Histology and immunohistochemistry of the gut-associated lymphoid tissue of the eastern grey kangaroo, Macropus giganteus

Mesenteric lymph nodes and gut-associated lymphoid tissue (GALT) from juvenile eastern grey kangaroos were investigated. The mesenteric nodes had a similar structure to that described for eutherian mammals. They contained distinct regions of medulla and cortex, with prominent follicles and germinal centres. Gut associated lymphoid tissue consisted of areas of submucosal follicles. These varied from areas of densely packed lymphocytes with darkly staining, prominent coronas to areas with no defined follicles. The distribution of T cells in these tissues was documented by use of species-crossreactive antibodies to the surface markers CD3 and CD5; B cells were identified by antibodies to CD79b. Within the lymph nodes T cells were located mainly in the paracortex and cortex, with limited numbers observed in the follicles; B cells were located on the marginal zone of the follicles. In GALT, T cells were located in the peripheral regions of the germinal centres of secondary follicles, while B cells were abundant in primary follicles. These observations are consistent with those made in a range of other marsupials (metatherian) and eutherian mammals and are indicative of the capacity to respond to antigens entering via the mouth.     

Localization of a protein antigen in the chicken spleen. Effect of various manipulative procedures on the morphogenesis of the germinal centre.

The time course of the localization of a protein antigen human serum albumin (HSA) into the chicken spleen after intravenous injection is analysed. Localization within seconds to the region surrounding the Schweigger-Seidel sheaths is accomplished by HSA complexes with chicken anti-HSA or by heat aggregated HSA. The localization of soluble HSA has to await the synthesis of sufficient chicken anti-HSA to accomplish localization to the same white pulp sites in the spleen at 25-30 hours after injection. By the use of complexes of HSA-anti-HSA in ten times antigen excess, the time for localization of HSA withing germinal centres was accelerated as compared with soluble HSA, so that newly formed centres containing antigen-bearing dendritic ells were seen at 48 hours instead of 72 hours after use of soluble HSA. Neonatally bursectomized and irradiated (Bx+Irr.) birds fail to localize HSA into germinal centres or to dendritic cells within the white pulp. Heat-aggregated human gamma-globulin (HGG) injected intravenously into Bx+Irr. birds rapidly localizes within seconds to the periphery of Schweigger-Seidel sheaths and at 24 hours can be seen attached to the surface of typical dendritic cells throughout the white pulp. Hence, heat-aggregated HGG can localize to dendritic cells in the absence of specific antibody. However, such localization to dendritic cells in Bx+ Irr. birds is not followed by segregation of the aggregated HGG-bearing dendritic cells within germinal centres--a further stage in the process which is presumed to require B cells and/or specific antibody. Localization of heat-aggregated HGG to white pulp dendritic ells was prevented by treatment with pepsin sufficient to destroy the ability of aggregated HGG to activate guinea-pig complement. Similary, in vivo decomplementation with a purified anticomplementary fraction (CoF) from the venom of Naja naja resulted in failure of intravenously injected HSA to localize to white pulp dendritic cells and failure of subsequent germinal centre formation. However, such decomplementation did not prevent the localization of aggregated HGG to white pulp dendritic cells. These facts are discussed in the light of hypotheses concerning germinal centre formation and the homeostasis of the antibody response in the bird.     

Phenotypically distinct subpopulations of T cells in domes and M-cell pockets of rabbit gut-associated lymphoid tissues.

Follicle epithelium and domes of gut-associated lymphoid tissue (GALT) contain populations of lymphocytes which first contact antigen taken up from the intestine. In order to study the association of lymphocytes with M cells in follicle epithelium, monoclonal antibodies (mAb) were generated by immunizing BALB/c mice with lymphocytes populating GALT domes from NZW rabbits, and their specificity was assessed by immunohistochemistry and flow cytometry. mAb 3C10 (IgM) and 3B6 (IgG3) recognized subpopulations of intraepithelial lymphocytes associated with M cells. mAb 3C10 also identified macrophage-lymphocyte clusters in domes and tangible body macrophages in germinal centres of GALT but did not react with cells in T-dependent areas (TDA) or B cells in follicles. mAb 3B6 recognized lymphocytes in domes and B cells in follicles but not T cells in TDA of GALT. The distribution of 3B6+ cells overlapped with, but was more restricted than, that of Ia+ cells. Analysis of lymphocytes in follicle epithelium showed that greater than 95% of lymphocytes associated with M cells were Ia+. T cells represented approximately 95% of intraepithelial lymphocytes in the appendix and approximately 65% in Peyer's patches. A majority of intraepithelial lymphocytes was recognized by mAb 3B6, but mAb 3C10 identified only approximately 30%. Because neither 3C10 nor 3B6 recognized lymphocytes in TDA of GALT, these results indicate that most lymphocytes associated with M cells are a distinct phenotype of Ia+ T cells.     

