Heterogeneity of the effectors of natural killer activity in CBA mice

The natural cytotoxicity of Percoll fractions of CBA mice splenocytes was tested against cells of human erythroblastosis suspension culture K-562 and murine C3HA hepatoma solid culture MGXXIIa. The 1.076 g/ml dense fraction was shown to have the highest activity in 3H-uridine cytotoxic test against the cell targets. Immunosera prepared by the Golub method against CBA mouse brains and exhausted supplementary by syngeneic thymocytes decreased the natural cytotoxicity of CBA mouse splenocytes against MGXXIIa tumor cells and, on the contrary, increased it against K-562 tumor cells.     

Influence of bacteria of Lactobaccilus genera on cytotoxic activity of splenocytes in experimental animals

Influence of lactobacilli on cytotoxic activity of splenocytes from mice of CBA line was studied in vivo and in vitro (after cultivation of splenocytes with chloroform-killed bacteria, homologous lysates obtained by ultrasound, and with native filtrates). Data on increase of splenocytes cytotoxic functions induced by lactobacilli has been obtained. Mechanisms of lactobacilli-mediated activation of cellular factors of antitumor immunity (cytotoxic lymphocytes and natural killers) were discussed. Information on the ability of cell wall components, cytoplasmic fractions, and substances secreted by lactobacilli to stimulate the immune response of a microorganism is presented.     

CC chemokine receptor 2 expression in donor cells serves an essential role in graft-versus-host-disease

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6-->bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4(+) T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-gamma-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4(+) T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2(-/-) CD4(+) T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4(+) T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2(-/-) splenocyte-->bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.     

Characterization of cytostatic effector lymphocytes during the development of a syngeneic lymphosarcoma in C3H mice: use of monoclonal reagents to identify T-cell subsets

The development of the in vitro cytostatic capacity of splenic lymphocyte subpopulations from C3H mice carrying the syngeneic Gardner tumor was examined at different times after intramuscular tumor injection. Most mice died between 3 to 6 weeks after tumor injection, while some rejected their tumors or survived longer than 3 months. Cell separation procedures and monoclonal antibodies against T-cell subsets were used to identify the cells responsible in anti-tumor immunity. Cytostatic capacity against tumor cells developed in the T-cell enriched subpopulation of splenocytes 3 days after tumor injection and was partly abrogated by anti-Lyt-1. Effector function of Lyt-2+ T cells and B cells developed later and peaked at around 10 days after tumor injection. Another cell population with cytostatic capacity which was not blocked by anti-Lyt-1, anti-Lyt-2, or anti-Ly-5 was noted to develop early after tumor injection and lacked both T-cell and B-cell markers ("null"). This subpopulation was eluted with T cells from nylon wool columns and comprised up to 50% of the T-enriched fraction of splenocytes in later stages of tumor growth. An interesting characteristic of these "null" cells was susceptibility to T-cell suppression both in early and later stages of tumor growth except in regressor mice which lacked suppressor T cells. The cytostatic capacity of the "null" cells could be restored either by removal of Thy-1+ cells from the T-enriched fraction by panning, or the addition of anti-Thy-1 or F(ab')2 fragments of anti-Thy-1 to the lymphocyte-tumor reaction mixtures. Most mice examined after 10 days of tumor growth were immunosuppressed to varying degrees. Unseparated splenocytes from these mice were not cytostatic but removal of T cells allowed the B cells to exert their cytostatic capacity. A strong underlying B-cell cytostasis was shown to be present in long survivor mice even though their unseparated spleen cells were only weakly cytostatic. T cells did not play a role in the regression of tumors or long-term survival of tumor bearer mice. Splenocytes from regressor mice were strongly cytostatic, their anti-tumor activity residing in the "null" and B-cell populations.     

