Prolonged effects of nitrogen mustard on mouse haematopoietic and lymphoid tissues.

Groups of mice have been autopsied at regular intervals during the period of lymphomyeloid tissue regeneration which follows the phase of hypocellularity induced by the i.v. injection of 100 mug (4 mg/kg bodyweight) nitrogen mustard. The marrow cellularity recovered to levels in the normal range by the 8th day and remained in this range up to the 40th day. Subsequently, the marrow showed a slight degree of hypocellularity up to day 120. Granulocytes were predominant during the initial phase of marrow regeneration during the second week post-injection. The thymus exhibited periodic size variations such that it was substantially larger than the thymus of age-matched controls from 20-30 d, 46-73 d, and 75-100 d after injection. The lymph nodes and lymphoid tissue of the spleen regenerated only slowly to reach control values by 40-50 d. Superimposed on the recovering lymphoid tissue of the spleen was a phase of erythroid hyperplasia lasting from 10-18 d post-injection. This coincided with a shift from granulocytosis to erythroid hyperplasia in the marrow. This erythroid hyperplasia lasted until day 30 when the cellular composition of the marrow and spleen returned to normal. A possible explanation of these results is that nitrogen mustard introduces a degree of synchrony into stem cell proliferation and differentiation. Additionally, these results emphasize the role of the haematopoietic microenvironment in the control of stem cell differentiation.     

Haematopoiesis in Busulphan-Treated Mice: A Comparison between Two Different Stem Cell Assays

The effect of busulphan on haematopoiesis was investigated in mice. Bone marrow and blood cells were sampled 2 h to 55 d after a single treatment with busulphan. The effect on progenitor cells was examined with the diffusion chamber (DC) technique and the spleen colony assay. Busulphan had a severe depressive effect on progenitor cells in bone marrow, and their number was reduced to 0.01–0.1 % of the control value on day 5–6. During the first 2 d the CFUs were reduced more than granulocyte progenitors as determined in the DC system. Histological examination of spleen colonies showed that in the same interval erythroid colony number was more depressed than granuloid colony number. This situation reversed after 4–6 d. Our interpretation is that busulphan affects all types of progenitor cells irrespective of their proliferative state. However, multipotent CFUs in G_o-phase are more depressed than granulocyte progenitor cells (DC), which have a higher proliferative rate. The selective effect on erythroid colonies during the initial period might indicate that busulphan impairs the ability of multipotent CFUs to erythroid differentiation. This damage was repaired within a few days. An abortive regeneration of bone marrow cells was observed on day 7–10. This can best be explained by an inflow from a cell pool intermediate between stem cells and early precursor cells, with some degree of ‘stem-cellness' (self-sustaining capacity).     

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.     

Animal model for immune dysfunction associated with adenosine deaminase deficiency.

An in vivo murine model for immunodeficiency of both B and T cells is produced by continuous intraperitoneal infusion of 2'-deoxycoformycin (DCF), a specific tightly binding inhibitor of adenosine deaminase (ADase; adenosine aminohydrolase, EC 3.5.4.4). After DCF infusion, ADase of thymus, spleen, and lymph nodes was inhibited to varying degrees ranging from 57% to 100%. Immunodeficiency under these conditions was indicated by: (i) a striking decrease in lymphocyte response to the T-cell mitogens concanavalin A and phytohemagglutinin; (ii) an impairment of delayed hypersensitivity measured by the footpad reaction; (iii) a decrease in antibody production measured in both in vivo and in vitro plaque-forming cell assay; (iv) a significant prolongation of mouse skin allograft survival after transplantation into the C57BL/6J (H-2b) strain of skin from BALB/c (H-2d) mice; and (v) a marked lymphopenia. Histological examination indicated lymphoid degeneration in the thymus, lymph nodes, and spleen with no alterations in other tissues including bone marrow, kidney, lung, gastrointestinal tract, and liver except for the occurrence of hepatitis. A decrease in the number of Thy-1-positive cells in both spleen and lymph nodes further supported the fact of cytotoxicity of DCF to T cells. Anorexia and weight loss were observed within 5 days of continuous DCF infusion at 0.4 mg/kg body weight per day. These data indicate that this method provides an experimental model for future studies on the biochemical mechanisms responsible for the genetically determined severe combined immunodeficiency disease in man.     