免疫组化技术分析人淋巴组织的多种分化抗原

用间接免疫过氧化物酶和PAP技术检测本室制备的31种抗人分化抗原单抗与淋巴组织的反应性.结果表明,CD3、CD5单抗与扁桃腺和淋巴结的T细胞、大部分成熟髓质胸腺细胞和脾白髓的中央动脉周围淋巴鞘呈非常强的反应.CD4~+细胞在扁桃腺的分布与CD3~+细胞类似,但数量稍少.只有少部扁桃腺和淋巴结T细胞与CD8单抗反应,CD8单抗主要染大部分胸腺皮质细胞,但抗CD8单抗与脾窦的内皮细胞呈强阳性交叉反应.Wu59单抗同时与扁桃腺、淋巴结和脾脏的T、B细胞呈非常强的膜染色,并与胸腺皮质和髓质细胞呈阳性反应,该单抗可能识别白细胞共同抗原或LFA-1.Wu 26.145单抗除与扁桃腺生发中心呈弱阳性反应外,还与脾红髓窦状结构内的血小板呈强阳性反应.此外,抗B细胞及其亚群单抗与扁桃腺、淋巴结、脾白髓生发中心呈强阳性反应.抗IL-2受体单抗与上述组织基本上呈阴性反应。     

The ontogeny of germinal centre forming capacity of neonatal rat spleen.

In non-specifically immunized rats, bred under conventional conditions, the first 'spontaneous' germinal centres were observed by 21 days after birth. Deliberate antigenic stimulation led to an earlier appearance of germinal centres in neonatal spleen: immunization with sheep red blood cells as early as 7 days after birth resulted in germinal centre formation in the spleen as observed 7 days later. By that time the first primary follicles could also be observed, in both immunized and non-immunized rats. Although 3-day-old rats upon antigenic stimulation failed to generate germinal centres in their spleen, transfer experiments of 3-day-old spleen cells to lethally X-irradiated syngeneic adult recipients indicated that 3-day-old spleens at least contained all the essential lymphoid elements (B and T cells) needed for germinal centre formation. These results strongly suggest that the failure to induce germinal centres in 3-day-old rats is most probably due to an immaturity of their splenic microenvironment. Immunohistochemical staining of frozen sections of neonatal rat spleen using mAb ED 5 and MRC OX-2 showed that follicular dendritic cells (FDC) were found as soon as primary follicles were found (i.e. by 14 days after birth). The appearance of FDC in neonatal spleens was not influenced by deliberate antigenic stimulation nor by the administration of adult spleen cells. We postulate that, during the development of FDC, a splenic microenvironment is created that allows primary follicle formation and the generation of germinal centres.     

Characterization of two distinct antigens expressed on either resting or activated human B cells as defined by monoclonal antibodies.

Two antigen systems (L29 & L30) expressed on two distinct human B cell subpopulations were identified by using BL1-4D6 and TB3-7D5 monoclonal antibodies, respectively. L29 was expressed on approximately one-third of B cells in human lymphoid tissues. These B cells associated with L29 were large activated B cells located in the germinal centres of lymphoid follicles. L30, on the other hand, existed on approximately two-thirds of B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. In addition, L30 was shared on mature granulocytes. With the use of polyclonal activators such as pokeweek mitogen (PWM) and protein A-bearing staphylococci (SAC), L29 antigen was inducible on PWM- or SAC-stimulated B cells in correspondence with the emergence of Tac and T10 antigens of these B cells. In contrast, L30 antigen on the B cells stimulated by the polyclonal activators was decreased in its expression and was finally lost from these B cells. Although none of L29 and L30 was expressed on normal, non-activated human thymus and peripheral T cells, L29 but not L30 was expressed on concanavalin A-activated T cells. Immunochemical studies showed that L30 consist of a single polypeptide with mol. wt of 40,000. L29 antigen is presently under study.     

