Ontogenetic development of lipopolysaccharide-reactive B cells against bromelain-treated mouse erythrocytes in mouse lymphoid tissues

In lymphoid tissues of mice, there exist LPS-reactive B cells which can differentiate to IgM-secreting plaque-forming cells (IgM-PFC) and PFC secreting antibodies against bromelain-treated mouse erythrocytes (BrMRBC) by LPS activation. In this study, four groups of LPS-reactive B cells in spleen, peritoneal cavity and mesenteric lymph nodes from 2- and 10-week-old mice were compared and enumerated as precursors of IgM-PFC and anti-BrMRBC PFC on days 1 and 2 after LPS activation in quantitative culture conditions. The induction of each of four PFC responses in peritoneal cells was sensitive to LPS and anti-mouse IgM antibodies as much as the induction of the respective PFC response in spleen cells. The ratios of four groups of PFC to each other were different among three tissues and between two ages. These findings support the view that the four groups of LPS-reactive B cells in each tissue are mostly in distinct subpopulations of B cells from each other, and the respective groups of different lymphoid tissues at different ages belong to the same subpopulation.     

Immunoglobulin-containing cells in different lymphoid organs of the CBA mouse during its life-span.

The number of cells containing cytoplasmic immunoglobulin (C-Ig cells) was determined in the spleen, mesenteric lymph nodes, bone marrow and Peyer''s patches of CBA mice of different ages. A rapid increase in the number of C-Ig cells at between 2 and 6 weeks of age was observed in spleen and gut-associated lymphoid organs. The absolute number of C-Ig cells in these organs decreases with advancing age. In the bone marrow, the number of C-Ig cells increases steadily with age up to one year. From one year on, the number remains approximately constant in the males. In female mice, the number of C-Ig cells, mainly of the IgA class, increases sharply around 1 year of age. The spleen is the major site of Ig synthesis up to about 6 months of age. In older animals, the relative contribution of the bone marrow increases with age, possibly due to a gradual shift in the individual animal from primary type responses to a pattern of secondary type responses. No indication of a decreased overall immunological activity in senescence was obtained.     

Effects of cyclosporin A on mouse lymphoid tissues.

Cyclosporin A (CsA) was administered s.c. to BALB/c and C3H mice for periods up to 25 days and the effects were monitored by the immune response to SRBC, HGG, and B. abortus. Whereas the rate of growth of BALB/c mice was only depressed after the 20th day, C3H mice lost weight from the start of treatment. In both strains there was no change in the weight of the spleen and mesenteric lymph node but the thymus weight decreased markedly. This decrease in thymus weight was accompanied by thymic involution with little or no change in spleen or mesenteric lymph node. An increase in Mott cells, as so strikingly demonstrated in the CsA-treated chicken (Nowak et al., 1982), was dependent on the degree of thymic involution. When the thymus was normal in appearance there was a slight increase in the number of splenic Mott cells, when compared with untreated controls; in the more usual case of thymic involution (or necrosis), there was no increase in the number of Mott cells.     

Electrophoresis of lymphoid cells. Characterization of human B and T cells in peripheral lymphoid tissues

Small lymphocytes from human blood, tonsils, spleen and lymph nodes showed a bimodal distribution on electrophoresis, the electrophoretic mobility (EPM) of one group being slow, the other fast. The difference in mean EPM between the slow and fast groups was about 30%. The percentage of slow cells in each organ was similar to that of cells with surface-bound immunoglobulin as judged by immunofluorescence studies. It was therefore concluded that a major proportion of B cells has a relatively low net surface charge while that of the T cells is higher.     

