Lymphoid Tissues Of Mice Research Articles

Turnover of dendritic cells in rat heart.

Taking advantage of the high class II (I-region-associated) antigen content of the tissue dendritic cells and monoclonal antibodies directed to the backbone determinants of the bone marrow donor strain, we have investigated the location and analysed the turnover rate of tissue dendritic cells in rat heart. Low numbers of class II-expressing, factor VIII-negative, non-phagocytic cells with dendritic appearance were observed between the heart muscle fibres. After irradiation with 960 rad, these cells were no longer visible but they reappeared (in lower numbers) on day 20, indicating that they are relatively radioresistant but the antigen expression is radiosensitive. Transplantation of allogeneic bone marrow demonstrated that similar cells appeared from the transplanted bone marrow on day 10, and that these cells populated the heart at a maximum of 20-25 days after transplantation. This indicates a relatively rapid turnover rate, comparable with the turnover rate of dendritic cells in rat kidney and mouse lymphoid tissue.

Activation of T lymphocytes results in an increase in H-2-encoded neuraminidase.

The endogenous neuraminidase activity of various mouse lymphoid subpopulations and tissue compartments was examined by a sensitive fluorometric assay. These analyses indicated that activated T lymphocytes possessed a significantly higher level of intracellular neuraminidase than activated B or resting T or B lymphocytes. Examination of the level of neuraminidase in bone marrow, thymus, lymph node, and unfractionated spleen indicated that these lymphoid tissues contained significantly less neuraminidase than was detected in stimulated T cells. Kinetic studies revealed that the majority of the increase in neuraminidase activity occurred between 24 and 48 h following stimulation. Analysis of activated T lymphocytes prepared from a panel of inbred mouse strains indicated that cells from mice of the H-2v haplotype, which possess the Neu-1a allele and are deficient in liver neuraminidase, exhibited a level of activity which was significantly lower than that detected in stimulated T cells from other mouse strains. These results indicate that the endogenous neuraminidase activity of T lymphocytes increases upon stimulation, and that the level of this enzyme activity in lymphoid cells is also controlled by the Neu-1 locus, which is located in the H-2 region of the major histocompatibility complex.

Selection of a fixative for identifying T cell subsets, B cells, and macrophages in paraffin-embedded mouse spleen

Fixation techniques were investigated for their ability to preserve morphology, esterase activity and cell surface antigens in paraffin-embedded mouse lymphoid tissue. The avidin-biotin-peroxidase system was used to stain antigens Thy-1.2, Lyt-1, Lyt-2, RA32C2 and F4/80. Conventional fixatives were compared with fixatives containing periodate and lysine plus paraformaldehyde and/or glutaraldehyde. Conventional fixatives preserved esterase activity but not many cell surface antigens. Periodate-lysine fixatives allowed better preservation of membrane antigens, but esterase activity was often lost at antigen-preserving concentrations of paraformaldehyde or glutaraldehyde. However, a periodate-lysine fixative containing both paraformaldehyde and glutaraldehyde preserved esterase and showed good to excellent staining of Lyt-1, Thy-1.2, RA32C2, and F4/80. Lyt-2 could not be stained with any fixative, but was well preserved in frozen material post-fixed with periodate-lysine based fixatives. We conclude that with proper fixation immune cell surface markers can be identified in paraffin-embedded tissue.

Chronic Dietary Copper Deficiency Alters Biochemical and Morphological Properties of Mouse Lymphoid Tissues

