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     

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 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.     

Dynamic analysis of lymphocyte migration into Peyer's patches of rat small intestine

Dynamic aspects of lymphocyte immigration into Peyer's patches were analyzed in rat small intestine using intravital fluorescence microscopy. Lymphocytes from intestine, lymph, and spleen were labeled with acridine orange in vitro and infused from the mesenteric artery of rats. There are three types of lymphocytes as characterized from their interaction with post-capillary venules of Peyer's patches immediately after infusion, and most of the lymphocytes passed through the microcirculatory bed without any interaction with venules. About 15 to 20 minutes later, the number of lymphocytes which stick to the Peyer's patches was increased and reached a maximum at 40 to 50 minutes. There was a difference in distribution pattern of infused lymphocytes between spleen cells and lymphocytes from intestinal lymph suggesting the complex control mechanism of lymphocyte migration into Peyer's patches.     

Radioautographic Studies of Bone Marrow Lymphocytes in Vivo and in Diffusion Chamber Cultures

Radioautography with tritiated thymidine has been utilized to examinethe turnover rate and origin of small lymphocytes in the bone marrow of theguinea-pig.

Very few marrow lymphocytes were initially labeled by a single injectionof tritiated thymidine, but thereafter the number of labeled lymphocytesrapidly increased to high maximum levels at 3 days. Analysis of the labelingcurves and grain counts indicates that the population of marrow lymphocytesis maintained in a dynamic steady state with an average turnover time of3 days or less.

Suspensions of bone marrow cells were isolated from the circulation withinintraperitoneal diffusion chambers after short-term labeling with tritiatedthymidine in vivo. Although very few small lymphocytes were labeled whenintroduced into the diffusion chambers, a considerable percentage becamelabeled during the subsequent culture period.

Tritiated thymidine was also administered intravenously whilst excludedfrom one hind limb by the application of an occlusive compression bandagefor 20 minutes. Very few labeled small lymphocytes were found after 72hours in the tibial marrow of the initially occluded limb, whereas the normalhigh percentage was labeled in the control tibial marrow.

These experiments do not demonstrate any large-scale influx of smalllymphocytes from the blood stream into the marrow parenchyma. They suggest that newly formed small lymphocytes appear in the marrow as a resultof the division of locally situated precursor cells, but the mechanism of intramedullary lymphocytopoiesis is uncertain. "Transitional" cells, intermediatein morphology between blast cells and small lymphocytes, synthesize DNAand are actively proliferative, but they do not appear to account fully for therate of lymphocyte production.

Certain large, undifferentiated labeled cells appeared in the bone marrowas a result of hematogenous migration. Some implications of these findingsare discussed.

Submitted on May 21, 1963 Accepted on August 12, 1963     

Naive recirculating B cells mature simultaneously in the spleen and bone marrow

We have recently demonstrated that IgD(hi) B cells can occupy an extravascular perisinusoidal niche in the bone marrow in addition to the well-established follicular niche in conventional secondary lymphoid organs. The spleen has long been considered to be the site at which newly formed B lymphocytes mature into IgD(hi) naive recirculating B cells, but the existence of mutant mice that have selectively lost mature B cells in the bone marrow prompted an examination of B-cell maturation at this latter site. Following a single pulse of BrdU in intact mice, sequential labeling of more mature B-cell populations in the bone marrow suggested ongoing maturation at this site. Further evidence for B-cell maturation in the bone marrow was obtained from analyses of transitional B cells in splenectomized lymphotoxin alpha-deficient mice that lack all secondary lymphoid organs. In these mice, antibody-secreting cells recognizing multivalent antigens were also observed in the bone marrow following an intravenous microbial challenge. These data suggest that newly formed B cells mature into IgD(hi) B cells simultaneously in the spleen and the bone marrow and establish in a stringent manner that humoral immune responses can be initiated in situ in the bone marrow.     

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.     

