Migration pattern of splenic lymphocytes after local labelling of the pig spleen with 3H-cytidine

In normal young pigs splenic lymphocytes were selectively labelled by injecting tritiated cytidine into a splenic artery. 10 h later several lymphoid and non-lymphoid organs were investigated for spleen-derived lymphocytes by autoradiography. The relative and absolute organ distribution of the labelled cells was determined. Labelled lymphocytes appeared rapidly in the peripheral blood reaching a mean labelling index of 4.8%. More splenic lymphocytes were found in thymus dependent areas than in thymus independent areas of lymphoid organs. Nearly 40% of all emigrated lymphocytes homed to lymph nodes and only about 1% in the thymus. A surprisingly high number of splenic lymphocytes were located in the bone marrow and the lung.     

In situ labelling of bone marrow lymphocytes with fluorescein isothiocyanate for lymphocyte migration studies in pigs

In normal young pigs, the femoral artery and vein were cannulated and after occluding other vessels to one hind leg they were connected to an extracorporeal perfusion system. Fluorescein isothiocyanate (FITC) was added to the perfusate to selectively label cells in the bone marrow. Large numbers (approximately 0.9 X 10(9] of labelled lymphocytes left the bone marrow of one leg within 1 d and migrated via the blood to the bone marrow in other bones, lymph nodes, spleen, Peyer's patches and even into the thymus. On average 1.7% of the lymphocytes in the blood and about 1% within the spleen were labelled. Peyer's patches and the thymus showed very low indices. Thus the bone marrow is an integral part of the migratory route of lymphocytes. Selective labelling of bone marrow cells in their normal microenvironment with FITC is a suitable method for studies of cell migration from the bone marrow.     

Migration of Long-lived Lymphocytes to the Bone Marrow and to Other Lymphomyeloid Tissues in Normal Parabiotic Guinea Pigs

Guinea pigs, in which cells with longlife span were selectively labeled (³H-thymidine), were joined in parabiosisto nonlabeled syngeneic litter mates ata time when label reutilization detectable by radioautography could be excluded. The distribution of labeledcells was investigated quantitativelyusing radioautography and liquid scintillation counting in the marrow andblood at the time of establishment ofparabiosis and again at its termination2 wk later, when the thoracic ductlymph, lymph nodes, spleen, and thymus were also examined. Single animals labeled in the same mannerserved as controls. Of all cells with aslow rate of turnover and long lifespan, only small lymphocytes enteredthe circulation and crossed the anastomosis in detectable numbers. As indicated by the similar percentages oflabeling in respective tissues, a complete intermixing of long-lived lymphocytes occurred in the bone marrow,lymph, lymph nodes, and spleen of theparabionts. The sum of the per centlabeled lymphocytes in two parabiontswas in agreement with the extent oflabeling in respective tissues of singlecontrols. The presence of a minor population of lymphocytes with a long lifespan was confirmed in the marrow.Ten to 30 times as many labeled long-lived lymphocytes migrated into thebone marrow of initially unlabeled animals as were found in an equal volumeof blood. The majority, if not all long-lived lymphocytes migrate to the marrow from the blood, and they also reenter the blood. They have a similarlife span and in parabionts equilibratein a similar manner as recirculatinglong-lived lymphocytes.

Submitted on October 22, 1971 Revised on January 3, 1972 Accepted on January 4, 1972     

Circulation and migration of small blood lymphocytes in the rat: II. Study of the lymphocyte selective migration by light and electron microscopic autoradiography

Fourteen male Wistar rats were the recipients of labeled small lymphocytes (1.5 x 10(7) each) collected from the peripheral blood of syngeneic donors. The migrating labeled lymphocytes were traced in the various organs from one to 60 minutes following their transfusion. Sections from the lymph nodes, bone marrow, spleen, thymus, ileum, liver, lung, and kidney were analyzed morphologically by autoradiographic studies. The results showed that some of the labeled small blood lymphocytes migrate to the lymph node and bone marrow as early as one minute following transfusion; their exodus from these two organs occurs within three minutes. In the case of the spleen, the lymphocytes did not migrate selectively to the marginal zone of the lymphoid follicles until ten minutes following transfusion. The electron microscopic study of the spleen and lymph node showed that the labeled lymphocytes selectively migrate to certain areas which consist of reticulum cells, macrophages, unlabeled lymphocytes, and plasma cells. The term "immunocompetent zones" is proposed for these areas because of the biological significance of this selective migration with reference to immunity.     