Chronic copper deficiency in mice impairs both humoral and cellmediated immunity, but the mechanisms are unknown. Copper deficiency was produced in C58 mice by feeding dams a diet low in copper throughout lactation and weaning the pups to this diet. Control mice were from dams fed the same diet but with copper supplementation in the drinking water. Six-week-old mice were sampled for biochemical and morphological studies. Compared to copper-supplemented mice, copper-deficient animals were smaller, anemic and exhibited hypoceruloplasminemia. The copper-deficient mice have small thymus glands, enlarged spleens, and livers equivalent in size to copper-supplemented mice. Thymic atrophy is not caused by elevated serum corticosterone. Liver, spleen, and thymus tissues from copper-deficient mice exhibit low cytochrome oxidase (56, 38, and 45%, respectively) and superoxide dismutase activities (61, 60, and 43%, respectively) compared to tissues from copper-supplemented mice, indicating a functional copper deficiency. Electron micrographs taken of thymus and spleen from copper-deficient mice demonstrate altered morphology characterized by abnormal mitochondria and misshapen nuclei. Chronic copper deficiency alters the size, biochemistry and morphology of primary (thymus) and secondary (spleen) lymphoid tissue.

Human dendritic cells. Enrichment and characterization from peripheral blood.

Previous studies demonstrated that lymphoid tissues of mice and rats contain small numbers (less than 1 percent of nucleated cells) of dendritic cells (DC) with special cytologic, surface, and functional properties. We show here that similar DC represent 0.1-0.5 percent of human peripheral blood mononuclear cells. DC can be enriched to 20-60 percent purity by a multistep procedure analogous to that used in mice. Adherent peripheral blood mononuclear cells are cultured overnight, and the released cells are depleted of monocytes and B cells by readherence to plastic, rosetting with erythrocytes coated with anti-human IgG, and centrifugation in dense albumin columns. Enriched DC have similar cytologic features to rodent DC by light and electron microscopy. DC express HLA, and HLA-DR and the leukocyte-common antigens. They lack phagocytic capacity, receptors for antibody-coated and neuraminidase-treated erythrocytes, surface and intracellular Ig, esterase, peroxidase, and azurophilic granules. DC do not react with several monoclonal antibodies directed to phagocytes (OKM 1, "mac-1," 63D3, and 61D3) and T cells (OKT 3, 6, 8). Unlike the mouse, human DC express complement receptors. When maintained in culture for 4 d, human DC did not give rise to either B cells or monocytes. Therefore, DC identified by cytologic criteria are distinct from other leukocytes. Enriched populations of DC have been compared to fractions enriched in monocytes, B cells, and T cells in three functional assays: stimulation of the primary allogeneic mixed leukocyte reaction, stimulation of the primary syngeneic MLR, and accessory function for the proliferation of periodate- modified T cells. In each case, the DC fraction was 10-fold or more active than other cell fractions. We conclude that DC circulate in man, and represent the principal cell type required for the initiation of several immune responses.

Incubation of trypanosome-derived mitogenic and immunosuppressive products with peritoneal macrophages allows recovery of biological activities from soluble parasite fractions.

This report describes further attempts to define the nature of the parasite product(s) responsible for the extensive changes in lymphoid tissue in mice during infection with Trypanosoma brucei. As previously described, potent mitogenic and immunosuppressive effects are induced by a trypanosome-derived crude membrane fraction in vivo. There was no enrichment in these activities when purified parasite surface membranes were used. Mitogenic activity can be recovered from soluble trypanosome material only when it is incubated with peritoneal macrophages before transfer into syngeneic recipients. Thus, by encouraging association with a critical target cell, soluble parasite products can be studied, and their active components can be separated by conventional methods. Preliminary fractionation of high-spin trypanosome supernatant over Sepharose 4B confined the mitogenic activity to the high-molecular-weight fraction, which is a macromolecular complex of proteins, glycoproteins, and lipid. Extracted lipid from this material was able to significantly suppress a primary immunoglobulin G anti-sheep erythrocyte response. The activity was periodate sensitive and pronase resistant. The use of macrophages in vitro may be a general method whereby important biological activities lost as a result of fractionation procedures can be recovered and the active components studied in greater detail.

Role of disulfide interchange enzyme in immunoglobulin synthesis.