Immunoglobulin- and J chain-producing cells associated with lymphoid follicles in the human appendix, colon and ileum, including Peyer''s patches.

Immunohistochemistry of B cells associated with normal human Peyer's patches and solitary lymphoid follicles of the ileum, colon and appendix mucosa showed that local accumulation of IgG-producing cells is a common feature of gut-associated lymphoid tissue (GALT). These immunocytes have strikingly down-regulated J-chain expression, indicating that they belong to mature memory clones. They are located mainly in the dome areas, alongside the follicles, and to a lesser extent in the germinal centres, and are accompanied by a much smaller number of J-chain negative IgA- and IgM-producing cells. It is concluded that B cells of mature memory clones are retained in GALT, whereas relatively early counterparts with a high J chain-expressing potential probably emigrate rapidly after stimulation and seed distant secretory sites where they undergo terminal differentiation to produce mainly J chain-containing dimeric IgA.     

Does the availability of either B cells or CD4+ cells limit germinal centre formation?

To determine whether the availability of either B cells or T cells regulates the number and size of germinal centres observed following antigenic challenge, irradiated PVG rats were reconstituted with limiting numbers of thoracic duct B cells (with excess T cells) or with limiting numbers of thoracic duct CD4+ cells (with sufficient B cells). Germinal centre formation was measured histologically in the spleens of reconstituted rats 7 days after immunization with 2 x 10(9) sheep erythrocytes (at the height of the germinal centre reaction). Although reconstitution with B cells and antigen alone, or with thymocytes and antigen alone, produced no germinal centres, saturating numbers of germinal centres on the order observed for normal, non-irradiated rats were observed in irradiated rats reconstituted with only 10(7) B cells and 5 x 10(6) CD4+ cells. Germinal centres in the spleens of minimally reconstituted rats were also comparable in size to those observed in normal rats. Reconstitution with either 4 x 10(7) thoracic duct lymphocytes (including 2 x 10(7) B cells, as well as CD8+ and CD4+ cells) or with 4 x 10(7) thoracic duct lymphocytes and 2 x 10(8) thymocytes also led to saturating numbers of germinal centres. It is concluded therefore that (i) CD4+ cells are sufficient for the T-cell contribution required for germinal centre formation, and (ii) some factor other than the availability of B cells or CD4+ cells normally limits germinal centre formation.     

Anatomical distribution of T and B lymphocytes identified by immunohistochemistry in the chicken spleen.

Antisera against chicken bursal and thymic lymphocytes were raised in rabbits. They were made specific for the target T or B cells by appropriat absorption and then used for immunohistochemical identification of T and B lymphocytes in tissue sections of chicken spleen, applying the unlabeled antibody enzyme method. Lymphocyte reacting with anti-chicken-thymus globulin predominated in the periarteriolar lymphatic sheaths and in the red-pulp cords. A few of these lymphocytes were localized between the periellipsoid lymphatic tissue and in germinal centers. The periellipsoid lymphocytes around the Schweigger-Seidel sheaths consisted of lymphocytes positive with anti-chicken-bursa-cell globulin. The majority of lymphocytes in the germinal centers were labeled also by anti-chicken bursa-cell globulin. Age-dependent changes in the anatomical localization of T and B lymphocytes are described between late embryonic development and an age of 2 years. The different conditions of avian and mammalian T and B cell distribution in the spleen were considered.     

Cell types required for anti-acetylcholine receptor antibody synthesis by cultured thymocytes and blood lymphocytes in myasthenia gravis.

In most young myasthenia gravis patients, the thymic medulla contains germinal centres. Thymocytes from these cases spontaneously synthesize anti-acetylcholine receptor autoantibody (anti-AChR) in culture; after irradiation they may also selectively stimulate anti-AChR antibody production by autologous blood lymphocytes. By depleting cortical or mature thymic T cells by complement killing, we now show that neither of these responses depends on thymic T cells, unlike the total IgG response to pokeweed mitogen which is T cell-dependent and shows T/B cell synergy. The results suggest that much of the spontaneous anti-AChR production is by autonomous thymic plasma cells, which may be HLA-DR-. The ability to stimulate autologous blood lymphocytes does not require viable HLA-DR+ thymic cells but appears to depend on rare antigen presenting cells from the germinal centres. In preliminary experiments, blood T cells were apparently also necessary.     