Growth of canine T-lymphocyte colonies in vitro.

Canine lymphocytes from peripheral blood, lymph nodes, thymus and bone marrow were stimulated with phytohemagglutinin-P (PHA) or concanavalin-A (CON-A) to form colonies in methylcellulose. Lymphocytes exposed to mitogens in liquid phase formed clumps the size of colonies. Lymphocyte clumping was eliminated by plating cells directly into methylcellulose, but high concentrations of mitogens (CON-A or PHA is greater than 10 mg/10(6) lymphocytes) were required in order to get subsequent colony formation. Thus, in contrast to published reports, exposure of lymphocytes to mitogen prior to plating was not required for cloning of canine peripheral blood lymphocytes. Colonies from thymus, lymph node, or peripheral blood consisted predominantly of T lymphocytes, whereas cultures from bone marrow also produced colonies with macrophage morphology and surface-adherent colonies with mesenchymal morphology.     

Insulin as a target antigen in autoimmune diabetes: a natural repertoire as the source of antibody response

A solid-phase immunoenzymatic technique with B1- or B29-biotinylated insulin coupled to avidincoated wells was used to characterize serum anti-insulin antibodies and to locate insulin antibody-producing B lymphocytes in different organs of mice. Low natural serum anti-insulin IgM and IgG antibodies were found in ten different healthy inbred strains of mice. Prediabetic nonobese diabetic (NOD) mice had significantly higher measurements than BALB/c mice (P<0.05). Anti-insulin IgM antibody-producing B lymphocytes were found in bone marrow and spleen of NOD mice and healthy strains of mice, but not in peripheral lymph nodes, thymus, blood or pancreas. B29-fixed insulin was more frequently recognized than B1-fixed insulin. There was no relationship to the MHC or to other immune markers. IgG insulin antibody-producing cells were not detected. IgG insulin antibody-producing cells appeared in the draining lymph node and in the blood 10 days after immunization with insulin. IgM insulin-recognizing cells in the spleen were reduced in number during the same period (P<0.05–0.01 for BALB/c, DBA2, B10.D2 and NOD), suggesting migration of these cells. This was tested by in vivo staining of spleens with the red-fluorescent membrane linker PKH-26 on day 7 after immunization. Cells from immunized lymph nodes were FACS-sorted on day 10. Insulin antibody-producing B lymphocytes with red-fluorescence were found, indicating a splenic origin. Examination of IgG subclasses showed preferential production of complement-fixing IgG2b in sera and lymph node cells of immunized NOD mice (P<0.05 vs BALB/c). We conclude that a natural repertoire of insulin recognition exists in bone marrow and spleen of mice. Hydrophobic epitopes around B1 (B29-fixed insulin) are more frequently recognized than hydrophilic epitopes around B29 (B1-fixed insulin), indicating a genetically fixed pattern of autorecognition. Insulin-recognizing cells from the spleen function as the source of insulin antibody response. Preferential occurrence of complement-fixing IgG2b in NOD mice could contribute to autoimmune-mediated -cell damage.     

Immunologic abnormalities in an animal model of chronic hypoplastic marrow failure induced by busulfan.

