The development of the spleen in the Australian lungfish, Neoceratodus forsteri Krefft, with special reference to its relationship to the "gastro"-enteric vasculature

The spleen of Neoceratodus forsteri Krefft grows in the shape of a " boomerang " on the left lateral-dorsal-right lateral part of the borderline between the foregut and the yolkgut . Development of the spleen follows the following stages: 1) appearance of the splenic primordium as a mesenchymal condensation in a limited portion of the region supplied by the third and fourth vitelline arteries on both sides, 2) development of the splenic sinuses within the primordium, 3) formation of the "gastric" and enteric splenic portal systems and 4) growth of the spleen along the anterior extremity of the spiral valve. The blood vascular dynamics of the foregut and the yolkgut are intimately involved with the formation of the spleen in this species. These processes were, therefore, compared with similar processes in other animals, and the characteristics of this organ common to vertebrates were investigated.     

Peripheral tolerance induced in lymph nodes by syngeneic spleen cells inhibits generation of CTLs to hapten-altered self antigens but not to alloantigens.

The immunological tolerance that is induced in lymph nodes that have been exposed to syngeneic spleen cells has been examined. Development of cytotoxic T lymphocytes was used to assess the immunological status of the lymph node cells. The tolerance was studied from the viewpoint of its induction, its activation, and its specificity. We had already reported that injecting either T or B cells of splenic origin into a regional lymph node environment a week prior to immunization for CTL to hapten-altered self antigens prevents development of the CTL. Here, we confirm that syngeneic splenic cells but not lymph node cells will induce the suppression provided that spleen cells are not coupled with hapten. We now report that splenic cells that cannot replicate or synthesize and secrete protein are capable of inducing the suppression. The data suggest a preformed surface marker peculiar to spleen cells and perhaps on cells that traverse the thymus induces local tolerance that is mediated by suppressor cells. Triggering the induced suppressor T cells (previously identified as CD8-) was achieved by syngeneic spleen cells as well as by H-2-compatible, Mls-disparate spleen cells but not by syngeneic lymph node cells or apparently by allogeneic spleen cells. Furthermore, triggering suppression was achieved by hapten-coupled syngeneic spleen cells whereas such cells would not induce the suppression. Thus, activating the suppressor cells requires reexposure to splenic cells of the proper MHC haplotype, unaltered or coupled with either TNP or FITC. Once triggered, the suppression was manifested toward CTL generation against hapten-coupled syngeneic antigens on either spleen or lymph node cells but not against allogeneic antigens. Thus, the specificity of the tolerance was directed to altered self antigens despite its induction by unaltered spleen antigen. Furthermore, for suppression to be seen the spleen antigen was not required to be on the hapten-coupled syngeneic cells used for the CTL immunization. The relationship of the splenic cell "antigen" to hapten-altered self antigens and to other surface markers and its site of acquisition within the body and its significance for cell homing have become intriguing questions of importance. This information has been discussed from the viewpoint of its applicability to autoimmune diseases as well as to cessation of inflammatory reactions that may be mediated by lymph node cells.     

Characterization of lymphocyte responsiveness in early experimental syphilis. I. In vitro response to mitogens and Treponema pallidum antigens

Lymphoid cells from spleens and lymph nodes of rabbits infected with T. pallidum respond by proliferation to concanavalin A (Con A) and T. pallidum antigens. Spleen cell responsiveness to treponemal antigens appears 6 days after infection, is 100 to 600 fold higher than the response of uninfected control rabbits, and is maintained throughout the 31-day observation period. Specifically responding cells in the inguinal and popliteal lymph nodes of infected animals are demonstrable on day 10, and the magnitude of the response increases throughout the observation period. Specific responsiveness to T. pallidum antigens in vitro is enhanced in purified T cell populations and is abolished by treatment with goat anti-rabbit thymocyte serum and complement. The response of spleen and lymph node cells to Con A is unaffected during syphilitic infection. These results are consistent with a role for T cell-mediated specific immunity to treponemal antigens early after infection and do not support a hypothesis of depressed cellular immunity during syphilitic infection.     

Phenotypic identification and development of distinct microvascular compartments in the postnatal mouse spleen.

