Normal Rat Spleen Research Articles

Ligandin Content of Normal and Carcinogen-Treated Rat Tissues

Ligandin was detected by immunofluorescence in tissue sections and determined by immunoquantitation in the cytosols of the liver, kidney and testes of normal and carcinogen-treated rats. Ligandin was not detected by either of these procedures in normal or carcinogen-treated rat lung, spleen, brain, and skeletal or cardiac muscle.

D-penicillamine: Mitogenic effect on mouse, rat and human spleen lymphocytes

The effect of D-penicillamine (D-Pen) on the proliferation of cultures of normal mouse, rat, and human spleen lymphocytes and peripheral blood lymphocytes was examined. D-Pen in concentrations of 2 x 10(-3) M to 8 x 10(-3) M in serum-free and in serum-containing medium resulted in a highly significant incorporation of 3H-TdR by normal mouse and rat spleen cells. Enhanced incorporation of 3H-TdR by normal human spleen cells only occurred in serum-containing medium. D-Pen in concentrations of 10(-4) M to 10(-3) M in serum-free and serum-containing medium resulted in significant inhibition of 3H-TdR incorporation by normal and mitogen-stimulated mouse and rat spleen cells. Doses of D-Pen greater than 2 x 10(-2) M strongly inhibited 3H-TdR incorporation by both normal and mitogen-stimulated mouse, rat, and human spleen cells and peripheral blood cells. The latter cells were not stimulated or inhibited at lower concentrations of D-Pen. Results from cell depletion and enriching procedures (specific antibody +C' cell killing, employment of athymic, nude spleen cells, adherent and phagocytic cell removal, E rosette cell separation procedures) suggested that target cells in the mouse spleen for D-Pen activation are non-adherent B cells whereas the D-Pen responsive cells in the human spleen probably are T cells.

The mechanism of K-cell (antibody-dependent) mediated cytotoxicity. III. The ultrastructure of K cell projections and their possible role in target cell killing.

The cytotoxic interaction between lymphoid K cells from normal rat spleen and antibody-coated P815 mastocytoma cells has been studied in conditions under which the number of cytolytic events occurring at the time of observation was at a maximum. Electron micrographs of material fixed during the first 15 min after contact between the target and effector cells had been initiated by centrifugation showed that the K cells produce long projections which push deeply into the P815 cells, causing infoldings of the plasma membrane and distortion of the nucleus. The plasma membranes of the effector and target cells, and the nuclear membrane, remain intact. Subsequently the target cells undergo violent cytoplasmic blebbing (zeiosis) which is the first stage of cell lysis. The evidence for the hypothesis that projections from lymphoid K cells develop as a result of contact between receptors on the K cell surface and antibody bound to the target cell, and that the projections are involved in the cytotoxic mechanism is discussed.

Analysis of synthetic RNA-directed DNA polymerase activities in the spleen of a rat with myelogenous leukemia induced by N-nitroso- N-butylurea

DNA polymerase activities from the spleen of an individual rat with myelogenous leukemia induced by N-nitroso- N-butylurea were examined in view of their possible association with RNA tumor viruses. Viral-like DNA polymerase was not detected when DNA polymerase activities of the cells were analyzed as follows: 1. (1) Phosphocellulose chromatography of the extract from leukemic spleen gave a single peak of poly(rA)·(dT) 12–18 directed activity which coincided with that of DNA polymerase γ obtained from normal rat spleen, whereas the chromatography of the extract from virus-producing cultured R 35 rat mammary tumor cells or from those viruses themselves gave a unique peak of activity different from that of DNA polymerase γ. These viruses appeared to be one of the rat endogenous viruses similar to the R 35 virus. 2. (2) The peak from the leukemic spleen showed several enzymatic properties similar to those of DNA polymerase γ obtained from normal rat spleen, but different from those of the R 35 viral DNA polymerase. 3. (3) The peak from the leukemic spleen responded to poly(rCm)·(dG) 12–18 , which is reported to be specific for viral reverse transcriptase. However, DNA polymerase γ fraction from normal rat spleen also responded to the template-primer to the same degree. 4. (4) Postmitochondrial fractions from the leukemic spleen gave poly(rA)·(dT) 12–18 -directed activity sedimented at a density of 1.17 g/cm 3 on sucrose. However, addition of nonionic detergent was not required for the activity. In the case of our R 35-like viruses banded at a density of 1.16 g/cm 3 , the detergent was required for detection of the activity.