The immunology of chronic hypoplastic marrow failure (CHMF, aplastic anemia) was studied in an experimental murine model of the disease induced by busulfan. B lymphocytes of peripheral blood, spleen, and bone marrow were reduced to 30%-40% and T lymphocytes of thymus, spleen, marrow, and blood were decreased to 20%-70% of control values. IgG and IgM antibody titer to sheep red blood cells were reduced to one-third of control levels, and splenic IgG, but not IgM, plaque-forming cells were fewer on day 7 after antigen stimulation. The proliferative responses to phytohemagglutinin or concanavalin A were reduced in cultures of peripheral blood lymphocytes, splenic lymphocytes, and thymocytes, and cutaneous delayed-type hypersensitivity induced by dinitrofluorobenze was not detected in mice with CHMF. The results demonstrate disturbance of a variety of cellular and humoral functions and suggest that the disturbance was due to quantitative and possibly qualitative abnormalities of the cell types subserving these functions. The results suggest that residual cell injury, the lesion underlying experimental CHMF, is not confined to the myeloid stem cell but also involved cells of the lymphoid series.     

Stem Cells and Small Lymphocytes of Congenitally Asplenic-Athymic Mice Quantitative and Autoradiographic Results

A new type of animal: Mice with congenital absence of both the spleen and the thymus (ASAT mice) have been investigated. Haemato- and lymphocytopoietic findings in these mice were compared to findings in congenitally asplenic (AS), congenitally athymic (AT), and normal mice. Major findings were: (1) Normal numbers of CFU-S in bone marrow of ASAT mice and presence of CFU-S in lymph nodes of these mice, (2) High numbers of theta-positive cells in the bone marrow of ASAT and AT mice, (3) Low numbers of bone marrow lymphocytes as well as thoracic duct lymphocytes in ASAT and AT mice, and (4) Comparable proliferative kinetics of small lymphocytes in all 4 groups of mice, an exception being a rapid appearance of newly-formed small lymphocytes in thoracic duct and lymph nodes of ASAT mice.     

Immunologically mediated aplastic anemia in mice: effects of varying the source and composition of donor cells

Experimental aplastic anemia (EAA) can be induced in CBA/J mice when they are sublethally irradiated and injected with lymph node cells (LNC) from C3H/He mice. Mice injected with LNC die of severe pancytopenia and marrow aplasia. In the present study, cells from other anatomical locations and subsets of LNC were examined for their ability to induce and to modulate EAA. Of peritoneal, splenic, and thymic cells, only cells from the thymus had EAA activity. C3H/He bone marrow cells did not induce any adverse effects in sublethally or lethally irradiated CBA/J mice. LNC, when depleted of B or phagocytic cells, retained EAA activity. In contrast, LNC depleted of T cells had significantly less EAA activity. Furthermore, when T cells of LNC were separated into peanut agglutinin (PNA)-receptor-positive and negative fractions, only the PNA- cells were able to induce EAA. EAA activity was lost when LNC were irradiated (1000 rad). The inability of splenic or bone marrow cells to induce EAA could have been due to the presence of cells that suppressed EAA activity. When splenic or bone marrow cells were coinjected with LNC, EAA was not induced. Coinjected irradiated splenic cells, but not bone marrow cells, were still able to inhibit EAA activity. Bone marrow cells seemed to inhibit EAA by replacing stem cells that were lost during the EAA process. On the other hand, splenic cells appeared to suppress EAA activity of LNC. Thus, radiosensitive PNA- T cells of lymph nodes or thymus were capable of inducing EAA, and their activity could be modulated by radioresistant splenic cells.     

Studies on T and B lymphocytes in rats bearing methylcholanthrene-induced tumor.

The authors have studied the influence of primary, methylcholanthrene-induced tumor on T and B lymphocytes of spleen, thymus, draining lymph nodes and peripheral blood of rats. Differences in weight of tumors were found to correlate with changes in proportionality of T and B lymphocytes of followed organs. Small tumors induced but insignificant changes. There was increased trapping of T lymphocytes in spleen and lymph nodes with simultaneous decrease in peripheral blood. The authors noted a high percentage of blasts. B lymphocytes showed a tendency to compensate for the loss of T cells. Large, progressively growing tumors caused evident exhaustion in the number of both cell types in lymph nodes and peripheral blood. In the spleen there was slower exhaustion. Reduction in the number of B lymphocytes correlated with the size of tumor. Blasts disappeared. Proportionality of T and B lymphocytes in thymus did not appear to be influenced by the size of tumor.     

Changes in the tissues of the immune system in dengue haemorrhagic fever.