Effects of cyclosporin A on mouse lymphoid tissues

Cyclosporin A (CsA) was administered s.c. to BALB/c and C3H mice for periods up to 25 days and the effects were monitored by the immune response to SRBC, HGG, and B. abortus. Whereas the rate of growth of BALB/c mice was only depressed after the 20th day, C3H mice lost weight from the start of treatment. In both strains there was no change in the weight of the spleen and mesenteric lymph node but the thymus weight decreased markedly. This decrease in thymus weight was accompanied by thymic involution with little or no change in spleen or mesenteric lymph node. An increase in Mott cells, as so strikingly demonstrated in the CsA-treated chicken (Nowak et al., 1982), was dependent on the degree of thymic involution. When the thymus was normal in appearance there was a slight increase in the number of splenic Mott cells, when compared with untreated controls; in the more usual case of thymic involution (or necrosis), there was no increase in the number of Mott cells.     

B and T cells in lymphoid tissues of human appendix.

Human appendix lymphoid cells (HAL) react very strongly to stimulation with concanavalin A, strongly to stimulation with phytohaemagglutinin, and weakly but definitely to stimulation with lipopolysaccharide. From the results of rosette formation assay, cytotoxicity tests with anti-T cell antiserum or anti-B cell antiserum, cell surface or intracellular immunoglobulin staining with fluorescein-conjugated rabbit anti-Fab of human immunoglobulin serum, and plaque-forming cell (PFC) assay, it was concluded that human appendix lymphoid tissue is a B cell pool but includes T cells. However, both direct and indirect PFC could not be significantly demonstrated against sheep red blood cells in a 5-day HAL culture.     

The distribution of Helix pomatia lectin receptors on mouse lymphoid cells and other tissues

The distribution of receptors for the Helix pomatia lectin on mouse lymphoid cells and other tissues was investigated. Using a sensitive resetting assay combined with immunofluorescence, lectin receptors were found on the membrane of approximately 90% of peripheral T lymphocytes, 75% of thymocytes, 30% of bone marrow cells, 20% of nude spleen cells, 15–50% of peritoneal exudate macrophages, and a subpopulation of peritoneal exudate mast cells. The Thy-1-positive nude spleen cells were predominantly Helix lectin receptor-negative. Approximately 5% of B lymphocytes were weakly positive, and neutrophils were negative. Receptors were present also on a subpopulation of cells of a fibroblast cell line and in acetone powder from the liver and, at a lower level, from the kidney and brain. Membrane receptors on all cell types were partially detectable without neuraminidase treatment of the cells. Two methods of fractionating Helix lextin-positive cells were employed, which gave significantly different results. By rosetting and depletion using density fractionation, T cell mitogen responses were abolished, while B cell mitogen responses, T cell cytotoxicity, and natural killer cytotoxicity were only slightly affected, if at all. Helix lectinagarose column fractionation seemed more sensitive, in that essentially all natural killer cells bound to the column, as well as a considerable number of B lymphocytes. Cytotoxic T cells were heterogeneous: roughly half were not bound, but the remainder were bound and eluted.     

Invasion of collagen gels by mouse lymphoid cells.

Small mouse lymphocytes from lymph nodes rapidly invaded three-dimensional collagen gels (in the absence of any added chemical attractant). In short-term assays (2-8 hr) this property was restricted to 20-25% of the cell population. Invasion was an active process involving cell locomotion. Time-lapse cinematography revealed that movement was erratic with frequent changes in cell speed. Tracks of cell paths within collagen gels demonstrated that lymphocytes made narrow angles of turn and thus showed a 'persistent random-walk' similar to other cell types moving on plane substrata. Analysis of lymphocyte movement within aligned collagen gels demonstrated that locomotion was biased in the axis of fibre alignment, i.e. lymphocytes showed contact guidance. Separated B lymphocytes invaded collagen gels at a slower rate than unseparated lymph node cells, as also did T cells purified by filtration through nylon wool columns. This latter anomaly implied that nylon wool filtration selectively depleted cells with invasive characteristics from a heterogeneous lymphocyte population. A comparison of Peyer's patch and lymph node lymphocytes showed that both populations invaded at the same rate but the latter cell type did this in greater numbers. This difference may reflect the different proportions of B and T lymphocytes in the two tissues. Lymphocytes from oxazolone-stimulated lymph nodes showed greatly increased movement into collagen matrices compared to unstimulated control lymph node lymphocytes. This increase was demonstrated to be a property of the blast cell population by separating the cells on Percoll gradients into lymphoblast-enriched and -depleted populations.     