Evidence for a relationship between lymphoid cells and osteoclasts: bone resorption restored in ia (osteopetrotic) rats by lymphocytes, monocytes and macrophages from a normal littermate.

AbstractOsteopetrosis in the ia rat is known to be due to reduced bone resorption and the absence of ruffled borders in osteoclasts. The disease can be cured by the temporary parabiotic union with, or by transfusion of spleen, thymus, or liver cells from, normal littermates. This investigation was pursued in an attempt to determine the cellular basis of this cure by testing the relative effectiveness of populations of mononuclear cells — lymphocytes, monocytes, and tissue macrophages, from spleen and thymus of normal rats — in curing the disease, compared with cell suspensions of the entire organs. The mononuclear fractions were separated from cell suspensions of spleen or thymus by an adaptation of the Ficoll‐Hypaque density‐gradient‐centrifugation procedure. The mononuclear isolates were evaluated by conventional histological procedures and non‐specific esterase histochemistry and found to contain approximately 97% lymphocytes and 3% monocytes from the spleen and 99% lymphocytes and less than 1% monocytes from the thymus.The results of these transplantations were evaluated radiographically and histologically three weeks after treatment. No difference in the rate of skeletal cure was observed in the ia rats that received cells from either whole spleen or thymus, or the mononuclear isolate from these organs. Osteoclasts from the ia animals cured by the transplantation of the mononuclear isolate were examined by electron microscopy and found to have ruffled borders.We conclude that the mononuclear cells isolated from either the spleen or thymus of normal littermates have positive effects on osteoclasts and bone resorption in the ia rat, either indirectly, by elaboration of some local factor that affects the mutant osteoclasts, or directly by transformation into osteoclasts.

On the mechanism of phenylhydrazine-induced hemolytic anemia

The thiobarbituric acid (TBA)-reactants have been measured in the circulating red blood cells (RBC) and RBC trapped in the spleens of normal and phenylhydrazine-treated rats. There was significant TBA-reactivity in the circulating RBC of phenylhydrazine-treated rats which was increased 3-fold in RBC obtained from the spleen. Since lipid peroxidation accompanies formation of TBA-reactive malonyldialdehyde, it is suggested that phenylhydrazine induces anemia as a consequence of peroxidation of RBC membrane lipids and this effect may be a result of the autoxidation of the drug and the interaction of oxygen radicals with membrane lipids.

Antibody dependent cell-mediated cytotoxicity of Trypanosoma cruzi: the release of tritium-labelled RNA, DNA and protein

The cytotoxicity of normal rat spleen cells to antibody-coated Trypanosoma cruzi epimastigotes has been studied by assaying the release of [3H]-labelled macromolecules from the parasites. The release of thymidine (DNA) is slower than the release of uridine (RNA), suggesting that the nucleus is broken down more slowly than the cytoplasmic membrane. Less than 50% of the leucine (protein) is released when the parasites are lysed, whereas uridine (RNA) is almost totally released. In practical terms these results show that the release of incorporated radioisotope-labelled uridine can be used as a sensitive assay for cytotoxicity of T. cruzi. Cytotoxicity by normal rat spleen cells is antibody dependent and proportional to the logarithm of effector cell number. The lag phase and the rate of RNA release is not altered by centrifuging the parasites and effector cells to enhance contacts between them.

Natural cell-mediated cytotoxicity in rats. II. In vivo augmentation of NK-cell activity.