Inverse Relationship between Splenomegaly and Stem Cell Compartment Size in Mice Treated with Nitrogen Mustard

Following the administration of similar doses of nitrogen mustard (4 mg/kg) to different strains of mice, wide variations in the subsequent degree of splenomegaly were observed, implying strain differences in the role of the spleen in the compensatory erythropoietic response to haematopoietic stress. This investigation was undertaken to determine whether or not these differences were related to the size of the haematopoietic stem cell compartment size in the various strains of mice. Groups of 4 different strains of mice (Swiss Webster, A/J, C57BL/6J and CS1/ASH) were injected i.v. with nitrogen mustard (4 mg/kg body weight) and autopsied at regular intervals up to 20 d post-injection. At autopsy, the wet weight of the spleen was determined. Subsequently, groups of the same 4 strains of mice were exposed to single doses of wholebody γ-irradiation in the range of 500–900 rads. 9 d after γ-irradiation the mice were autopsied, their spleens removed, and the number of endogenous spleen colonies determined. The greatest degree of splenomegaly was observed in the C57bl/6J mice. The Swiss Webster mice showed no splenomegaly during the time period studied. There existed a linear inverse relationship between the maximum degree of splenomegaly observed and the dose of wholebody γ-irradiation required to completely eliminate endogenous spleen colonies. This data is in accord with the hypothesis that there exists an inverse relationship between the extent of splenomegaly observed following haematopoietic stress and the haematopoietic stem cell compartment size.     

Hematopoiesis of hereditarily asplenic-athymic (lasat) mice.

The hematopoiesis of athymic-asplenic (lasat) mice was compared with that of normal, asplenic, and athymic littermates with the same strain background. Erythrocyte blood volume, number and survival time were normal when related to the body weight of the animals. Peripheral blood showed leukopenia with absolute and relative lymphopenia, resembling the athymic rather than the asplenic pattern. The bone marrow was hypocellular as a consequence of a decrease in both lymphocytes and erythroid precursors, while thrombocytopoiesis and granulcytopoiesis-monocytopoiesis were essentially normal. Although the percentile value of femoral stem cells was high, their absolute number was, in fact, reduced by 35% as a result of the bone marrow hypocellularity. When lasat bone marrow cells were injected into normal, lethally irradiated mice, a rapid erythropoietic recovery was observed, whereas the restoration of the granlocytic compartment was impaired. It was concluded that: 1) lasat mice depict a normal hematopoiesis in spite of the congenital absence of the thymus and the spleen; 2) bone marrow stem cells may be defective when administered to lethally irradiated hosts; and 3) the athymic status predominates over the asplenic one.     

Stem Cell Migration and Proliferation During Severe Anemia

The pluripotential stem cell (CFU) compartment of marrow and spleen wasevaluated in mice subjected to an intense erythroid stimulus associated withphenylhydrazine-induced anemia. Erythroid hyperplasia occurred in both marrow and spleen. CFU in the marrowgradually declined to approximately 50per cent of control levels (day 5) whiletheir numbers in the spleen increased(fourfold) by day 3 and were maintainedat this level for several days. Thesechanges in numbers of marrow andsplenic CFU were not associated withCFU proliferation. Thereafter, CFU inthe marrow, but not in the spleen, entered active cell cycle. The data suggestthat CFU migrate from marrow to spleenduring the demands of severe anemia.The induction of marrow CFU into cyclefurther suggests a negative feedback,which, perhaps through cell-cell interaction, maintains stem cells at a criticalcompartment size. The failure of splenicCFU to cycle may reflect the converseeffect, i.e. an inhibition on stem cell proliferation in the wake of an expandedstem cell pool.

Submitted on March 17, 1970 Revised on May 14, 1970 Accepted on June 9, 1970     

Tissue Localization of Lymphocytes Bearing a Membrane Receptor for Antigen-Antibody-Complement Complexes

To determine the tissue localization of lymphocytes provisionally termed "complement-receptor lymphocytes," which are characterized by having a membrane receptor for antigen-antibody-complement complexes, we investigated the adherence of sensitized and nonsensitized sheep red cells to frozen sections of mouse lymphoid organs. Nonsensitized erythrocytes became bound exclusively to sinus-lining cells of spleen and lymph nodes, whereas erythrocytes sensitized with antibody and complement adhered to lymphocytes in the follicular areas and the marginal zone of the spleen and in the true cortex of lymph nodes. However, the doubly sensitized erythrocytes failed to bind to the "thymus-dependent" areas of peripheral lymphoid organs or to the thymus itself. We suggest that complement-receptor lymphocytes are of extrathymic origin and that they contribute substantially to follicular antigen localization, which appears to be complement-dependent.     