Reticular cells in peripheral lymphoid tissues express the phosphatidylinositol-linked BP-3 antigen.

The murine BP-3 antigen was initially described as a variably glycosylated cell surface protein of Mr 38,000 to 48,000 on lymphoid and myeloid cells. In the present experiments we found that this antigen is released from the surface of pre-B cells and macrophages by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting a glycosyl-phosphatidylinositol (GPI) linkage with the plasma membrane. When the tissue distribution of the BP-3-reactive cells was examined by immunohistology, high levels of the antigen were observed on brush borders of the intestinal epithelial cells, within collecting tubules of the kidney and on a subpopulation of reticular cells located on lymph nodes. Peyer's patches and the white pulp areas of the spleen. In contrast, reticular cells located in the thymus, bone marrow and splenic red pulp did not express the BP-3 antigen. Ontogenic studies revealed that BP-3 was expressed by the reticular cells in peripheral lymphoid tissues in the neonatal period near the time of lymphocyte immigration into these organs. BP-3+ reticular cells were observed in the collapsed periarterial lymphatic sheaths of adult mice depleted of T and B cells by cyclophosphamide treatment and in mice with severe combined immunodeficiency (scid), indicating that development of this reticular network is lymphocyte independent. The BP-3 antigen on the splenic reticular cells was also GPI anchored but its glycosylation pattern differed from that of the BP-3 molecules on pre-B cells. A specific subpopulation of reticular cells is thus marked by the BP-3 antigen, and the distribution and biochemical properties of the molecule make it an attractive candidate for a role in lymphocyte-stromal interactions in the peripheral lymphoid tissues.     

Heterogeneity of lymphoid tissue inducer cell populations present in embryonic and adult mouse lymphoid tissues

Lymphoid tissue inducer (LTi) cells have a well established role in secondary lymphoid tissue development. Here, we report on the heterogeneity of LTi cells based on their CD4 and chemokine receptor expression. The CD4⁻ LTi-cell population has a similar phenotype to the CD4⁺ population, with similar chemokine-receptor-expressing subsets. In both embryonic and adult spleen the CD4⁻ LTi-cell population is comparable as a proportion of total splenocytes to its CD4⁺ counterpart. In contrast, different proportions of CD4⁺ and CD4⁻ LTi cells are found in different lymph nodes. Both CD4⁺ and CD4⁻ LTi cells share the anatomical location and are associated with vascular cell adhesion molecule-1-positive stromal cells in spleen and lymph nodes. The numbers of both CD4⁺ and CD4⁻ LTi cells in adult spleen are augmented in the presence of B cells. With the exception of CD4, there is a strong correlation coefficient (0·89) for gene expression between the two populations. Polymerase chain reaction analysis of individual CD4⁺ and CD4⁻ LTi cells shows that a similar proportion in embryonic and adult spleen co-expressed both CXCR5 and CCR7 or CXCR5 alone: 84·6% for adult CD4⁺ and 87·6% for adult CD4⁻; 95·3% for embryonic CD4⁺ and 91·5% for embryonic CD4⁻. Consistently fewer CCR7 single-positive cells were found in the CD4⁺ and CD4⁻ fractions in the embryo.     

Selective effects of cholera toxin on the activation of mouse B cells by different polyclonal activators

Murine B cells were stimulated in vitro with anti-immunoglobulin (Ig) antibodies, lipopolysaccharide, or with various combinations of phorbol dibutyrate and ionomycin. Very low concentrations (ca. 10(-14) M) of cholera toxin inhibited anti-Ig-stimulated DNA synthesis, while the response to LPS was only abrogated by 2 X 10(4)-10(5)-fold greater concentrations of the toxin. Earlier responses in anti-Ig-stimulated B cells, such as increases in Ia antigen levels, were not affected by the toxin. Protein kinase C-activating phorbol esters, together with Ca2+ ionophores, are believed to stimulate DNA synthesis in lymphocytes by mimicking the two second messengers resulting from ligation of the antigen receptors. However, concentrations of cholera toxin which totally abolish anti-Ig-induced B cell proliferation significantly enhanced DNA and RNA synthesis induced by phorbol dibutyrate plus ionomycin. The results are discussed in terms of possible effects of cholera toxin on guanine nucleotide-binding (G) proteins controlling receptor coupling to second messenger-generating systems in B cells.     