In this paper we report the development of the sinus network of mouse spleen during the first postnatal month as studied with a set of new rat monoclonal antibodies (mAbs) against mouse splenic endothelial cell subpopulations. One of the new mAbs (IBL-7/1) also stained B-cell lineage cells in the spleen shortly after the birth as confirmed by three-color flow cytometry. This B-cell staining in the primordial follicles vanished by the fourth postnatal week, so that the expression of IBL-7/1 antigen was restricted to the marginal sinus endothelium and some red pulp sinuses and a minor B-cell subset in the spleen, presumably distinct from the follicular B-cell compartment. The other mAb (IBL-9/2) selectively labeled the sinusoids of the deeper part of the red pulp, without any reactivity against hemopoietic cells. The IBL-9/2-reactive cells in newborns appeared as isolated elements throughout spleen, and during the segregation of white and red pulps they formed an extensive network in the red pulp outside the marginal zone. Double-labeling immunofluorescence revealed that most of these sinusoids also stained weakly with IBL-7/1 mAb, whereas the strongly IBL-7/1-positive vessels of this region were IBL-9/2 negative. Neither of these mAbs reacted with the central artery. The comparative phenotypic analysis of the various vascular segments indicates that the splenic sinusoids of the marginal zone and red pulp, respectively, are lined with a heterogeneous array of endothelium. For the precise identification, isolation, and characterization of the possible homing function of these endothelium subsets these region-specific mAbs may be of potential value.     

Immunodiffusion analysis of water-soluble rat bone marrow antigens

Antisera were obtained against six electrophoretic fractions of the rat bone marrow extract. With their help, 18 tissular antigens and 11 antigens immunologically similar to the blood serum proteins were revealed in the rat bone marrow. All tissular antigens are divided in five groups by the degree of organ specificity: 1) bone marrow organospecific antigens (4 antigens), 2) antigens present in the bone marrow, spleen and lung extracts (2), 3) "granulocytic" antigens (4), 4) antigens common for many rat organs, but not found in the extracts of blood formed elements, skeletal muscle, heart, brain, eyes (3), 5) antigens present in all the organs studied (5). The bone marrow organospecific antigens may be specific antigens of hemopoietic cell precursors. The possibility of utilization of antisera against the bone marrow water soluble proteins for labelling hemopoietic cells of different lines of differentiation is discussed.     

B-Lymphocyte differentiation in lethally irradiated and reconstituted mice. II. Recovery of humoral immune responsiveness.

The recovery of humoral immune responsiveness was studied in lethally irradiated, fetal liver-reconstituted mice. By means of both membrane fluorescence and antibody formation to sheep red blood cells (SRBC) as a functional assay, the rate of recovery of the compartments of B and T lymphocytes was determined in various lymphoid organs. The recovery of the immunoglobulin-positive (B) cell compartment after irradiation and reconstitution started in the spleen. This organ was also found to be the first in which the recovery of the B-cell population was completed. The interval between the recovery of the B-cell population in the spleen and that in the other organs tested was found to increase when the irradiated mice were reconstituted with spleen colony cells instead of fetal liver cells. This proved to be caused by the number and nature of the reconstituting hemopoietic stem cells. The immunoglobulin-positive (B) cells were found to appear before SRBC-reactive B cells could be demonstrated in spleen, lymph nodes, and Peyer's patches. The appearance of T lymphocytes in the various lymphoid organs required even more time. By means of cell transfer experiments, a sequential appearance of the precursors of anti-SRBC IgM-, IgG-, and IgA-plaque-forming cells could be demonstrated in spleen, bone marrow, lymph nodes, and Peyer's patches.     

Mathematical Modeling Reveals Kinetics of Lymphocyte Recirculation in the Whole Organism