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.     

HEMATOPOIESIS IN PANTOTHENIC ACID-DEFICIENT RATS

Rats fed a purified diet low in pantothenic acid developed granulocytopeniaand anemia singly or in combination. In the former, the marrow showed markeddepletion of granulocytes, particularly of the more mature cells, and a slight increase in erythroid cells. In combined granulocytopenia and anemia the granulocytes of the marrow were still further reduced and the erythroid cells were alsodepleted. Marked reduction in the number of megakaryocytes occurred both inthe granulocytopenic and in the granulocytopenic and anemic rats. Purpura wasnoted grossly in about 25 per cent of the rats of both groups. In anemia withoutaccompanying granulocytopenia the marrow granulocytes showed slight tomoderate depletion, whereas the erythroid index (mean) was not significantlylowered. Megakaryocytes were moderately reduced.

Lymphoid tissue—spleen, thymus, and cervical nodes—showed atrophy ofvariable degree, most marked in the thymus.

Adrenal glands showed marked depletion of cortical lipoids and rarely hemorrhage and necrosis.

Following treatment with combined folic acid, pantothenic acid, and niacinamide, granulocytopenic rats responded by showing a prompt rise in lymphocyteand polymorphonuclear leukocyte count, marked granulocyte response of thebone marrow, increased splenic hematopoiesis, lymphoid hyperplasia, and increased lipoid content of the adrenal glands.

     

The organization of lymphoid tissue in relation to function.

Organized lymphoid tissue is found in the thymus, spleen, lymph nodes; lining the respiratory and alimentary tracts; and also occurring at sites of chronic inflammation. Apart from the thymus which is involved in the regulation of T-cell function, the other tissues are organized into T-cell and B-cell areas. Lymphocytes in T-cell areas respond by proliferation in cell-mediated immunity and by the production of suppressor cells and helper for antibody formation. B-cell areas are involved in the humoral antibody response. B-cells are segregated into lymph follicles where they form germinal centers and are found at the corticomedullary junction where they differentiate into plasma cells. The role of lymph follicles in becoming germinal centers is poorly understood, but these areas are known to be the site of antigen trapping in primed animals. The particular function of the spleen as a localized area of lymphoid tissue along the course of the blood vascular system is discussed, particularly with respect to its ability to respond to soluble antigen released from sites of localized antigen deposition such as tumors.     

Lymphoproliferative changes induced by infection with a lymphotropic herpesvirus of guinea pigs

The effects of experimental infection with guinea-pig herpes-like virus (GPHLV) on hematologic and histopathologic parameters were studied in Hartley strain guinea pigs inoculated intraperitoneally with 10(3) or 10(6) 50% tissue culture infectious doses of GPHLV. The total leukocyte count, the percentage of cells that were mononuclear, the number of spontaneous rosette-forming cells, and hematocrit values were determined after various intervals. Cells from spleens, cervical lymph nodes, and bone marrow of animals killed at various times were assayed for infectious virus, and tissues were prepared for histological examination. Spleen-to-body weight ratios were also determined. GPHLV infection resulted in a mild, asymptomatic, transient lymphoid hyperplasia. The infected animals experienced a relative and absolute peripheral-blood lymphocytosis lasting for seven to eight weeks. The spleens showed evidence of immune stimulation for four to six weeks. Despite the transient nature of the histological and peripheral-blood changes, virus was recovered by cocultivation from the spleen, bone marrow, and lymph nodes for up to eight months (the time of the last test). Infectivity titers were highest in the B-lymphocyte subpopulation during latent infection. These findings of GPHLV persistence after a mild, acute mononucleosis-type syndrome were compared with observations during human Epstein-Barr viral infection.     