Responsiveness of lymphoid precursors to polyclonal B-cell activators.

We have shown previously that in the differentiation of fetal liver cells to mature B cells in irradiated hosts, these cells sequentially gain responsiveness to the polyclonal B-cell activators dextran-sulphate (DxS), lipopolysaccharide (LPS), and purified protein derivative from tuberculin (PPD), in that order. In this paper we show that both fetal liver cells and adult bone marrow cells responded with proliferation to DxS, but not to LPS OR PPD. However, neither fetal liver nor bone marrow cells gave rise to detectable numbers of high-rate antibody-secreting cells on short-term stimulation by polyclonal B-cell activators. The lack of LPS and and PPD responses of fetal liver and bone marrow cells could not be ascribed to the presence of inhibitory cells, and the DxS-induced response in these cell populations was not dependent on adherent cells. However, LPS could inhibit the DxS response of fetal liver cells, possibly indicating that DxS-responsive cells are precursors to B cells. Direct evidence was provided that DxS activated B-cell precursors in bone marrow. Thus, this cell population became responsive to LPS after DxS prestimulation, as measured by DNA synthesis. Bone marrow cells, sequentially stimulated with DxS and LPS, contained increased numbers of cells with surface immunoglobulin, although no significant increase in numbers of antibody-secreting cells was obtained. These data indicate that DxS had the capacity to increase the rate of differentiation of B-cell precursors. Finally, we show that the sequential appearance of responsiveness in B-cell differentiation to polyclonal B-cell activators is not due to lack of accessory cells during early stages in maturation.     

L-selectin expression differentiates T cells isolated from different lymphoid tissues in cattle but does not correlate with memory.

L-selectin (LECAM-1, LAM-1) was expressed by a high proportion of CD4+ and CD8+ T cells, as well as almost all of the gamma delta T-cell receptor (TcR)+ (WC1+) T cells, isolated from blood, lymph nodes or tonsils. CD4+ T cells in the lamina propria of the gut villi and CD8+ T cells in the villous epithelium as well as the majority of WC1+ T cells in the gut mucosa were L-selectin-. The proportion of T cells from Peyer's patches that synthesized the molecule was intermediate between the value for blood and gut mucosa. Expression of L-selectin therefore marks T cells in cattle with a distinct tissue distribution that correlates with its function as the peripheral node homing receptor. The proportion of CD4+ and CD8+ T cells in the circulation that were L-selectin+ decreased with age. Unlike CD45R, expression of L-selectin was not related to CD4 T-cell memory as judged by proliferation in transformation assays to soluble antigen. Three-colour immunofluorescent staining demonstrated four subpopulations of CD4 and CD8 T lymphocytes in peripheral blood mononuclear cells (PBMC) that were CD45R+, L-selectin+; CD45R+, L-selectin-; CD45R-, L-selectin+; CD45R-, L-selectin-. CD4(4) memory cells were CD45R- and L-selectin+ or L-selectin-. Taken with earlier studies the reported observations demonstrated that only one of the four phenotypes of the CD4+ T cells in blood is present in the lamina propria of the gut villi and these are CD45R-, L-selectin-. Two of the four phenotypes of CD8+ T cells were present in the gut epithelium; these were CD45R+, L-selectin- or CD45R-, L-selectin-. Expression of the bovine molecule was not rapidly down-regulated on T cells following activation by exposure to phorbol myristate acetate.     

Effect of cyclic nucleotides on DNA synthesis in mouse lymphoid cells.