The Localization of Antigen in Lymph Node Follicles of Congenitally Athymic Nude Rats

We have examined the postulated dependence on T cells of follicular retention of antigen by studying antigen retention in the draining lymph nodes of congenitally athymic, nude rats after local injections of horseradish peroxidase (HRP). The lymphoid tissues of these rats contained germinal centres and follicular dendritic cells (FDC) that were ultrastructurally identical to those seen in euthymic rats and expressed the differentiation antigen MRC OX2. Nude rat FDC captured and retained locally injected antigen on their surfaces, but as with euthymic rats, only in the presence of previously injected anti-HRP antibody. This demonstrates that the FDC mature both morphologically and functionally in the absence of a thymus or T cells. However, in contrast to euthymic rats, there was no detectable antigen retention in nude rats that had been actively immunized by repeated intraperitoneal injections with HRP for 3 months. The lower number of germinal centres observed in athymic animals compared with their euthymic littermates could thus be explained by deficient production of specific antibody of the isotype necessary for follicular localization of environmental antigens.     

Localization of 125I-labelled antigen in germinal centres of mouse spleen: effects of competitive injection of specific or non-cross-reacting antigen

Studies were performed on localization of ¹²⁵I-human γ-globulin in spleen lymphatic tissue germinal centres during the primary and secondary immune response as influenced by competitive injections of specific or non-cross-reacting antigens. Isologous mouse 7S serum protein labelled with ¹²⁵I was used as the control. The results of these studies support the following conclusions:

(1) Antigen retention in germinal centres during the primary immune reaction is a dynamic process. For some antigens there may be opsonins available at the the time of injection which promote initial localization in germinal centres. However, the continued localization of antigen over weeks and months is a function of specific antibody production.

(2) For some period of time, germinal centres are specific to the antigen that stimulated their development, and eventually these centres will respond to a different antigen.

(3) Antigen persisting in germinal centres is functional in the development of the secondary immune potential.

     

双价志贺菌苗滴鼻免疫对小鼠NALT、GALT和脾淋巴细胞的影响

本文是探讨滴鼻免疫对NALT、GALT和脾淋巴细胞中特异性抗体分泌细胞及淋巴细胞表型变化的影响。将Balb/c小鼠随机分为 3组 ,每组 2 0只。FSM 2 117或FS 5 416 4× 10 7CFU/只经滴鼻途径免疫小鼠 ,间隔 2周 ,3次免疫后 7d活杀 ,分离NALT、鼻通道、脾、小肠PP及MLN淋巴细胞 ,采用BA ELISPOT法检测其中特异性抗福氏、宋内LPSIgA和IgG分泌细胞 ;采用流式细胞术检测其淋巴细胞表型的变化。结果是两株菌苗经鼻内免疫都诱发了NALT、NP、GALT内 (PP、MLN、LPL )以及代表系统免疫反应的脾淋巴细胞中特异性抗福氏、宋内LPSIgA、IgG ASC的显著增加 (P <0 0 1) ;NALT、NP和PP淋巴细胞中CD3+T细胞显著增加 ,其中以CD4+T细胞增加为主 ;FSM 2 117免疫组的脾细胞中B2 2 0 +细胞显著增加 (P <0 0 1) ,而FS 5 416免疫组的脾细胞中CD3+T细胞显著增加 (P <0 0 1)。说明两株菌苗经鼻粘膜免疫均可诱导鼻粘膜局部、GALT及系统免疫反应的发生 ,是一个安全有效的免疫途径     

Changes in Lymphoid Tissue After Treatment with a Gonadotropin Releasing Hormone Antagonist in the Neonatal Marmoset (Callithrix jacchus)