The hematopoietic stem cell compartments in mice during and after long-term inhalation of three doses of benzene

Female BDF1 mice were exposed for 16 weeks to airborne concentrations of 100, 300, and 900 ppm of benzene, 6 h per day, 5 days per week. Bone marrow hemopoietic stem cell compartments and peripheral blood cell counts were studied using clonal assays and standard methods. Dose-dependent depressive effects were observed on all stem cell compartments. Only the erythroid colony-forming units (CFU-E) compartment was depressed during exposures to 100 ppm; CFU-E were more sensitive than the erythroid burst-forming units (BFU-E), spleen CFU (CFU-S), or G-M CFU (CFU-C) during exposure to 300 ppm or 900 ppm. Lymphocytopenia was observed in the peripheral blood. After benzene-free intervals, a regeneration of lymphocyte numbers and slow normalization of stem cell numbers was seen. Complete recovery from the 16 weeks exposure to 300 ppm was seen between 73 and 185 days.     

Some Factors Involved in the Effect of X-Irradiation on the Phosphatase Activity of Hematopoietic Tissues

Factors which modify lymphoid distribution of tissues were found to modifythe adenosine triphosphatase activity of these tissues. Starvation or cortisoneinjection, which produces destructive changes in lymphoid tissues, was foundto increase the enzyme activity of spleen and thymus tissues. The greater increment of enzyme activity of the thymus as compared to that of the spleenwas correlated with its normally higher content of lymphoid tissue.

The increase in adenosine triphosphatase activity of hematopoietic tissuesappears to be associated with the type of cells present in the assay medium.With respect to peripheral blood leukocytes of the rat, the cell type is confined largely to lymphocytes and granulocytes. The increase in adenosinetriphosphatase activity of the leukocytes after total-body x-ray was seen toparallel the increase in granulocytes present in the assay medium. The ratioof granulocytes to lymphocytes is not appreciably altered in dog peripheralblood after exposure to total-body x-ray; the adenosine triphosphatase activitysimilarly was not significantly altered. After total-body x-ray (390 r and 780 r),cells isolated from the rat bone marrow displayed a fivefold increase inadenosine triphosphatase activity. This increase was seen to correspond withan increase in the ratio of segmented leukocytes and reticuloendothelial cellsand a decrease in the immature forms of the erythroid and myeloid cells.

The heterogeneous cell mixtures used for our assay procedures permit theobservation that total-body x-irradiation results in an increased enzyme activityof the isolated cells of the peripheral blood, bone marrow and spleen tissue ofthe rat. The increased enzyme activity was associated with the increased ratioof cells with high enzyme activity present in the assay medium.

Submitted on September 22, 1958 Accepted on November 27, 1958     

THE THYMUS: EXPERIMENTAL PHYSIOLOGY,AND PATHOLOGY IN MAN

The thymus is an integral part of the immunological system. It is a site of intense lymphopoiesis, especially in early life. Neonatal thymectomy in mice causes runting and death due to gross immunological deficiencies. These deficiencies are determined by lymphopenia, and by lack of a lymphotrophic hormone secreted by the epithelial cells of the medulla; this hormone confers on lymphocytes the capacity to respond to antigenic stimulation. The thymus may be the main source of lymphoid cells carrying new or primary patterns of immune reactivity; it is thus “first-level” or “central” lymphoid tissue, which seeds cells to “second-level” or “peripheral” lymphoid tissues in the lymph nodes and spleen. Pathological lesions of the thymus in man include aplasia, hyperplasia, dysplasia and neoplasia. Gross aplasia characterizes the immunological deficiency diseases of infancy, including the lymphopenic type of congenital agammaglobulinæmia. Hyperplasia accompanies thyrotoxicosis. Dysplasia refers to the lymph follicle-germinal centre development in myasthenia gravis, probably an autoimmune disease, and to the proliferation in the medulla of spindle-epithelial cells in lupus erythematosus, an autoimmune disease. Neoplasia occurs as benign thymoma, which may be accompanied by extrathymic diseases which are possibly autoimmune in origin; these include myasthenia gravis, red cell aplasia, polymyositis, agammaglobulinæmia and lupus erythematosus. These diseases may in some way be caused by the thymoma; alternatively, the thymoma may represent the result of continuing hyperplasia of the thymus provoked by a primary autoimmune process. The place of thymectomy in the treatment of autoimmune disease is discussed. It is an established procedure in myasthenia gravis, and has been successful in two cases of autoimmune hæmolytic anæmia in infancy. We review our experience with thymectomy for three patients with systemic lupus erythematosus.     