The role of disulfide interchange enzyme in protein biosynthesis was evaluated by studying the enzyme from mouse lymphoid tissue. The enzyme isolated from lymphoid cells was shown to have no tissue-specific characteristics. It was identical with the enzyme synthesized by mouse liver in its biochemical and immunological properties and its capacity to promote both disulfide bond formation and insulin degradation. In contrast to liver, the levels of enzyme in lymphoid tissues were found to vary with immunoglobulin secretory activity, Assays of lymphoid cells and their transformed counterparts showed that the enzyme contents of cells actively secreting immunoglobulin were 1-2 orders of magnitude higher than that of unstimulated B cells or non-immunoglobulin-producing T cells. The increase in enzyme levels paralleled the increase in immunoglobulin synthesis after antigen or mitogen stimulation and was independent of the class of immunoglobulin produced. This correlation indicated that the enzyme plays a critical role in the formation of intramonomer bonds common to all immunoglobulin molecules. Supporting data were obtained by assaying the ability of the enzyme to promote the polymerization of mouse pentamer IgM in vitro. The enzyme was found to catalyze the formation of the interchain bonds required for monomer IgM assembly but not the formation of the intermonomer bonds required for pentamer assembly. The sum of these results provides strong evidence that disulfide interchange enzyme functions in the in vivo synthesis protein disulfide bonds.

Incomplete tolerance to MHC antigens in irradiation chimeras: implications for MHC restriction and self tolerance.

SUMMARY AND CONCLUSIONS (1) Chimeras of the type (P, X P2)Fi-Pi have a fragile state of T cell tolerance toP2 antigens. Though no ongoing response to P2 antigens can be detected in thespleen at the time of its removal, anti-P2 MHC responses can be provoked invitro (Tc cell generation), or in vivo (skin graft rejection). These results imply thatanti-P2 T cell precursors are present in these mice but are usually suppressed.If the idiotypes of anti-P2 T cells crossreact with those of anti-P2 + X T cells,then this could contribute to the observed bias of H-2 restricted responses to Rantigens of Pi type,(2) Lymphomyeloid cells of (PiXP2)Fi origin that repopulate the lymphoidtissues of irradiated Pi mice can apparently express less P2 MHC antigens thannormal Fi cells. If this contributes to the fragile state of T cell tolerance to P2antigens in the secondary lymphoid tissues of chimeras, it implies that inductionor maintenance of self tolerance to MHC antigens is quantitatively dependentupon MHC antigen expression,(3) The amount of H-2 antigen expressed on lymphomyeloid cells apparentlydepends not only on the genotype of the stem cells from which they are derived,but on the environment in which lymphomyeloid differentiation takes place.

Immunohistological detection of B lymphocytes in the rat lymphoid organs by using anti-rat B lymphocyte serum.

Anti-rat B lymphocyte serum (ARBS) was prepared by immunizing rabbits with purified B cells from rat spleen and by absorbing the rabbit serum with rat red blood cells and syngeneic sarcoma cells. The specificity of ARBS for rat B cells was confirmed by various cytologic and immunofluorescence studies. By indirect immunofluorescence on tissue sections of rat spleen and lymph nodes, anatomical localizations of B cells in lymphoid organs were lymphoid follicles of the lymph node and lymphocyte corona surrounding periarteriolar lymphoid sheaths of the spleen; while cells located in the thymus-dependent area were essentially devoid of immunofluorescence or no cytoplasmic immunofluorescence was observed in plasma cells. Thus, the B cell distribution in tissue sections was clearly demonstrated with ARBS. When the cross-reactivity of ARBS with mouse B cells was studied by complement-dependent cytotoxicity and immunofluorescence, it was found that mouse B cells shared at least one determinant of rat B lymphocyte specific antigens (RBLA) with rat B cells, and that the distribution pattern of the cross-reacting antigens in mouse lymphoid tissues was essentially the same as that of RBLA in rat lymphoid organs.