The effect of eight cyclic purine and cyclic pyrimidine nucleotides on DNA synthesis on mouse lymphoid cells was investigated. Two out of eight compounds tested, namely 2', 3'-cyclic guanosine monophosphate (2', 3'-cGMP) as well as 3', 5'-cyclic guanosine monophosphate (3', 5'-cGMP), stimulate thymidine incorporation in all types of lymphocytes tested. The stimulatory activity of the cyclic guanosine nucleotides as well as the effects of lectins could be antagonized by 3', 5'-cyclic adenosine monophosphate (3', 5'-cAMP). 2', 3'-cGMP seems to stimulate preferentially mature T-cells while 3', 5'-cGMP preferentially acts on B-cells.     

Self-reactive T cells are present in the peripheral lymphoid tissues of cyclosporin A-treated mice.

Cyclosporin A (CsA) prevents most immature thymocytes from progressing to a mature phenotype and blocks the deletion of T cells that express self-specific TCR in the small population of cells that achieve maturity. The latter effect may explain the paradoxical observation that this immunosuppressive drug can induce autoimmunity in irradiated rodents and humans if administered while new T cells are developing in the thymus. This study shows that the repopulation of the periphery with mature T cells is delayed in irradiated CsA-treated mice, presumably because CsA blocks T cell development in the thymus. The peripheral repertoire of these mice contained self-reactive IL-2 producing T cells that could be detected in a sensitive limiting dilution assay. In addition, self-reactive T cell hybridomas were isolated from the IL-2 receptor+ population present in CsA-treated mice. One of these hybridomas expressed a TCR V beta chain that is normally expressed on thymocytes that are deleted via recognition of self-antigens. Despite the presence of self-reactive T cells that had escaped clonal deletion, CsA-treated mice rarely developed lethal autoimmune disease, implying that a peripheral mechanism of tolerance can prevent the onset of autoimmune disease.     

Development of B-lymphocyte colony-forming cells in foetal mouse tissues.

In CBA and (CBA X C57B1)F1 mice, cells forming B-lymphocyte colonies in agar culture were first detected in the 17-day foetal liver and the following day in the spleen, bone marrow and peripheral blood. Colony-forming cells were not detected in the yolk sac or foetal thymus. Adult levels of colony-forming cells were achieved within 3 days of birth. In organ cultures of 15-day foetal liver or spleen, B-lymphocyte colony-forming cells developed during a 5-day incubation period, indicating that both organs can function as bursal analogues. Foetal liver colony-forming cells were of small size and generated colonies of cells with a pattern of membrane immunoglobulin similar to colony cells generated by cells from adult animals.     

Distribution of MEL-14+ cells in various lymphoid tissues

The distribution of MEL-14+ lymphocytes was investigated by both fluorocytometric analysis and complement-dependent-cellular-cytotoxicity (CDCC) tests in which rabbit anti-rat Ig was added with complement at a secondary step. When CDCC was employed to detect MEL-14+ cells, almost half of the thymocytes were found to be MEL-14+ in various strains of mice. This high proportion of MEL-14+ cells stands in striking contrast to prior reports. Furthermore, when determined by fluorocytometric analysis, MEL-14+ cells were found to comprise more than 80% of the cells in the thymus. The MEL-14+ thymocytes comprised both immature subsets (CD4-8-, CD4+8+) and mature subsets (CD+8-, CD4-8+). MEL-14 brightly positive (MEL-14high) cells, however, were located mainly in mature T cell subpopulations within the thymus. The MEL-14high thymocytes appeared to be susceptible to the CDCC method. Most of MEL-14+ cells present in spleens and lymph nodes were shown to be included in the MEL-14high population. The MEL-14+ cells susceptible to treatment with MEL-14, rabbit anti-rat Ig plus complement in the spleen and lymph node were restricted to cells of the T-lineage. These data suggest that T cells may change from cells with low expression of the MEL-14 antigens at their surface to cells with high MEL-14 antigens in the process of differentiation. Furthermore, these findings indicate that MEL-14 molecules may be used as a surface marker to characterize an important T cell subpopulation.