B-cell type (35 cases) did not express CD100. The neoplastic cells in 3 of 11 cases of predominantly large-cell-type follicular lymphoma did express CD100. In contrast, all five cases of high-grade, small non-cleaved (Burkitt-like) B-cell lymphoma were immunoreactive for CD100 expression, as were 18 of 20 cases (90%) of malignant T cell neoplasms. Northern blot analysis of CD100 expression correlated with immunohistochemical findings. Absence of expression of CD100 by neoplastic follicular center B cells is a common feature in follicular lymphomas, but expression of CD100 by T cells is maintained in T-cell lymphoproliferative disorders.

Follicular Lymphomas Contain a Clonally Linked But Phenotypically Distinct Neoplastic B-Cell Population in the Interfollicular Zone

AbstractFollicular lymphomas are thought to arise from the follicle center B cells and are characterized by follicular structures that recapitulate many features of normal secondary lymphoid follicles. The neoplastic B cells of follicular lymphoma reside not only in follicles but also in the interfollicular zone in which they form a diffuse infiltrate. We have investigated the frequency, extent, and biological characteristics of this interfollicular component in 30 cases of follicular lymphoma. An interfollicular B-cell infiltrate of variable extent (minimal, moderate, or prominent) was present in all cases. Morphologically interfollicular neoplastic B cells were small centrocyte-like cells with lower grade cytology and lower proliferation fraction compared with the neoplastic follicles. The neoplastic phenotype of these cells (CD20+, light chain restricted) was confirmed in 18 cases. Clonal identity between the follicular and interfollicular components was shown in five cases using microdissection and PCR amplification of immunoglobulin heavy chain genes. Analysis of Ig heavy chain gene sequences showed identical variants of tumor subclones in both follicular and interfollicular compartments, indicating active tumor cell traffic between the two. In six cases in which frozen tissue was available, the immunophenotype of follicular and interfollicular tumor cells were compared using immunohistochemistry. Activation markers such as CD10, CD38, and CD95 and T-cell costimulatory molecules CD80 and CD86, which were expressed by neoplastic follicles, were either downregulated or absent in the interfollicular component in most of the cases. The low-grade cytological features, low proliferation fraction, and downregulation of activation markers in the interfollicular neoplastic B cells suggests that these are resting cells analogous to memory B cells of normal lymphoid tissues. The presence of such a resting tumor cell subpopulation in the majority of follicular lymphomas may partly account for the remarkable resistance to therapy of this disease.

Follicular Lymphomas Contain a Clonally Linked But Phenotypically Distinct Neoplastic B-Cell Population in the Interfollicular Zone

Follicular lymphomas are thought to arise from the follicle center B cells and are characterized by follicular structures that recapitulate many features of normal secondary lymphoid follicles. The neoplastic B cells of follicular lymphoma reside not only in follicles but also in the interfollicular zone in which they form a diffuse infiltrate. We have investigated the frequency, extent, and biological characteristics of this interfollicular component in 30 cases of follicular lymphoma. An interfollicular B-cell infiltrate of variable extent (minimal, moderate, or prominent) was present in all cases. Morphologically interfollicular neoplastic B cells were small centrocyte-like cells with lower grade cytology and lower proliferation fraction compared with the neoplastic follicles. The neoplastic phenotype of these cells (CD20+, light chain restricted) was confirmed in 18 cases. Clonal identity between the follicular and interfollicular components was shown in five cases using microdissection and PCR amplification of immunoglobulin heavy chain genes. Analysis of Ig heavy chain gene sequences showed identical variants of tumor subclones in both follicular and interfollicular compartments, indicating active tumor cell traffic between the two. In six cases in which frozen tissue was available, the immunophenotype of follicular and interfollicular tumor cells were compared using immunohistochemistry. Activation markers such as CD10, CD38, and CD95 and T-cell costimulatory molecules CD80 and CD86, which were expressed by neoplastic follicles, were either downregulated or absent in the interfollicular component in most of the cases. The low-grade cytological features, low proliferation fraction, and downregulation of activation markers in the interfollicular neoplastic B cells suggests that these are resting cells analogous to memory B cells of normal lymphoid tissues. The presence of such a resting tumor cell subpopulation in the majority of follicular lymphomas may partly account for the remarkable resistance to therapy of this disease.