A total of 100 post-mortems were done on patients clinically diagnosed as dengue haemorrhagic fever from Rangoon Children's Hospital. Histopathological changes in bone marrow, thymus, spleen, lymph nodes and other associated tissues of the immune system were analysed and correlated with the clinical picture and serology results. The major changes in cases with a positive serology result for secondary dengue infection consist of hypoplasia of the bone marrow, acute atrophy and wasting of the thymus, atrophy and depletion of cells in the periarterial lymphatic sheaths of the spleen and the paracortical areas of the lymph nodes. The tissues affected are the thymus-dependent areas of the spleen and lymph nodes, and the thymus itself. Thymus-independent areas of the secondary lymphatic tissues are also affected but to a lesser extent. The pathological observations suggest that immunodepression may be an integral part of the pathophysiology of dengue haemorrhagic fever.     

Composite splenic marginal zone lymphoma and classic Hodgkin lymphoma -- an unusual combination

The simultaneous occurrence of Hodgkin lymphoma with a variety of B-cell Non-Hodgkin lymphomas (composite lymphoma) has been described. We report the first case of composite Hodgkin lymphoma and splenic marginal zone lymphoma occurring simultaneously in the same lymph node of a 64-year-old man who presented with cervical and axillary lymphadenopathy and massive splenomegaly. He had a peripheral blood lymphocytosis and a bone marrow infiltrated by small lymphocytes. However, cervical lymph node biopsy showed classic Hodgkin lymphoma. His splenomegaly showed only a partial response to six cycles of ABVD chemotherapy so he underwent splenectomy with biopsy of remaining nodes. Histology of the spleen and nodes showed splenic marginal zone lymphoma. Review of the original biopsy confirmed the presence of both diseases in the original lymph node.     

The histopathology of splenic lymphoma with villous lymphocytes

Whereas the hematologic, immunophenotypic, and molecular characteristics of splenic lymphoma with villous lymphocytes (SLVL) have been well documented, the histologic features of the spleen and lymph nodes remain uncharacterized. We have reviewed the histopathology of the spleen in 37 cases of SLVL and of involved splenic hilar lymph nodes in 6 cases. Tissue immunophenotyping was performed in 24 cases, 6 of which had frozen tissue available, and the results were compared with the membrane immunophenotype of the circulating villous lymphocytes and cells extracted from spleen and lymph nodes. In the spleen, SLVL is characterized by involvement of the white pulp follicles, which may be surrounded or replaced by the lymphoma cells. In the red pulp, the cells form small nodules and infiltrate diffusely with sinusoidal invasion. The cytologic appearance of the neoplastic cells varies from a resemblance to small mantle-zone--like lymphocytes to that of marginal-zone cells and there are scattered blast forms. In involved lymph nodes, the infiltrate again centers on the follicles that are usually replaced, but occasionally show preservation of follicle centers; sinuses are often preserved. The tissue immunophenotype is similar to that of marginal-zone B cells. Membrane immunophenotyping may give different results in some cases and may vary depending on the compartment from which the cells are obtained. SLVL in the peripheral blood is a histologically homogeneous entity identical to the condition previously characterised by histopathologists as splenic marginal-zone lymphoma.     

Radioautographic Study of Cellular Migration Using Parabiotic Rats

Leukocyte exchange between thehemopoietic tissues of parabiotic ratswas studied subsequent to giving multiple injections of ³H-thymidine to onemember of each pair while arrestingthe cross-circulation. Cell types thatmigrated from one parabiont to theother were segmented granulocytes,small, medium and large lymphocytes,immunoblasts, monocytoid cells, macrophages or their immediate precursors, and plasma cells. Evidence for thetransformation of circulating cells toother cell types was rarely seen. Thelong-lived small lymphocytes wereequilibrated between parabionts, suggesting that this is a single pool ofcells with respect to kinetic behaviorand recirculation. There was no evidence for a trephocytic function oflymphocytes. A small number of bonemarrow lymphocytes coursed directlyto lymph nodes and spleen. Evidenceis given for a limited recirculation ofshort-lived lymphocytes of thoracicduct lymph (TDL), as well as for long-lived cells. Only a few immunoblasts ofTDL recirculated. The majority of cellsthat entered the white pulp of thespleen were long-lived small lymphocytes, while the majority of immigrantcells to the red pulp were monocytoidcells and granulocytes. Many smalllymphocytes originated in splenic redpulp and entered the blood. No immigrant cells to the thymic cortex werenoted, although some small lymphocytes and monocytoid cells enteredthe medullary areas. Immigrant cells tothe bone marrow (less than 2% of thecells in marrow) included monocytoidcells, small lymphocytes, and plasmacells. Evidence for the direct transformation of a circulating cell into a committed blast, based on reduction ingrain count, was noted only in bonemarrow.