Natural cell-mediated cytotoxicity in rats as well as in mice has been shown to vary consistently with age, with peak levels detectable at 5-10 weeks. The levels of cell-mediated cytotoxicity against tumor cells could be augmented in strains of inbred rats with either high or low levels of natural reactivity, by IP injection of a variety of agents, including C. parvum, LCMV, KRV, and poly I:C. The specificity of the augmented cytotoxicity appeared to be the same as the specificity of natural killer cells which are found in normal rat spleen cells. Similarly, the cells mediating the augmented cellular cytotoxicity were small, non-adherent, esterase-negative lymphocytes with Fc receptors, as are rat NK cells. The kinetics and organ distribution of the augmentation of NK activity by poly I:C and C. parvum were compared and the kinetics were found to differ, with a shorter time course of augmented activity seen after inoculation with poly I:C. These data indicate that interferon may play a central role in the augmentation of NK activity in vivo.

Suppression of the Plaque-Forming Cell Response by Macrophages Present in the Normal Rat Spleen

Depletion of phagocytic and/or adherent cells from normal rat spleen cell preparations permitted the generation of PFC to SRBC in vitro. Suppressive activity was associated with the presence of macrophages in the normal rat spleen, since irradiated unfractionated or irradiated adherent cells suppressed the PFC response of macrophage-depleted cells. Whereas depletion of macrophages from normal rat spleen cells permitted the PFC response to SRBC to occur, comparable treatment of mouse spleen cells eliminated the PFC response. These results are discussed in relation to the requirement for macrophages in the generation of PFC with rat cells in vitro.

Tryptophan force-feeding: Changes in the activities of acetylcholinesterase in various tissues of well-fed normal and adrenalectomized rats

The activity of acetylcholinesterase in the liver, heart, spleen, lungs and kidneys of well-fed normal and adrenalectomized rats was measured following a single tube-feeding of tryptophan. In well-fed normal rats, 30 min after tryptophan force-feeding, the enzyme activity in the heart and lungs was stimulated by 28 and 25% as compared to the water-fed control while in well-fed adrenalectomized rats acetylcholinesterase acticity in the heart, liver spleen and lungs was 40, 31, 22 and 15% increased, respectively over that of the corresponding control. In both groups of rats the enzyme activity in the kidney was unaffected by tryptophan. In the liver, spleen and heart of well-fed adrenalectomized rats the pattern of response for acetylcholinesterase to a tryptophan dose, over a period of 24-hr. was found to be biphasic. In well-fed adrenalectomized rats the tryptophan-mediated stimulation of acetylcholinesterase activity in the heart was found to be insensitive to actinomycin-D. The tryptophan-mediated stimulation of acetylcholinesterase activity in the heart of well-fed normal and adrenalectomized rats could not be related to the presence of an activator.

Macrophages Suppress CTL Generation in Rat Mixed Leukocyte Cultures

The generation of CTL in rat MLC was actively suppressed by a cell population present in spleen cell preparations from normal rats. These suppressor cells were characterized by using a variety of cell fractionation techniques. Suppressor cells were removed by passage of spleen cells through nylon wool columns or by treatment with carbonyl iron. Suppressive activity was present in the mononuclear cell fraction of spleen cells obtained by Ficoll-Hypaque density gradient centrifugation. After velocity sedimentation at unit gravity, enrichment of suppressive activity was demonstrated in the fractions containing large cells as compared to the fractions containing small cells. Populations rich in macrophages were shown to have similar suppressive activity upon CTL induction in MLC. These studies suggest that macrophages present in normal rat spleen cell preparations account for the difficulty in generating CTL in MLC prepared with rat cells.

Cytolipin R from rat spleen.

Normal rat spleen contained a large amount of cytolipin R, N-acetylgalactosaminyl(beta1 leads to 3)-galactosyl(alpha1 leads to 3)galactosyl(beta1 leads to 4)glucosyl ceramide, together with a lesser amount of cytolipin K, N-acetylgalactosaminyl(beta1 leads to 3)galactosyl(alpha1 leads to 4)galactosyl(beta1 leads to 4)glucosyl ceramide, as in the case of rat kidney. Cytolipin R was confirmed to be not a rat tumor-dependent, but a species-specific glycolipid.