Oncostatin M transforms lymphoid tissue function in transgenic mice by stimulating lymph node T-cell development and thymus autoantibody production

Oncostatin M (OM) is a member of the IL-6 subfamily of cytokines that is expressed in primary lymphoid tissues such as bone marrow and thymus, as well as in secondary lymphoid tissues and activated leukocytes. We produced transgenic mice that overexpressed the human, bovine, or mouse OM genes and compared their relative ability to modulate lymphopoiesis. Each species of cytokine induced a similar extrathymic pathway of T-cell development involving the accumulation of immature T cells within lymph nodes. Reconstitution experiments utilizing lethally irradiated athymic mice indicated that OM had caused hematopoietic precursors within fetal liver and bone marrow to initiate lymph node T-cell development in the absence of a thymic environment. Breeding experiments with IL6-/- and IL-7r(alpha)-/- deficient mice, indicated that induction of this extrathymic pathway by the OM transgene occurred in the absence of IL-6, but was strictly dependent on IL-7 receptor signaling. Separately, OM stimulated the accumulation of immature B cells within the transgenic thymus and caused the subcapsular regions of the thymus to expand with mature B and T cells. This thymus conversion to secondary lymphoid tissue was responsible for a lethal autoimmune-like disease marked by high titers of circulating autoantibodies, proteinuria, and glomerulonephritis. The conserved phenotypes elicited by these three forms of OM indicate that this potent hematopoietic cytokine can regulate lymphoid tissue function and morphogenesis.     

Prolactin receptor expression by lymphoid tissues in normal and immunized rats

Prolactin receptor (PRLr) expression and distribution in thymus, spleen, bone marrow, lymph nodes, and peripheral blood lymphocytes from young adult Lewis rats are analyzed using single-color flow cytometry and a well-characterized monoclonal antibody directed against the rat liver PRLr. The in vivo effects of regional immunization on PRLr expression are also examined. PRLr is found to be widely distributed among cells of the immune system and demonstrates lymphoid tissue-specific patterns of expression. Footpad immunization caused the rapid, but transient, induction of PRLr expression in the draining lymph node, with only modest effects on PRLr expression in other distant lymphoid tissues. These studies indicate that PRL may be capable of direct interaction with the immune system through differential expression of the PRL cell surface receptor on select lymphoid target cell populations.     

On the Origin of Foà-Kurloff Cells

The thymic and splenic release of Foà-Kurloff cells into the blood was studied in estradiol-treated male guinea pigs by comparison between the cellular content in afferent and efferent blood. The amount and distribution of such cells in thymus, spleen, lymph nodes, and bone marrow was investigated. The treatment with estradiol caused involution of the thymus and splenomegaly. An abundance of Foà-Kurloff cells was found in the red splenic pulp and a considerable release of such cells from the spleen into the blood was demonstrated. At the same time the output of lymphocytes from the spleen was reduced, suggesting that the Foà-Kurloff cells are transformed lymphocytes. The spleen contained an increased amount of erythroblasts, indicating a stimulation of splenic erythropoiesis by estradiol. In the bone marrow and the thymus the number of Foà-Kurloff cells was much smaller than in the spleen and no emigration of such cells from the thymus into the blood was demonstrated. A very small amount of Foà-Kurloff cells was found in the lymph nodes and very few occurred in the thoracic duct lymph. Thus, the Foà-Kurloff cells of the blood do not originate in the lymph nodes and do not recirculate between blood and lymph. It is concluded that the spleen is the major producer of Foà-Kurloff cells and that they are released from the spleen into the blood.     

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)     

A transplantable myeloid hamster leukemia with high peripheral leukocyte counts and C-particles. II. Histology and cytochemistry (author's transl)]

Results on morphologic and hematologic characterization of a hamster leukemia capable of both cellular and cellfree transmission are described. Solid tumors removed in the course of several passages of leukemia animals, after producing blood smears, and spleen, liver, lymph nodes, bone marrow, thymus, kidney and lung were investigated histologically and histochemically. The morphological picture of the hamster leukemia has not changed during several transplantation generations. In addition to solid tumours, typical leukemic infiltration were detected histologically in spleen, liver, lymph nodes, bone marrow, kidneys and lung. No leukemic proliferation was noticed in the thymus. The final stage of the disease is characterized by an abrupt occurrence of high leukemic cell counts. The demonstration of alkaline phosphatase, but especially of naphtol-AS-D-chloroacetate esterase, in the leukemic cells is interpreted as indicating malignization of cells of the granulocytic line.