Submitted on April 14, 1971 Revised on August 9, 1971 Accepted on August 17, 1971     

Simian immunodeficiency virus-specific cytotoxic T lymphocytes and cell-associated viral RNA levels in distinct lymphoid compartments of SIVmac-infected rhesus monkeys

Major histocompatibility class I-peptide tetramer technology and simian immunodeficiency virus of macaques (SIVmac)-infected rhesus monkeys were used to clarify the distribution of acquired immunodeficiency syndrome virus-specific cytotoxic T lymphocytes (CTL) in secondary lymphoid organs and to assess the relationship between these CTL and the extent of viral replication in the various anatomic compartments. SIVmac Gag epitope-specific CD8(+) T cells were evaluated in the spleen, bone marrow, tonsils, thymus, and 5 different lymph node compartments of 4 SIVmac-infected rhesus monkeys. The average percentage of CD8(+) T lymphocytes that bound this tetramer in all the different lymph node compartments was similar to that in peripheral blood lymphocytes in individual monkeys. The percentage of CD8(+) T cells that bound the tetramer in the thymus was uniformly low in the monkeys. However, the percentage of CD8(+) T cells that bound the tetramer in bone marrow and spleen was consistently higher than that seen in lymph nodes and peripheral blood. The phenotypic profile of the tetramer-binding CD8(+) T lymphocytes in the different lymphoid compartments was similar, showing a high expression of activation-associated adhesion molecules and a low level expression of naive T-cell-associated molecules. Surprisingly, no correlation was evident between the percentage of tetramer-binding CD8(+) T lymphocytes and the magnitude of the cell-associated SIV RNA level in each lymphoid compartment of individual monkeys. These studies suggest that a dynamic process of trafficking may obscure the tendency of CTL to localize in particular regional lymph nodes or that some lymphoid organs may provide milieus that are particularly conducive to CTL expansion. (Blood. 2000;96:1474-1479)     

Thymus-Derived Lymphocytes in Blood,Lymph and Lymphoid Organs after Intrathymic Labelling with 3H-Thymidine

Guinea-pigs were investigated 2, 4 and 8 days after intrathymic or intraperitoneal labelling with tritiated thymidine. By the comparison between the content of lymphocytes in afferent and efferent thymic blood, an export of heavily labelled lymphocytes from the thymus was demonstrated in the intrathymically labelled animals after 2 and 4 days but not after 8 days. By use of liquid scintillation tritiated thymidine was localized to the spleen in a significantly higher amount 2 days after intrathymic than after intraperitoneal labelling. By autoradiography heavily labelled lymphocytes derived from the thymus were found in the spleen after 2 days and also to a lesser extent after 4 and 8 days, and in the mesenteric lymph nodes at all times after labelling. In the thoracic duct lymph the frequency of heavily labelled lymphocytes was the same as in the blood after intrathymic labelling, but less than in the blood after intraperitoneal labelling. The result indicates that thymus-derived lymphocytes are recirculating between blood and lymph.     

Changes in expression of murine leukemia virus antigens and production of xenotropic virus in the late preleukemic period in AKR mice.