The kinetics of recirculation of naive lymphocytes in the body has important implications for the speed at which local infections are detected and controlled by immune responses. With a help of a novel mathematical model, we analyze experimental data on migration of ⁵¹Cr-labeled thoracic duct lymphocytes (TDLs) via major lymphoid and nonlymphoid tissues of rats in the absence of systemic antigenic stimulation. We show that at any point of time, 95% of lymphocytes in the blood travel via capillaries in the lung or sinusoids of the liver and only 5% migrate to secondary lymphoid tissues such as lymph nodes, Peyer''s patches, or the spleen. Interestingly, our analysis suggests that lymphocytes travel via lung capillaries and liver sinusoids at an extremely rapid rate with the average residence time in these tissues being less than 1 minute. The model also predicts a relatively short average residence time of TDLs in the spleen (2.5 hours) and a longer average residence time of TDLs in major lymph nodes and Peyer''s patches (10 hours). Surprisingly, we find that the average residence time of lymphocytes is similar in lymph nodes draining the skin (subcutaneous LNs) or the gut (mesenteric LNs) or in Peyer''s patches. Applying our model to an additional dataset on lymphocyte migration via resting and antigen-stimulated lymph nodes we find that enlargement of antigen-stimulated lymph nodes occurs mainly due to increased entrance rate of TDLs into the nodes and not due to decreased exit rate as has been suggested in some studies. Taken together, our analysis for the first time provides a comprehensive, systems view of recirculation kinetics of thoracic duct lymphocytes in the whole organism.     

Lymphomyeloid organs of amphibia. II. Vasculature in larval and adult Rana catesbeiana

There were no lymphatic vessels and lymph nodes demonstrable in adult and larval Rana catesbeiana by a method that adequately demonstrated the same in mammals. Although the parenchymal arrangement in the lymphomyeloid organs is not exactly the same as in mammalian hemal nodes, nonetheless the vascular patterns of the lymph glands and jugular bodies are prima facie evidence that they function as blood-filtering organs among other probable functions. The vascular pattern of the lymph gland is that of a rete mirabile, particularly a venous portal system, inasmuch as the afferent and efferent vessels are venous in character and interposed between them is a labyrinth of sinusoids. This is not the case, though, in the adult organs. The vascular pattern of the jugular bodies is very much like the spleen, viz., artery-capillary-sinusoid-vein sequence. It is doubtful, however, if the propericardial and procoracoid bodies ever filter blood, because the smallest blood vessels in them are capillary in type Because of the absence of a well-defined capsule in some parts of the propericardial body, similarly to lymphoid follicles, especially in the mammalian gastrointestinal tract, it is probable that it filters tissue fluid. The last two organs are apparently mainly blood cell-forming organs. It is inferred from the vascular connections of the larval and the adult lymphomyeloid organs that they are not genetically related. This aspect was analyzed from earlier developmental data, but actual follow-up of the larval organs to the adult stage is still in progress.     

Splenic lymphocytopoiesis and migration pattern of splenic lymphocytes

The spleens of young pigs were selectively labeled with tritiated thymidine ([³H]-TdR) and the relative and absolute numbers of labeled lymphocytes found 24 hr later in different lymphoid and nonlymphoid organs were determined autoradiographically. It was deduced that about 4.6 × 10⁹ lymphocytes (that is, about 15% of all splenic lymphocytes) are produced by the spleen per day and about 17% of the newly formed lymphocytes leave the spleen within the first day of labeling. Spleen-derived lymphocytes could be found in relatively high numbers in the lymph nodes, blood, gut-associated lymphoid tissues, and, surprisingly, in the bone marrow, whereas the concentration in the thymus was very low. In a second series, pigs were labeled with [³H]TdR and only the spleen was excluded from labeling. The labeling index of splenic small lymphocytes was about 10% 1 day later, indicating a high rate of influx of newly formed lymphocytes into the pig spleen. The spleen of the young pig is an important lymphocytopoietic organ and exports and imports newly formed lymphocytes at high rates.     

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.     

Comparison of local and systemic responsiveness of lymphocytes in vitro to Bovicola ovis antigen and Concanavalin A in B. ovis-infested and naive lambs

The in vitro proliferation assay was used to determine lymphocyte responsiveness to soluble antigen of B. ovis and to Concanavalin A (Con A) in peripheral blood, spleen and various lymph nodes from B. ovis-infested and naive lambs. From March to July, an assay of monthly blood samples showed generally higher proliferative responses to antigen and Con A in B. ovis-infested than naive lambs. The proliferative response of cells from the skin-draining prescapular lymph nodes to B. ovis antigen was significantly higher in B. ovis-infested than naive lambs. Responses of cells from the medial iliac, mediastinal and mesenteric lymph nodes (which do not receive lymph from the skin) and spleen showed no significant differences between groups. Within the B. ovis-infested lambs, the response of cells from the prescapular lymph node was significantly higher than that from any other lymphoid organ examined. Responsiveness of the prescapular, medial iliac and mesenteric lymph node and spleen cells to Con A was not significantly different between groups, while mediastinal lymph node cells showed a significantly higher response in B. ovis-infested lambs. The data indicate that the antigen-specific cellular immune response is operating mainly locally, at the level of the skin and draining lymph nodes. Responses to the T cell mitogen Con A did not support non-specific immunodepression as reported in other ectoparasite/host systems.     