Distribution of 51Cr labeled leukemia cells in mice: comparison with representative normal cells

Cells of two transplantable leukemias of mice, one myeloid and one lymphoid, were labeled with 51Cr in order to follow their distribution in hemopoietic and parenchymatous organs and blood of syngeneic recipients. Distribution of myeloid leukemia cells was compared with that of regenerating bone marrow cells and normal spleen cells. The organ distribution of myeloid leukemia cells was essentially different from that of cells of regenerating bone marrow, and both were different from that of normal spleen cells. Cells of lymphoid leukemia, which are presumably of B-lymphocyte origin, were compared with a B-lymphocyte enriched population, obtained from the lymph nodes of so-called TIR mice (thymectomized, irradiated, and reconstituted with syngeneic bone marrow), and with spleen cells of normal mice. The three patterns of organ distribution were different. It is concluded that the two leukemias studied each have a specific and characteristic distribution.     

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)     

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.     

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.     

A mouse model to study organ homing behaviour of haemopoietic progenitor cells reveals high selectivity but low efficiency of multipotent progenitors to home into haemopoietic organs

To study the homing behaviour of an enriched multipotent primitive haemopoietic progenitor cell (HPC) population in mice, undifferentiated murine factor-dependent multipotent HPCs (FDCP-mix), stably transfected with the green fluorescence protein gene, were intravenously injected into congenic mice. After 2 or 24 h, cell suspensions were prepared from bone marrow, spleen, lung, liver, muscle, colon, kidney, brain or blood of the mice and analysed by flow cytometry. Using direct quantifiable determination of total HPC numbers homed per organ and a method to estimate the degree of organ contamination by HPC that were present in blood vessels within the organs before preparation, the highest absolute numbers of HPC were detected in the liver and lungs at 2 h but this was sharply decreased at 24 h, whereas HPC selectively accumulated in the bone marrow and spleen at 24 h after transplantation. Only a few HPC were detected in other organs. The seeding efficiency of homed FDCP-mix HPC to the bone marrow and spleen was approximately 1.5% and ranged between that of primary whole bone marrow cells and lineage-depleted freshly isolated bone marrow cells. Pretreatment of HPC with inhibitors of signal transduction indicated that short-term homing of multipotent HPC into haemopoietic organs is an active process requiring co-ordinated intracellular signalling through Rho family small GTPases and protein kinases. Thus, short-term homing of FDCP-mix HPC into haemopoietic organs is of low efficiency but high selectivity, and provides a system to analyse the mechanisms and manipulation of primitive HPC which saves large numbers of donor animals.     

脾边缘带淋巴瘤的临床及肿瘤细胞特征的研究

目的 提高对脾边缘带淋巴瘤（SMZL）的认识和诊疗水平,方法 报告1例典型SMZL,外周血,骨髓及脾脏标本分别采用光镜,相差显微镜,扫描电镜,免疫组化染色,流式细胞术,G显带核型分析及PCR技术研究其肿瘤细胞的生物学特征。结果 本例患者肿瘤细胞为B淋巴细胞,不伴有绒毛,表达CD20,HLA-DR、CD45RA和bcl-2,无异常核型,肿瘤细胞主要浸润脾脏白髓致边缘带明显扩大,脾门淋巴结受累,骨髓和外周血与脾脏有相同的单克隆IgH重排基因,治疗7个月后转为多克隆重排,结论 脾大,外周血或骨髓淋巴细胞比例增高而无淋巴结肿大和白细胞增高患者应疑及SMZL,单克隆IgH基因重排有助于SMZL的诊断,对可疑病例应尽早切脾以明确诊断及防止恶性转化。     

Kinetics of Small Lymphocytes in Normal and Nude Mice after Splenectomy

Autoradiography and various quantitations on lymphoid tissues have been used to evaluate the kinetics of small lymphocytes in normal (+/nu or +/+) and congenitally athymic nude (nu/nu) NMRI mice 1 month after splenectomy or sham-splenectomy. The results indicate that splenectomy causes depressed thymic activity and diminished numbers of T lymphocytes in peripheral lymphoid tissues. The total number of cells in these tissues as well as the blast cell activity, were within normal limits. Bone marrow lymphocyte numbers and kinetics as well as blood lymphocyte levels in splenectomized and sham-splenectomized normal animals were comparable. Blood lymphocyte numbers were at normal levels in splenectomized nude mice, in spite of reduced numbers of bone marrow and thoracic duct lymphocytes. It is suggested that increased number of newly-formed lymphocytes, found in lymph nodes and blood of splenectomized mice, are released from the lympho-myeloid organs in compensation for the loss of long-lived, thymus-derived cells.     