PROBLEM: The effect of neonatal treatment with a gonadotropin releasing hormone (GnRH) antagonist on the morphology and distribution of lymphocytes in lymphoid tissue of the infant marmoset was examined. METHOD OF STUDY: From a screened panel of antihuman antibodies for specific immune cells, antibodies for the CD20 and CD3 antigens showed excellent reactivity with marmoset tissue. Five sets of marmoset twins were treated with either the GnRH antagonist or a vehicle from birth, and were euthanized at 7 to 9 (3 sets) or 16 to 20 weeks (2 sets) of age. The spleen, thymus, and inguinal lymph nodes from each animal were processed for immunocytochemistry, and the number of cells expressing the CD20 and CD3 antigens were quantified. RESULTS: Control twins exhibited high plasma levels of testosterone, characteristic of the neonatal period, whereas testosterone concentrations were reduced (P = 0.001) to detection limits in the GnRH antagonist-treated twins. Microscopic evaluation suggested that treatment reduced the volume and cellularity of the thymic cortex, resulting in a decrease in the cortical-to-medullary ratio. Treatment reduced (P = 0.046) the number of thymocytes expressing the B-cell antigen (CD20) and marginally lowered (P = 0.067) the number expressing the T-cell antigen (CD3) in the thymic medulla. In the spleens of treated animals, periarterial lymphatic sheaths were less prominent on microscopic examination, and there were marginally fewer (P = 0.064) CD3+ cells. Numbers of CD20+ lymphocytes in the peripheral white pulp of the spleen and in the germinal centers of the lymph nodes, or CD3+ cells in the paracortex and germinal centers of the lymph nodes, were not altered by treatment. CONCLUSION: Neonatal treatment with a GnRH antagonist may alter maturational processes for B and T cells in the thymus and spleen of the marmoset and may deprive the immune system of its normal sensitivity to GnRH at a potentially critical time in development.     

Antigens in immunity: XVI. A light and electron microscope study of antigen localization in the rat spleen*

This paper describes the ultrastructural location of labelled antigens and carbon in the spleens of rats from 4 minutes to 5 days after injection. Particular attention was focused on the sites of deposition 4 minutes after intra-arterial injection of microgram quantities of ¹²⁵I-labelled Salmonella flagellar antigens, crayfish haemocyanin and BSA, using colloidal carbon for comparison. The combination of radioautography with both light and electron microscopy showed the importance of antigen binding by lymphocytes in the marginal zone of the spleen. Macrophage sequestration of antigens was not prominent in the spleen, although it occurred in the liver with the flagellar antigens and haemocyanin.

In the spleen marginal zone, avid antigen-binding cells were found in situ 4 minutes after the injection of labelled haemocyanin. These appear to be the counterpart in vivo of antigen-binding lymphocytes prepared in vitro. Such cells also occurred infrequently after the injection of labelled polymerized flagellin, but were not found with either BSA or carbon.

The apparent movement of flagellar antigen from the marginal zone to the white pulp between 1 and 2 hours after injection was seen to involve lymphocyte-associated antigen. The follicular antigen localization occurring from 1 day onwards after injection was on the dendritic reticular cells of germinal centres, as has been described in lymph nodes after subcutaneous injection.

Carbon particles were rapidly sequestered in macrophages of the spleen and liver, although some particles were found between cells in the marginal zone for as long as 2 hours after injection. By 2 and 5 days, however, all the carbon was in phagocytes, even in the white pulp. Differences between the localization of antigens and carbon were clear, even in the ultrastructural sites of their location in tingible body macrophages of germinal centres.

The unexpected emphasis of lymphocyte association with labelled antigens in the spleen marginal zone has allowed a revison of the mechanism previously proposed for the movement of antigens within the microenvironments of the spleen.

     

The immune tissues of the endangered red-tailed phascogale (Phascogale calura)

The lymphoid tissues of the red-tailed phascogale (Phascogale calura) were examined using histological and immunohistochemical techniques. The distribution of immune cells in the tissue beds was documented using antibodies to surface markers CD3 and an MHC Class II antigen (equivalent to HLA DRII). Spleen, gut-associated lymphoid tissues (GALT), lung, bronchus-associated lymphoid tissue (BALT) and liver were examined. The spleen had defined areas of red and white pulp, with follicles containing tingible-bodied macrophages. Anti-CD3 and anti-HLA DRII antibodies revealed the presence of T cells in areas of white pulp and around the peri-arterial lymphatic sheaths. GALT and BALT were detected and appeared as scattered areas of lymphocytes in the tissues beds. This is the first study to report on the lymphoid tissues of this endangered species of marsupial and the first report of the capacity of anti-human antibodies to a surface MHC molecule to react with Dasyurid cells.     