A syndrome of anaemia, immunodeficiency and peripheral ganglionopathy in Fell pony foals

Fell pony foals developed a syndrome of anaemia, immunodeficiency and peripheral ganglionopathy. They became ill in the second or third week, and died in the second or third month of...

Immunohistochemical analysis of in vivo patterns of TRAF-3 expression, a member of the TNF receptor-associated factor family.

An immunohistochemical approach was used to explore the in vivo expression of TNF receptor-associated factor 3 (TRAF-3), a putative signaling protein that binds to the cytosolic domains of CD30, CD40, and lymphotoxin-beta receptors. TRAF-3 immunostaining was detected in many types of cells throughout the human body. TRAF-3 immunostaining was only rarely present in thymocytes but was found in the thymic epithelioreticular cells. Lymphocytes in the bone marrow were also typically TRAF-3 immunonegative, whereas myeloid progenitor cells and megakaryocytes were often TRAF-3 positive. Peripheral blood lymphocytes were mostly TRAF-3 immunonegative, while granulocytes were TRAF-3 immunopositive. Monocytes were strongly immunostained for TRAF-3, but macrophages in nodes typically contained little or no TRAF-3 immunoreactivity. Some lymphocytes within the germinal centers of secondary lymphoid follicles in normal and reactive nodes were TRAF-3 immunopositive, as were occasional interfollicular lymphocytes in the T cell regions of these organs, but most lymphocytes appeared to be TRAF-3 immunonegative or stained only weakly. Plasma cells, however, were strongly TRAF-3 positive. Stimulation of PBLs with anti-CD3 Ab induced marked increases in the steady state levels of TRAF-3 protein in vitro as determined by immunoblotting, while levels of TRAF-2 were unchanged, implying a dynamic regulation of TRAF-3 expression. The findings establish for the first time the cell type- and differentiation-specific patterns of expression of a member of the TRAF family of proteins.

Gap-junction communication pathways in germinal center reactions.

Intercellular channels called gap junctions enable multicellular organisms to exchange information rapidly between cells. Though gap junctions are held to be ubiquitous in solid tissues, we have only recently found them in the lymphoid organs. Functional direct cell-cell communication has now been confirmed by us and other groups in bone marrow, thymus, and in secondary lymphoid tissues. What functions do they serve in the lymphoreticular system where, so far, only cytokines/growth factors and adhesion molecules have been considered as regulators? Here we show evidence for and refer to published work about functional direct cellcell communication through gap junctions in germinal center reactions and make proposals for their role in the immune response. We found a large amount of the connexin43 (Cx43) gap junctions in the germinal centers of secondary lymphoid follicles. Ultrastructurally and immunohistologically, most of the junctions were detected on the processes of follicular dendritic cells (FDC) enveloping nondividing centrocytes in the light zone of germinal centers where B-cell selection is thought to take place. Further support for this finding came by revealing the Cx43 mRNA in situ at the same location as the protein. On antigen challenge, gap junctions appeared on the FDC as they formed meshworks in germinal centers. In order to find out which germinal center cells communicate directly, we separated FDC-rich, low-density, B-cell fractions from human tonsil. In culture, we injected single FDC with the low-molecular-weight fluorescent dye, Lucifer Yellow (Mr 457 Da), which passed between adjacent FDC and sometimes from FDC to B cells. Based on these findings and their assigned functions in other tissues, gap junctions may contribute to germinal center reactions in the following ways: (1) they may regulate follicle pattern formation by controlling FDC growth, (2) they may be involved in FDC-B-cell signaling contributing to the final rescue of selected B cells from apoptosis, and (3) they may enable FDC to work as a functional syncytium providing a cellular internet for integrating germinal center events. Data supporting these interpretations are briefly discussed.

Proliferation and apoptosis of follicular lymphocytes: relationship to follicular dendritic cell-associated clusters.