Inhibition of rat mixed lymphocyte cultures by suppressor macrophages

Normal rat spleens contain suppressor cells which can inhibit proliferative and cytotoxic responses of lymphocytes to alloantigens in vitro. The suppressor cells are adherent, phagocytic, resistant to treatment with ATS and C, radioresistant, resistant to treatment with mitomycin C, apparently absent from the thymus, and found in very high concentrations in peritoneal exudates. These characteristics indicate that the suppressor cell is a macrophages and not a T cell. When suppressor cells were removed from spleen cell suspensions, strong in vitro proliferative and cytotoxic responses to alloantigens could consistently be observed.

Determination of some trace elements in different organs of normal rats

In our biological investigations some trace elements have been determined by instrumental radioactivation analysis in organs of normal rats. The results of trace element distribution of Cr, Zn, Br, Sb, Fe, Co, Sc, Ag and Na in liver, spleen, brain, lung, kidney and heart of normal rats are presented in the paper.

Gross‐virus‐induced lymphoma in the rat. V. Natural cytotoxic cells are non‐t cells

The cytotoxic cells from the spleens of normal rats, which lyse Gross-virus-induced lymphoma target cells in a short-term 51Cr release test, are predominantly small to medium-sized cells sedimenting at 4-5 mm/h as shown by velocity sedimentation analysis. Their cytotoxic activity is relatively resistant to gamma-radiation, 50% surviving 1,000 rads and 30% remaining after 5,000 rads, and to heat, since 20 min incubation at 48-51 degrees C is required for its abolition. In these properties the natural killer (NK) cells are very similar to the cytotoxic T cells from tumour-immune rats, and they share in addition a requirement for Ca++ ions for cytolysis. They differ in that they are non-T cells as defined by their resistance to anti-T-cell antiserum and complement, and by their presence in T-cell-deprived rats. They lack detectable surface Ig, Fc receptors and phagocytic or adherence properties and belong therefore to that small proportion of lymphoid cells lacking the surface markers of T or B lymphocytes, as do the comparable NK cells of mice. Cytotoxicity appears to involve an autonomous, papain-sensitive recognition structure on the surface of NK cells, rather than acquired cytophilic antibody operating through an antibody-dependent mechanism.

In vitro inhibition of lymphoproliferative responses to tumor associated antigens and of lymphoma cell proliferation by rat splenic macrophages

Spleens from W/Fu rats bearing a syngeneic progressively growing (C58NT)D tumor contain cells which can inhibit lymphoproliferative responses in a mixed lymphocyte-tumor interaction designed to demonstrate suppressor activity. Spleens from rats having rejected (C58NT)D tumors also contained suppressor cells but to a lesser degree. The growth inhibition assay, which measures inhibition of proliferation of tumor cells, was evaluated as a simple assay system to screen for suppressor cell activity. The effector cells in both assays had the same characteristics, indicating a predominant role of macrophages. Normal rat spleens were found to contain growth inhibition activity which led to the demonstration of suppressor cell activity in spleens of normal animals. Removal of suppressor cells from the spleens of immunne rats results in consistently higher lymphoproliferative responses to tumor associated antigens on the tumor cells.

Trapping and destruction of blood-borne syngeneic leukaemia cells in lung, liver and spleen of normal and leukaemic rats.

Leukaemic cells from rats with a lymphoid (HRL) or myeloid (SAL) leukaemia were labelled with 125IUDR and injected i.v. into either normal or leukaemic syngeneic recipients. The fate of the injected cells was studied in terms of the radioactivity in various tissues at various times up to 24 h later. In normal animals the leukaemia cells were destroyed rapidly in the reticulo-endothelial (RE) system; immediately after injection most recoverable activity was in the lung, with smaller amounts in the blood, spleen and liver but by 24 h only 20-30% of the injected activity could be recovered. In leukaemic recipients with high numbers of blasts in the blood the amount of activity recoverable from the lungs and bone-marrow was markedly reduced, while that in the blood was doubled. Nonetheless, the overall rate at which radioactivity was eliminated was not significantly different from that found in normal rats, in spite of the fact that the RE system was extensively infiltrated by leukaemia cells.