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     

Differences of immunoglobulin secreting cells in bone marrow from those in circulation: relevance to their development

The surface markers of immunoglobulin secreting cells (ISC) in bone marrow and peripheral blood were analysed. Circulating ISC bear surface Ig and Ia-like antigens. However, these markers were not detectable on ISC in bone marrow. Fc and complement receptors were not present on circulating ISC. The areas of plaques corresponding to Ig secretion by bone marrow cells were always larger than those of peripheral blood cells. Although the majority of ISC were typical plasma cells, plasmacytoid lymphocytes were observed in peripheral blood. These findings seem to indicate that ISC in the peripheral blood are less advanced in their differentiation and maturation pathway from B lymphocytes to plasma cells than those in bone marrow. ISC in mesenteric lymph nodes exhibited nearly the same phenotype as peripheral cells.     

Simultaneous multiorgan presence of human herpesvirus 8 and restricted lymphotropism of Epstein-Barr virus DNA sequences in a human immunodeficiency virus-negative immunodeficient infant

Because a profound dysregulation of the immune system occurs in primary immunodeficiencies, viral infections are not uncommon. Human herpesvirus (HHV)-8 DNA was detected by polymerase chain reaction (PCR) analysis, Southern blotting, and in situ hybridization (ISH) in peripheral blood mononuclear cells and lymphoid organs (bone marrow, spleen, and lymph nodes) and endothelial and epithelial cells and macrophages from several organs (skin, lung, esophagus, intestine, choroid plexus [but not in brain or cerebellum], heart, striated muscle, liver, and kidney) of a human immunodeficiency virus-negative infant with DiGeorge anomaly who died of disseminated infection. Epstein-Barr virus DNA sequences were detected in the spleen and lymph nodes (by PCR and ISH) and in bone marrow (only by ISH) but not in blood or nonlymphoid organs. This report is believed to be the first of multiorgan dissemination of HHV-8 in a primary immunodeficiency.     

Distribution and differentiation of murine mast cell progenitors determined with soft agar culture

We have examined the distribution and differentiation of mast cell progenitors (mast-CFC) using a sensitive semisolid agar culture stimulated with STIL-3 (leukemic T cell line)-conditioned medium as a source of interleukin 3. The number of mast-CFC in the bone marrow of normal (+/+) mice was much higher than in previously reported data, although the number was almost the same in the spleen and peripheral blood as in previously reported data. Although mast-CFC were detectable in genetically anemic W/Wv mice as well, the concentration in the bone marrow was significantly lower than that of the +/+ mice. In the bone marrow there were more immature mast-CFC forming large-sized colonies (greater than 500 cells), whereas these were very few in the spleen and peripheral blood. Most mast-CFC were in the resting state. We conclude that mast-CFC differentiate to some extent in the bone marrow and then migrate in the peripheral blood to some organs where they proliferate and differentiate.     

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.     

Effect of thymectomy on tumor development and on T and B lymphocytes in tumor-bearing rats.

Tumor growth and changes in T and B lymphocyte ratio in spleen, draining lymph node and peripheral blood of thymectomized, irradiated rats, reconstituted with syngeneic bone marrow transplanted at various time intervals with MC-1 fibrosarcoma cells were followed. Control nonthymectomized or "sham" operated rats were transplanted an equal dose of tumor cells. Thymectomy and irradiation reduced the numbers of T lymphocytes in all lymphoid organs, while the enhanced numbers of B cells are probably related to reconstitution with cells of syngeneic bone marrow. The time interval between thymectomy, irradiation and transplantation of tumor cells proved to be a limiting factor for tumor growth and changes in T and B cell ratio. Early transplantation of tumor cells (7 days after irradiation) resulted in an enhanced resistance to tumor development, a reduced tumor growth rate and a progressing decline in the number of T cells. If the interval between thymectomy and tumor cell transplantation lasted 4 weeks, the T cell population became partially regenerated, and tumors grew progressively in correlation with a continuing T lymphocyte depletion. The results are discussed in terms of the role of various T cell subpopulations and the significance of residual, thymectomy- and irradiation-resistant T lymphocyte population, vital for a preservation of T cell immunological functions.