Experiments were designed to determine the in vivo cell-cycle phase of lymphocytes in secondary lymphoid follicles, and whether B-cell proliferation and apoptosis occur within follicular dendritic cell (FDC)-associated clusters. Using frozen serial sections of human tonsils, lymphoid follicles were stained to reveal histone H3 mRNA, as an S-phase marker, using in situ hybridization, and stained immunohistochemically with antibodies against cyclin E as a late G1 phase marker, cyclin B1 and p34cdc2 as S-G2-M phase markers, and Ki-67 as a marker of cycling cells. Each LF was divided into five zones: mantle zone, outer zone, apical light zone, basal light zone and dark zone, with the help of haematoxylin and eosin staining, and a CD23 immunostain. The rate of occurrence of positively labelled cells was calculated by dividing the number of positive cells by the number of all cells in each zone. The cells that were positive for cyclin E, histone H3 mRNA, cyclin B1, p34cdc2, and Ki-67 were found most frequently in the dark zone (54.5 +/- 6.6%, 22.0 +/- 5.7%, 36.7 +/- 14.5%, 40.0 +/- 10.2%, and 59.0 +/- 13.4%, respectively), followed by the outer zone (52.7 +/- 7.8%, 14.9 +/- 4.1%, 22.9 +/- 9.7%, 24.9 +/- 7.9%, and 44.6 +/- 12.3%, respectively), showing that both the outer zone and the dark zone contain many proliferating lymphocytes. Furthermore, FDC-associated clusters and free lymphocytes were obtained from enucleated germinal centres, using enzymatic digestion. The rates of occurrence of cells that were positive for cyclin B1 and Ki-67 within the clusters (7.2 +/- 1.9% and 37.9 +/- 10.5% respectively) were significantly lower than those of free lymphocytes outside the clusters (22.2 +/- 4.0% and 62.8 +/- 14.0%, respectively). The rates of occurrence of apoptotic bodies and cells within the clusters, as detected by in situ tailing or in situ nick translation (0.2 +/- 0.4% and 0.4 +/- 0.4%, respectively) were significantly lower than those outside the clusters (1.1 +/- 0.3, 1.6 +/- 0.5%, respectively). These results suggest that FDC-associated clusters are not the site of proliferation, and that they rarely contain apoptotic bodies and cells of B lymphocytes.

Mantle cell lymphomas.

MCLs are thought to arise from a subset of B cells that normally express the CD5 antigen and that reside in the mantle zone of secondary lymphoid follicles. Although expression of the CD5 antigen is also seen in small lymphocytic lymphoma and chronic lymphocytic leukemia, MCL differs from SLL/ CLL in several ways. Whereas trisomy of chromosome 12 is the hallmark cytogenetic abnormality of SLL/CLL, the translocation (11;14) (q13q32) is the most frequent karyotypic abnormality in MCL. The histologic pattern of MCL is most frequently diffuse. However, this lymphoma can grow in a unique pattern called 'mantle zone MCL,' indicating that the malignant cells expand the mantle of the follicle and grow around a normal germinal center. If the germinal center is also replaced by the malignant cells, but the follicular architecture remains, the pattern appears nodular. The clinical presentation of MCL is usually only seen with advanced disease stage, particularly in patients with diffuse MCL. The bone marrow is the most frequently affected extranodal site, followed by the gastrointestinal tract. The histologic pattern of disease in the lymph nodes correlates with clinical outcome. Patients with diffuse MCL have poor response to frontline combination chemotherapy including doxorubicin, whereas patients with mantle zone MCL have excellent complete remission rates. The therapeutic response correlates in turn with worse survival outcome for patients with diffuse MCL compared to mantle zone MCL. The few patients with nodular MCL had clinical behavior similar to diffuse MCL. The chemotherapeutic response of diffuse and nodular MCL, however, is quite poor, and we would propose that new investigational approaches be considered in the front-line therapy of these disorders.

Morphology, function and pathology of follicular dendritic cells.