Intracellular localization of anti-tumor immune RNA

Syngeneic spleen cells from normal, non-immune Fischer 344/N rats and allogeneic spleen cells from normal Wistar-Furth rats became cytotoxic, in vitro, to chemically induced Fischer rat sarcoma (MC3-R) target cells following incubation with xenogeneic Immune RNA (I-RNA) extracted from spleens of guinea pigs immunized with MC3-R tumor cells. I-RNA extracted from intact spleen cells or from the cytoplasmic fraction of spleen cells were equally active. RNA extracted from isolated spleen cell nuclei was inactive, as were all RNA fractions from spleen cells of nonspecifically immunized guinea pigs. Syngeneic I-RNA extracted from intact spleen cells or the cytoplasmic fraction of cells from spleens of Fischer rats bearing growing MC3-R transplants mediated cytotoxic reactions against MC3-R target cells when incubated with normal Fischer rat spleen cells. RNA from the nuclei of spleen cells of rats bearing MC3-R tumors was considerably less active. All RNA fractions from spleen cells of normal non-immune Fischer rats were inactive. The immunologically active component of xenogeneic and Syngeneic I-RNA, therefore, were found to be localized in the cytoplasm of specifically sensitized lymphoid cells.

Characterization of the normal lymphocyte population cytolytic to Burkitt's lymphoma cells of the EB2 cell line.

The normal human lymphocyte population which exhibits "spontaneous" cytolysis of EB2 Burkitt's lymphoma cells has been characterized. The effector cell has EA and EAC' receptors but lacks E receptors and probably surface Ig. "Spontaneous" anti-EB2 cytotoxicity was not reduced by preincubation of the effector cells with plastic or iron carbonyl or by passage through cotton wool or agarose columns but was reduced by passage through nylon wool columns. Thymocytes were not cytotoxic to EB2 cells, and chronic lymphocytic leukaemia cells (of B cell characteristics) had reduced cytotoxicity compared with normal lymphocytes. Cells from various lymphoid organs of rats and guinea-pigs were also cytotoxic to EB2 cells with reactivity in spleen greater than or equal to blood greater than lymph nodes. Spleen cells from neonatally thymectomized rats had increased cytotoxicity compared with normal rat spleen cells, suggesting that T lymphocytes are not essential. The effector cell in rat spleen did not adhere to cotton wool or agarose columns, indicating some resemblance to its counterpart in human peripheral blood.

Metabolic activities of histones in rat liver and spleen.

Synthesis and turnover of histone I and II in normal rat liver and spleen were studied by Amberlite CG 50 column chromatography. Histone I was separated into three or four subfractions, each of which showed a different rate of incorporation of [3H]lysine. This was verified by a more shallow gradient chromatography developed by Kinkade and Cole [3] for very lysine-rich histone (F1), which showed tissue specific differences between liver and spleen in both the elution pattern and synthetic rates. These subfractions were distinguished from each other by dodecylsulphate electrophoresis. The turnover, or disassociation of histone I and II in chromatin was measured by double-labelling of normal rat liver with [3H] and [14C]lysine. A good correspondence was found between the synthesis and turnover patterns of individual histone I fractions, while the histone II synthesized was conserved for over a month. From consideration of the turnover in relation to the cell population of normal liver tissue, which consists of a very small fraction of growing cells and a very large fraction of resting ones, it was concluded that turnover of histone I must occur even in resting cells. When DNA synthesis in the spleen was completely inhibited by hydroxyurea, the synthesis of histone II was inhibited but that of histone I was only partially inhibited. The remaining synthesis seemed to occur in cells in the resting state. It was concluded tentatively, the continuous replacement of very lysine-rich histones of chromatin must occur even in resting cells in which DNA synthesis has ceased. The biological significance of disassociation of histones from chromatin was discussed.