Splenic hemopoiesis of the platypus (Ornithorhynchus anatinus): evidence of primary hemopoiesis in the spleen of a primitive mammal

The generation of blood cells has been observed in the spleen and in the bone marrow of the platypus. Hemopoiesis was found to be far more active in the spleen than in the bone marrow judging by the number of proliferating hemopoietic elements within a unit area of tissue from each organ. Granulocytes, erythroblasts, and megakaryocytes, with the related immature forms for each cell line, were noted in the spleen. In contrast, there were very few examples of immature forms of these cell lines and a complete absence of mature megakaryocytes in the bone marrow. These findings suggest that the spleen is the primary hemopoietic organ in the platypus. Since the platypus is one of two species representing the most primitive existing mammals, it seems likely that the spleen may be the primary hemopoietic organ in mammalian evolution.     

Preliminary observation of blood cells and hematopoietic organs in Lutjanus herenbergi and Lutjanus flaviflammus

The morphology of differentiated and differentiating cells of the red and white series in Lutjanus herenbergi and in Lutjanus flaviflammus is described. Early stages of red and white blood cells may be found only in smears of hemopoietic organs. Polychromatic erythroblasts, myelocytes and lymphoblasts may also occasionally be found in blood smears. Mature blood cells may be found both in blood smears and in hemopoietic organs. Differential white cell counts seem to demonstrate that the granulocytic series elements are the most common leukocytes in blood smears. Almost all granulocytes may be classified in the first three Arneth classes. An analysis of hemopoietic organs in these species was also performed. It was found that the only organs carrying on a hemopoietic function are the kidney and the spleen. The kidney is essentially a site of granulocytic differentiation while the spleen is a lymphopoietic organ. An erythropoietic activity may generally be observed in the kidney although weak erythropoietic activity may at times be found in the spleen.     

A syngeneic splenic cell antigen induces a suppressor T cell in lymph nodes that controls cytotoxic T-cell and primary antibody responses

The nonspecific suppression of immunological responses that is generated within host popliteal lymph nodes upon exposure to syngeneic normal spleen cells has been examined. The suppression, which had previously been described as being capable of preventing initiation of cytotoxic T lymphocytes (CTLs) to hapten-altered self antigens, arises within 3 to 7 days after injecting the spleen cells. Suppression was shown to be attributable to an induced T cell that was functional when transferred intravenously. Although the cell surface marker(s) on both splenic B and T cells that stimulates appearance of Ts has not yet been identified, the cells possessing the marker were not required to be viable to cause the induction. We have shown here that the Ts is fully functional when it is put in the antigenic site used for CTL immunization. The induced Ts has been identified as bearing the Lyt 2.1 cell surface marker. Furthermore, it has been shown to be insensitive to cyclophosphamide (CY), thus differentiating it from the naturally occurring Ts cell (TS0) that is known to be CY sensitive. In addition to preventing induction of CTLs toward hapten-altered self antigens, exposing popliteal lymph nodes to syngeneic spleen cells induced Ts capable of suppressing the primary IgM antibody response to sheep red blood cells. The Ts cells that suppressed the primary antibody response possessed the same Lyt cell surface markers and CY insensitivity as the Ts that mediated suppression of the CTL response. Thus, evidence that two dissimilar immunological reactions may be down-regulated by the same suppressor mechanism has been provided. Results of a kinetic study showed that the Ts prevented development of both the humoral and the cell-mediated immune responses by affecting their inductive phases. Possible targets for suppression that more than likely would have to be common to the two widely different immune responses have been indicated.     

Splenic regulation of lymphocyte trapping in lymph nodes draining tumor grafts.