The precise ultrastructural morphology and functions in reactive conditions of lymphoid follicles (LF) and dendritic cells, including follicular dendritic cells (FDC) are reviewed; as well as the pathognomonic role of FDC in some disease conditions and finally, the cellular origin of FDC. In reactive conditions, FDC in each of the five follicular zones have distinct ultrastructural features, reflecting the different three-dimensional structures and functions of these zones. The FDC framework may be supported by some characteristic factors, including desmosome-like junctions between FDC and the expression of fibronectin and laminin receptors and caldesmon on FDC. FDC, especially in the light zone, express various cytokine receptors, but produce only one cytokine, TGF-beta. The outer zone may not only be a cellular pathway in the LF, but may also provide a site for germinal center B cell proliferation, and the FDC-lymphocyte cluster is not the site of germinal center B cell division. In patients with auto-immune diseases, such as Hashimoto's thyroiditis and rheumatoid arthritis, FDC may be in a hyperfunctional state, whereas those in patients with immunosuppressive disorders, such as Kimura's disease and AIDS, may be in a dysfunctional state. FDC may be derived from fibroblastic reticulum cells in lymphatic tissues rather than in bone marrow cells. The data discussed in this review provide fascinating insight into the roles of FDC, which are intimately related to the migration, proliferation, cell selection and differentiation of B cells in secondary LF.

Follicular Dendritic Cells Adhere to Fibronectin and Laminin Fibers Via Their Respective Receptors

The expression of adhesion molecules on human tonsillar follicular dendritic cells (FDCs) in the secondary lymphoid follicle (LF) in vivo and in vitro was investigated using cryostat sections and cytospin preparations of FDCs isolated with a magnetic cell sorter, respectively. FDCs were immunochemically positive for Mac-1 (CD11b), sialyl-Lex (CD15s), CD22, integrin beta 1 (CD29), CD40, very late activation antigen (VLA)-alpha 3 (CD49c), VLA-alpha 5 (CD49e), VLA-alpha 6 (CD49f), intercellular adhesion molecule (ICAM)-3 (CD50), ICAM-1 (CD54), B7 (CD80), and vascular cell adhesion molecule (VCAM)-1 (CD106). With respect to ligands on B cells for these adhesion molecules, the CD11b-CD54, CD50-leukocyte function-associated molecule (LFA)-1 (CD11a/18), and CD106-VLA-4 (CD49d/29) interactions in the apical light (ALZ) and basal light (BLZ) zones; the CD15s-L-selectin (CD62L) and CD106-CD49d/29 interactions in the mantle zone; and the CD54-CD11a/18 interaction in the entire LF may participate in FDC-B cell adhesion. Namely, the adhesion molecules participating in FDC-B cell interactions may differ in each of the five zones. Furthermore, the immunochemical evidence that FDCs were fibronectin (VLA-5, CD49e/29) and laminin (VLA-6, CD49l/29) receptor-positive discussed above was confirmed by immunoelectron microscopy and binding assays. Immunoelectron microscopy revealed fibers surrounded by cytoplasmic FDC extensions that were CD29-, CD49e-, and CD49f-positive. In the binding assays, the numbers of FDCs bound to fibronectin- and laminin-coated dishes and LFs of cryostat sections of human tonsils were reduced markedly by pretreatment with monoclonal antibodies against CD29, CD49e, and CD49f. These data indicate clearly that FDCs bind to reticulin and laminin fibers in LFs via their respective receptors.

Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie.

The scrapie-associated form of the prion protein (PrPSc) accumulates in the brain and lymphoid tissues of sheep with scrapie. In order to assess whether detecting PrPSc in lymphoid tissue could be used as a diagnostic test for scrapie, we studied the localization and distribution of PrPSc in various lymphoid tissues collected at necropsy from 55 sheep with clinical scrapie. Samples collected from the spleen, palatine tonsil, ileum, and five different lymph nodes were immunohistochemically stained for PrPSc. PrPSc was found to be deposited in a reticular pattern in the center of both primary and secondary lymphoid follicles. In addition, granules of PrPSc were seen in the cytoplasm in macrophages associated with the lymphoid follicles. In 54 (98%) of the 55 scrapie-affected sheep, PrPSc was detected in the spleen, retropharyngeal lymph node, mesenteric lymph node, and the palatine tonsil. However, only in the palatine tonsils was PrPSc present in a consistently high percentage of the lymphoid follicles. PrP was not detected in any of the lymphoid tissues of 12 sheep that had no neurohistopathological signs of a scrapie infection. We conclude that the tonsils are the best-suited lymphoid tissue to be biopsied for the detection of PrPSc in the diagnosis of clinical scrapie in living sheep.