Immunomodulatory activity of a methionine aminopeptidase‐2 inhibitor on B cell differentiation

Methionine aminopeptidase‐2 (MetAP‐2) inhibitors have potent anti‐angiogenesis activity and are being developed for the treatment of solid tumours. The recently observed specific expression of MetAP‐2 in germinal centre B cells suggests that it has a role in regulating B cell function. We have demonstrated a potent MetAP‐2‐dependent inhibitory effect on the antibody secretion from B cell receptor and CD40 co‐stimulated primary human B cells in the presence of interleukin‐21. The effect of MetAP‐2 inhibition on antibody secretion was due to a block in differentiation of B cells into plasma cells. Immunohistochemical analysis of germinal centres from human, mouse and marmoset spleen showed a similar expression pattern of MetAP‐2 in the marmoset and man, whereas mouse spleen showed no detectable expression. In a marmoset, T dependent immunization model, the MetAP‐2 inhibitor suppressed an antigen‐specific antibody response. Furthermore, histological analysis showed loss of B cells in the spleen and disrupted germinal centre formation. These results provide experimental evidence to support a novel role for MetAP‐2 in immunomodulation. These effects of MetAP‐2 are mediated by disruption of the germinal centre reaction and a block in the differentiation of B cells into plasma cells.     

Detection of T and B cell-specific heteroantigens on electrophoretically separated lymphocytes of the mouse

Mouse spleen lymphocytes were electrophoretically separated into pools of T and B lymphocytes. Heteroantisera were raised in rabbits against these cell pools, the IgG fractions were isolated and their lymphocytotoxicity tested. After appropriate absorption, the antisera were specifically directed against lymphocyte antigens. Further cross-absorption with T and B lymphocytes made the antisera specific for antigens which were mutually exclusively present on T and B cells and were related to neither alloantigens nor immuno-globulins. These anti-B and anti-T cell antisera killed antibody-forming cells and graft-versus-host reactive cells, respectively, in vitro. Furthermore, anti-B cell antiserum was cytotoxic to lymphocytes of low electrophoretic mobility in bone marrow and peripheral organs, except in the thymus, whereas anti-T cell antiserum was cytotoxic to lymphocytes of high electrophoretic mobility in all organs and, in addition, killed all thymocytes of low electrophoretic mobility, which had not been affected by anti-B cell antiserum. This distribution of lymphocytes in the electrophoretic distribution profiles, together with the described properties, gives evidence that heteroantigens were found to be exclusively present on T and B cells. They were designated “mouse B cell-specific” and “mouse T cell-specific” antigens and are part of mouse lymphocyte-specific antigen.     

Characterization of rat T helper cell clones specific for Bacteroides gingivalis antigen.

During the past several years, much interest has been directed towards delineating and characterizing different subsets of T helper (Th) cells in order to understand their roles in immune processes. In this study, we report the generation of antigen-specific rat Th cell clones and their characterization in terms of phenotype, function, and lymphokine production. The clones were derived by culturing purified splenic T cells from rats immunized with the pathogen Bacteroides gingivalis with equivalent numbers of irradiated spleen cells from nonimmune rats and B. gingivalis whole-cell antigen. The clones required antigen stimulation but not exogenously added interleukin-2 for growth and were maintained in culture for approximately 6 months. The cloned T cells proliferated in response to the mitogen concanavalin A and to B. gingivalis whole-cell antigen but not to other microbial antigens. Phenotypic characterization of the cloned T cells for cell surface markers demonstrated that these cells were OX19+ W3/25+ OX8- OX22- and therefore probably represented a mature subpopulation of CD4+ Th cells. These cloned T cells were positive for interleukin-2 receptor expression. Culture supernatants from the Th cell clones which were collected at various times after antigen stimulation exhibited low interleukin-2 activity and high gamma interferon activity. This in vitro study provides evidence of a rat Th cell subset that could represent an important population in regulating immune responses to microbial antigens.