Mode of action of methylating carcinogens: comparative studies of murine and human cells

Murine and human cells (mainly lymphocytes) were ethylated in vitro with either N-[14C]methyl-N-nitrosourea (MNU) or di[14C]methyl sulphate (DMS) and the extents of methylation of DNA at O-6 and N-7 of guanine and N-3 of adenine were determined. The cytotoxic action of MNU was also compared with that of DMS, as assessed by their effects on cell division following stimulation of these lymphocytes in culture by concanavalin A (Con A). The overall extent of methylation of the DNA of human cells was about 70% of that of murine cells after exposure to MNU and DMS. Mouse cells, responding to Con A in culture, were found to be much more sensitive to both agents than could be accounted for by these differences in overall extent of methylation of the DNA. Significant differences were found between cells in their ability to rapidly remove O6-methylguanine from DNA. Normal human lymphocytes were always proficient, but some human lymphoid lines were deficient in this respect, while all the murine cells tested were deficient, as found in vivo for mouse lymphoid tissues. No correlation has yet been found between the susceptibility of various mouse strains to the carcinogenic action of MNU and their ability to remove methylated bases from DNA. The cytotoxicity studies, showing that normal human lymphocytes were relatively more resistant than murine cells to methylation by both MNU and DMS, suggested that the ability to remove O6-methylguanine (which is produced in a very low proportion by DMS) was not of prime importance in conferring resistance in this type of assay.

Mitogen responsiveness of various immune tissues: Heterogeneity of accessory cells and susceptibility to suppression by macrophages

The present study characterizes accessory cell dependency of mitogenic responses to PHA and Con A of murine spleen, lymph node, and peripheral blood lymphocytes. Depletion of macrophages by carbonyl iron from peripheral blood and lymph node lymphocytes remarkably reduced their mitogenic response, whereas that of spleen cells after iron phagocytosis was occasionally reduced, but generally enhanced or not changed. However, the further purification of spleen cells by nylon-filtration always reduced the mitogenic response. The adherent but not phagocytic cells (NPAC), which were purified from the macrophage-depleted spleen cells by the adherent techniques, effectively restored the mitogenic response of highly purified spleen, as well as lymph node and peripheral blood lymphocytes, as did the purified peritoneal macrophages. These results indicate that the major accessory cells in lymph node and peripheral blood were phagocytic macrophages, while the spleen contained a population of adherent but not phagocytic cells which play a role as an accessory cell in the mitogenic response. The Con A response of purified lymphocytes either from spleen, lymph node, or peripheral blood was fully restored by addition of 1 or 2% peritoneal macrophages. However, when mixed with a larger number of macrophages, the mitogenic responses of spleen and lymph node cells did not further increase. Addition of more than 3% macrophages remarkably suppressed the response but had little effect on peripheral blood lymphocytes. Thus, the mitogenic response of spleen and lymph node cells appears to be suppressed by macrophages more easily than that of peripheral blood cells. The heterogeneity of accessory cells and different susceptibility to suppression by macrophages may account for many of the observed variations in mitogen responsiveness of different lymphoid tissues in mice.

Tissue distribution of IgA receptor-bearing cells in mouse and guinea pig with special reference to the lymphoid population of the gastrointestinal tract

The presence and relative number of cells bearing IgA and IgG receptors in various mouse and guinea pig lymphoid tissues was determined using rosetting techniques. Both types of cells were found to be widely distributed, with cells binding IgG (range 10 to 45%) being more common than cells binding IgA (range 2 to 6%) in all tissues examined. In the mouse, cells bearing IgA receptors were not more prevalent in the intestinal lymphoid organs, Peyer's patches and mesenteric lymph nodes, as compared to the systemic lymphoid organs, spleen and peripheral lymph nodes, although PP contained the highest percentage. In addition, the percentage of cells with IgA or IgG receptors in a given murine tissue did not directly correlate with the capacity of lymphocytes from that tissue to synthesize immunoglobulin of the same isotype upon LPS stimulation. In the guinea pig, cells with IgA receptors were found in both lamina propria and intraepithelial lymphocyte populations isolated from small intestinal mucosa. However, their number there was only marginally higher than that found in other guinea pig tissues, such as spleen. Thus, cells bearing IgA receptors are not preferentially localized in tissues which are active in the secretory (IgA) immune system.

Different labelling patterns in mouse lymphoid tissues with [3H]deoxycytidine and [3H]thymidine.