へん桃２次リンパろ胞におけるＢリンパ球増殖の場とろ胞樹状細胞の役割 細胞周期マーカーによる解析

The proliferation site of B lymphocytes in secondary lymphoid follicle of the tonsil was evaluated. Five follicles were selected to detect histon H3 mRNA as marker of cells in S-phase of the cell cycle by in situ hybridization. Moreover, 11 follicles were selected and stained immunohistochemically with serial frozen sections with cyclin B1 as a G2-M phase marker, p34cdc2 as a marker mainly of G2-M phase, cyclin E as a marker mainly of late G1 phase, and Ki-67 as a marker of cells in the cell cycle. Secondary lymphoid follicles were divided into five zones by the HE stain and the expression of CD23 (Fc ε receptor II) to calculate the positive rate of histon H3 mRNA and these proteins. The positive rates of histon H3 mRNA, cyclin B1, and p34cdc2 were the highest in the dark and the outer zones. The result implies that the outer zone as well as the dark zone is a proliferatation site of B cells.Nextly, the phase of the cell cycle of B cells in the follicular dendritic cell (FDC)-lymphocytes clusters, which were obtained from the germinal center of the secondary lymphoid follicle of human tonsils, is evaluated. The rate of cyclin B1-positive lymphocytes in the cluster (4.6%) is lower than that of the lymphocytes out of the cluster in germinal center (27.7%) and, moreover, the rate of Ki-67-positive cells in the cluster (35.9%) is lower than that of cells out of clusters (61.5%). The result shows that few G2-M phase lymphocytes are retained in the FDC-lymphocytes clusters and support that the B lymphocytes actively do not proliferate in the clusters.

Follicular Dendritic Cells and Germinal Centers

Follicular dendritic cells (FDCs) are stromal cells unique to primary and secondary lymphoid follicles. Recirculating resting B cells migrate through the FDC networks, whereas antigen-activated B cells undergo clonal expansion within the FDC networks in a T cell-dependent fashion, thereby generating germinal centers. Here, B cells undergo somatic mutation, positive and negative selection, isotype switching and differentiation into high-affinity plasma cells and memory B cells. Since the discovery of FDCs by electron microscopy as long-term antigen-retaining cells 30 years ago isolation of FDCs and generation of FDC-like cells lines and of FDC-specific monoclonal antibodies have been achieved. FDCs express all three types of complement receptors as well as Ig-Fc receptors, through which antigen-antibody immune complexes are retained. However, the mechanism that prevents FDCs from internalizing the antigens and retaining them in native form for long periods of time remains obscure. Substantial evidence derived from cultures in vitro indicates that FDCs contribute directly to the survival and activation of peripheral B cells. The adhesion between FDCs and B cells is mediated by ICAM-1 (CD54)-LFA-1(CD11a) and VCAM-VLA-4. T cells may interact with FDCs in a CD40/CD40-ligand-dependent fashion. Whether FDCs originate from hematopoietic progenitors or from stromal elements is still a controversy. New evidence suggests the presence of two types of dendritic cells within human germinal centers: (i) the classic FDCs that express DRC-1, KiM4, and 7D6 antigens represent stromal cells; and (ii) the newly identified CD3-CD4-CD11c- germinal center dendritic cells (GCDC) represent hematopoietic cells that may be analogous to the antigen-transporting cells described in mice. Finally, FDCs appear to be involved in the growth of follicular lymphomas and in the pathogenesis of HIV infection.