We recently reported that thymocytes from 6-month-old preleukemic AKR mice express higher levels of murine leukemia virus (MuLV)-related antigens that thymocytes from 2-month-old mice. We have now found that the level of xenotropic MuLV (defined operationally as MuLV able to infect mink cell cultures) is also markedly increased in thymus of 6-month-old AKR mice and that this increase in virus correlates closely with increased MuLV-antigen expression. There is no increase of MuLV antigen or xenotropic virus in spleen or lymph nodes. Production of ecotropic MuLV remains unchanged with age in thymus, lymph nodes, and spleen. Thymic grafts from 6-month-old AKR mice, but not from 2-month-old mice, induce both amplified MuLV-antigen expression and xenotropic virus production in the thymus of young AKR recipients. Experiments with lethally irradiated AKR mice reconstituted with syngeneic bone marrow cells indicate that age-related changes in the thymus rather than in bone marrow precursor cells are responsible for MuLV-antigen amplification.     

Junin virus infection of guinea pigs: immunohistochemical and ultrastructural studies of hemopoietic tissue

An association between viral antigens, cytopathic effect (CPE) and viral titers in blood and lymphoid tissues suggests a direct CPE of Junin virus on the lymphopoietic organs of guinea pigs infected with 10(3) 50% lethal doses of the XJ prototype strain. After seven days of infection, all lymphoreticular organs had infectivity titers higher than those for blood. Virus was recovered from bone marrow and lymph nodes at day 5 after infection; peak titers were obtained from bone marrow, spleen, and lymph nodes after day 10. Granular specific fluorescence was detected in the cytoplasm of reticular monocytes after day 7; megakaryocytes showed positive fluorescence, but specific staining of other lymphoid cells was not observed. Necrosis of bone marrow, lymph nodes, and spleen was observed after day 9. CPE consisted of overdevelopment of reticuloendoplasmic cisterne of reticulomonocytes and myeloblasts. Typical Junin virus particles were observed. Reticular cells were gradually destroyed, and simultaneous necrosis of surrounding lymphoid cells was observed.     

Clonable progenitors committed to the T lymphocyte lineage in the mouse bone marrow; use of an extrathymic pathway

We searched for clonable committed T cell progenitors in the adult mouse bone marrow and isolated rare (approximately 0.05%) cells with the Thy-1hiCD2-CD16+CD44hiCD25-Lin- phenotype. In vivo experiments showed that these cells were progenitors committed only to reconstituting the T cell lineage of irradiated Ly5 congenic hosts. Reconstitution of the thymus was minimal compared with that of the bone marrow, spleen, and lymph nodes. At limiting dilutions, donor T cell reconstitution of the spleen frequently occurred without detectable donor cells in the thymus. Progenitors were capable of rapidly reconstituting athymic hosts. In conclusion, the clonable bone marrow progenitors were capable of T cell reconstitution predominantly by means of an extrathymic pathway.     

Lymphocytosis Induced in Mice by Supernatant Fluids of Bordetella pertussis Cultures: A Histopathological Study

Intravenous injection into mice of phase IBordetella pertussis culture supernatantsproduces a marked lymphocytosis. Theevolution of lymphocytosis was correlatedwith histopathological alterations in thelymphoid organs. The early phase, 17 hr today 3, was associated with massive depletion of both lymphocytes from the whitepulp of the spleen and of cortical thymocytes. There was a corresponding profounddecrease of splenic and thymic weights.The later phase was associated with amarked decrease in lymphocytes of lymphnodes while concomitantly the spleen andthymus appeared to be repopulating. Boththymic-dependent and thymic-independentlymphocyte populations were mobilizedfrom spleen and lymph nodes. There wasa striking diminution in the number oflymphocytes contained within the walls ofpostcapillary venules (PCVS) of all lymphnodes between 2 and 7 days. The datasuggest that initially the lymphocytosis isdue predominantly to release of spleniclymphocytes and to a lesser extent ofthymocytes into the circulation. Subsequently, the lymphocytosis is sustainedby the depopulation of the lymph nodes.Lymph node depopulation is maintained bythe failure of circulating lymphocytes toemigrate from the blood through thePCVS.

Submitted on January 26, 1973 Revised on April 2, 1973 Accepted on April 12, 1973