We measured the trapping of 51chromium-labeled splenic lymphocytes in the lymph nodes and spleens of tumor-inoculated mice at various intervals after inoculation. Both histocompatible and incompatible tumors were studied. Significant trapping occurred in the draining lymph nodes over the entire course of the experiments. The splenic trapping response commenced very early after transplantation and abated after a few days. The magnitude of the lymph node trapping response correlated with the magnitude of antigenic difference between tumor and host. Splenectomy before tumor inoculation had bidirectional effects; it caused a marked augmentation of the trapping response in the early period after tumor inoculation, which declined with time so that late after tumor inoculation, splenectomized animals trapped less well than intact controls. Tumor resection always led to a rapid fall in the trapping response in normal mice. However, tumor resection at the time of the peak trapping response of splenectomized mice produced no change. These results support the notion that lymphocyte trapping is a valid and useful monitor of the immune response to tumors. They also serve to reemphasize the important regulatory role of the spleen plays in the immune response to tumor-associated antigens by demonstrating a new parameter of splenic control; regulation of lymphocyte traffic in response to antigenic stimuli.     

Chemokine/cytokine production by mononuclear cells from human lymphoid tissues and their modulation by Mycobacterium tuberculosis antigens

The majority of knowledge about the role of cytokines and chemokines in controlling Mycobacterium tuberculosis infection mainly derives from animal models. In humans, this knowledge is still mainly limited to the blood compartment or accessible lymphoid organs, such as tonsils. Here, we studied cytokine and chemokine production and their modulation by M. tuberculosis antigens in mononuclear cells from human blood, spleen and hilar lung lymph nodes. Results show that the kinetics and magnitude of cytokine and chemokine production varied according to the tissue of cell origin. Mycobacterium tuberculosis antigens enhanced cytokine and chemokine production in blood, but the enhancement was restricted in spleen and hilar lung lymph node cells. We show, for the first time in humans, differences in cytokine and chemokine microenvironments according to lymphoid tissues, and suggest that these differences may affect the way cells respond to M. tuberculosis infection.     

Stimulation of extramedullary hemopoiesis by dextran sulfate

The spleen of normal dogs (beagles) shows only slight hemopoietic activity, characterized by the presence of megakaryocytes in mitosis and small groups of erythroblasts scattered throughout the red pulp of the organ. Repeated intravenous injection of dextran sulfate, at a dose of 15 mg per kg body weight, produced markedly enhanced erythropoietic and megakaryocytopoietic activity in the splenic red pulp, without concomitant increase in splenic granulopoiesis. The probable existence of a micro-environment adequate for erythro- and megakaryocytopoietic differentiation of stem cells is discussed.     

An antigen expressed by avian neuronal cells is also expressed by activated T lymphocytes.

A monoclonal antibody, anti-BEN, initially characterized by its reactivity with an epitope present on the surface of avian bursa epithelial cells and neurons, also reacts with membrane molecules on some hemopoietic cells. In this study we examine BEN expression on lymphoid cells in thymus, spleen, and blood. We demonstrate that BEN is an activation antigen on mature T lymphocytes. It is not expressed on peripheral blood or splenic lymphocytes, but following mitogenic or allogeneic stimulation of blood lymphocytes it appears rapidly on a T cell subpopulation in parallel with the appearance of IL-2 receptors. BEN is also expressed on III-C5 cells, an avian IL-2-dependent permanent T cell line, and on immature CD4+CD8+ thymocytes. BEN is not expressed by resting or actively proliferating B cells. Biochemical analyses of the BEN protein on T lymphoblasts shows that the molecule is similar in size to the BEN molecules on bursa epithelial cells and on neurons. The physicochemical properties of the BEN protein and its tissue distribution differs from other known avian and mammalian T cell activation markers, differentiation antigens, and integrins. Thus BEN is a novel marker of activated T cells in birds.     

Suppressive effect of normal lymphoid cells on manifestations of immunologic memory

The authors studied the influence of the cells of normal lymphoid organs on the level of immunological response in the recipients of splenic cells from the suppressed animals. The organ cells were mixed with the suppressed ones and were administered to the recipients together with the reimmunizing dose of the antigen. Cells of the spleen, of the lymph nodes, the thymus or of the bone marrow suppressed the capacity of the memory cells to the realization of the immunological response to sheep red blood cells and egg albumin. The spleen cells of one and a half month old mice were more active than the cells of young or old animals. The suppressor activity persisted after the administration to donors of various doses of cortisone or heating of the cells transferred at 56 degrees C. Treatment with T-antiserum or heating at 80 degrees C led to reduction of the suppressor action of normal cells.