The percentages of labelled lymphocytes in smear preparations of mouse thymus were higher than those in similar preparations of mesenteric lymph nodes with either generally labelled tritiated deoxycytidine, [3H]CdR, or tritiated thymidine, [3H]TdR. Lymphocytes in the thymus cortex and in germinal centres of mesenteric lymph nodes were intensely labelled with [3H]CdR, whereas with [3H]TdR lymphocytes in the peripheral region of thymus and medullary cords of mesenteric lymph nodes were heavily labelled. The majority of lymphocytes in thymic cortex and germinal centres of mesenteric lymph nodes were labelled weakly with [3H]TdR. Thus, labelling patterns with [3H]CdR differed from those with [3H]TdR in lymphoid tissues of the mouse. Mouse lymphocytes can utilize [3H]CdR as a precursor molecule for cytosine and thymine in DNA. The ratio of radioactivity of thymine to that of cytosine was measured biochemically in DNA extracted from lymphocytes labelled with [3H]CdR. This radioativity ratio in thymus was higher than that in mesenteric lymph nodes. These results suggest that the metabolic activities of utilizing CdR for DNA synthesis differ within lymphocyte populations in various lymphoid tissues in the mouse.

Peanut lectin binding properties of germinal centres of mouse lymphoid tissue.

Peanut (Arachis hypogaea) lectin (PNL) has been shown to agglutinate the 90% of cells from murine thymus which are supposed to be immature cortical thymocytes. Further studies on the numbers of thymocytes binding fluorescein isothiocyanate conjugated PNL (FITC-PNL) confirmed the large proportion of PNL binding cells. In other organs such as bone marrow, spleen and peripheral lymph nodes, smaller proportions of PNL positive cells have been recorded. PNL-positive cells outside the thymus have been reported to be either Thy 1-positive or null cells. It has also been suggested that PNL binding may be a marker for immaturity not only in relation to T lymphocytes but also amongst haematopoietic stem cells. Thus PNL binding as an aspect of lymphocyte differentiation is a matter of considerable interest. The current study describes the distribution of horseradish peroxidase-conjugated PNL (HRP-PNL) on frozen sections of mouse lymphoid organs. It seems that PNL binds to cells in germinal centres but not to those in some other areas containing activated lymphocytes. There is good correlation between the presence of PNL-binding germinal centres in frozen sections of lymphoid organs and the number of PNL-binding cells counted in cell suspensions from the same organs.

Actin-Like Protein in Chicken and Mammalian Lymphoid Tissue Demonstrated by Reactivity with Human Smooth Muscle Autoantibody

The reactivity of chicken, human, rat and mouse lymphoid tissues with human serum containing smooth muscle antibody was studied by indirect immunofluorescence. In bursal follicles, strong fluorescence was observed predominantly in the lymphocytes and reticular epithelial cells of the medulla, the basement membrane and a row of undifferentiated epithelial cells at the corticomedullary junction. Reticular epithelial cells in lymphocyte-depleted bursas from cyclophosphamide-treated chickens and cultures of bursal and thymic reticular epithelial cells also reacted with smooth muscle antibody. The thymus of all species showed only medullary staining; lymph nodes, spleen and gut-associated lymphoid tissue showed extensive cell staining of both T and B lymphocyte dependent areas, with weak reactivity in germinal centres. Embryologically, the time of appearance of bursal and thymic lymphocyte staining coincided with the development of medullary lymphocytes. In organ imprints and cell smears of bursa, thymus, lymph node and spleen, and in cell smears of bone marrow and peripheral blood lymphocytes, immunofluorescent staining was restricted to cells in close contact with each other. Suspensions of dispersed viable lymphoid cells were negative. Specificity of the staining reaction was established by its prevention after neutralization: absorption of the serum with homogenates of smooth muscle or actin derived from the same source. It is concluded that an actin-like protein is present in mature B and T lymphocytes and in reticular epithelial cells and that its expression in lymphocytes appears dependent on cell-to-cell contact. The presence of the actin-like protein in mature lymphocytes may provide a means of lymphocyte motility <i>in vivo.</i>

Macrophages and lymphoid tissues in mice with concomitant tumour immunity.

The growth in mice of subcutaneous isografts of any of 5 methylcholanthrene-induced fibrosarcomas was associated with macrophage stimulation, reflected in an increased incidence of DNA-synthesizing cells among marcophages in the uninjected peritoneal cavity. This occurred at some stage with 4 tumours that induced concomitant immunity and one that did not. Some degree of splenomegaly also occurred with all 5 tumours. The spleens of all the tumour-bearing mice showed histological evidence of increased haemopoietic activity. Histological changes in the lymphoid elements of the spleen were very different with different tumours, ranging from lymphoid stimulation to lymphoid atrophy. The lymph nodes draining the sites of primary isografts which induced concomitant immunity showed signs of stimulation in the paracortical areas, followed by plasmacytopoiesis in the medullary areas. Stimulation of the paracortical areas was not detected in the nodes draining sites of injection of a tumour not inducing concomitant immunity. Nodes draining the sites of challenge isografts in mice exhibiting concomitant immunity showed plasmacytopiesis.

Rat T Lymphocyte-Specific Antigens and Their Cross-Reactivity with Mouse T Cells

Anti-rat T lymphocyte serum (ATLS)2 was prepared by immunizing rabbits with purified T cells from rat mesenteric nodes and absorbed with rat red cells and syngeneic sarcoma cells. The specificity of ATLS for rat T cells was confirmed by the following reasons: a) ATLS was not toxic for bone marrow cells but lysed most of the thymocytes and a number of spleen and lymph node cells, which were inversely correlated to the percentage of cells with B cell characteristics in respective organs; b) anatomical localization of ATLS-reactive cells in lymphoid organs coincided to the thymus-dependent areas, i.e. the paracortex of lymph node and the periarteriolar region of spleen; c) spleen cells treated with ATLS and complement failed to respond to phytohemagglutinin but normally responded to bacterial lipopolysaccharide; d) those cells treated with ATLS and complement could not induce a graft-vs-host reaction in F1 hosts, whereas the same treatment did not affect direct plaque-forming cells. All of these data confirm the specificity of ATLS and indicate that ATLS recognizes rat T lymphocyte-specific antigens (RTLA). Absorption studies showed that RTLA were present in higher concentration on medullary thymocytes and peripheral T cells than on cortical thymocytes, but absent from bone marrow, liver, and brain tissues. When the cross-reactivity of RTLA with mouse T cells was studied by C-dependent cytotoxicity and immunofluorescence, it was found that mouse T cells shared at least one determinant of RTLA with rat T cells, and that distribution pattern of the cross-reacting antigens in mouse lymphoid tissues was essentially the same as that of RTLA in rat lymphoid organs.

Effect of a single administration of testosterone on the immune response and lymphoid tissues in mice

Female mice were given a single intraperitoneal injection of testosterone immediately after irradiation and marrow reconstitution. Thirty days later testosterone had no suppressive effect on the recovery of thymus and spleen weights. Testosterone had no effect on the graft-versus-host reaction. Testosterone had no influence on the survival of the skin homografts. However, the plaque-forming cell response to sheep erythrocytes in the spleen was dramatically suppressed by testosterone. Histological observations revealed marked inhibition of lymphoid regeneration selectively in the thymus-independent areas of the peripheral lymphoid tissues. These results suggest that testosterone would act mainly on the differentiation of stem cells toward the population of bone marrow-derived B lymphocytes. The immune response to sheep erythrocytes was restored completely 90 days after testosterone administration. Testosterone given to normal adult mice can also have suppressive activity on the immune system 30 days after a single intraperitoneal injection.

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.

Cross-reactivity between human thymus and mouse lymphoid tissues, as revealed by rabbit antiserum against human brain.

Rabbit anti-human brain antisera were found cytotoxic for mouse thymus, lymph node, and spleen cells. Absorption with human thymus cells removed some of the cytotoxic antibodies. Cross-reactions were also detected among human brain and thymus and mouse brain, liver, and erythrocytes.