Monocyte-mediated augmentation of human natural killer cell activity: conditions, monocyte and effector cell characteristics

The characteristics of the effector cells and monocytes, and conditions required for the monocyte-mediated augmentation of human natural killer (NK) cell activity were investigated. Enriched null cell populations were further fractionated by Percoll centrifugation and used as effector cells. The LGL-enriched fraction was less susceptible than either the unfractionated cells or the other Percoll fractions to the monocyte augmentation when mixed with monocytes in the chromium-release assay and when precultured with monocytes for 12 hr, retrieved by carbonyl iron treatment, and tested for NK activity against K562. This differential susceptibility was reflected at the single cell level. The LGL-enriched Percoll fraction did not display the increase in target-binding cells with lytic activity that was exhibited by the other effector cell preparations after culture with monocytes. No differences in Leu-7 and Leu-11 phenotypes were detected between enriched null cells that had been cultured with and without monocytes for 12 hr. At the monocyte level, it was shown that pretreatment of the monocytes with LPS did not alter their NK-augmenting activity appreciably. Glutaraldehyde-fixed monocytes were not effective, and actinomycin D-treated monocytes were less effective than untreated or irradiated monocytes when mixed with enriched null cells in the assay. Actinomycin D-treated monocytes did not augment and possibly suppressed NK activity tested after 12-hr culture, and irradiated monocytes were less effective for augmenting NK activity than untreated cells. Monocyte-mediated augmentation could be detected when the medium used for null cell-monocyte coculture was supplemented with a) different lots of fetal bovine serum, b) human AB serum, c) autologous serum, or d) no serum. Polymyxin B and indomethacin did not alter the monocyte effect. Finally, the monocyte-mediated augmentation of human NK was not MHC restricted, since allogeneic combinations were also effective. These results suggest that 1) lymphocytes other than LGL participate in the monocyte-mediated augmentation of NK activity, 2) the augmentation is probably activational rather than maturational, 3) the monocytes must be viable to be effective when mixed with null cells during the assay, 4) de novo RNA and/or protein synthesis by the monocytes is required for the monocytes to induce augmented activity in null cells after 12-hr coculture, 5) prostaglandin synthesis and endotoxin are probably not involved in the augmentation, 6) the phenomenon is not MHC restricted, and 7) monocytes may express augmentative and suppressive activities concurrently.     

Augmentation of murine lymphokine (rIL-2)-activated killer cell activity by indomethacin

The effect of indomethacin on murine lymphokine-activated killer (LAK) cell activity was investigated using a natural killer-resistant, spontaneously developed, weakly immunogenic, and highly tumorigenic syngeneic murine mammary adenocarcinoma, mimicking that of human disease, as the target. When used in combination with human recombinant interleukin-2 (rIL-2), indomethacin was found to augment LAK cell activity, which was generated from culture of the normal mouse splenocytes with rIL-2, as compared to that with rIL-2 alone. This increase in LAK cell activity was shown to be indomethacin dose-dependent, and was demonstrated only when indomethacin was added to the rIL-2-containing medium at the beginning of culture. The enhancement of LAK cell activity by indomethacin was abrogated when the nylon-wool nonadherent "macrophage-poor" splenocytes were incubated with rIL-2 plus indomethacin. These results indicated that the rIL-2-induced LAK cell activity generated from murine splenocytes could be augmented by indomethacin, and the macrophages may be involved as the mediator.     

Host resistance to cyclosporine induced syngeneic graft-versus-host disease. Requirement for two distinct lymphocyte subsets

Cyclosporine is crucial for the prevention of organ allograft rejection and allogeneic graft-vs-host disease (GVHD). Despite its potent immunosuppressive activity, cyclosporine elicits a T cell-mediated autoimmune syndrome after autologous or syngeneic bone marrow transplantation, which has been termed syngeneic GVHD (SGVHD). Recent studies have shown that for disease manifestation, a cytoxan and radiation-sensitive T cell dependent host resistance mechanism must be eliminated, allowing the clonal expansion of autoreactive cells. This report characterizes the autoregulatory lymphocyte population, present in normal animals, capable of inhibiting the adoptive transfer of SGVHD. First, twice the number of unfractionated splenocytes from normal animals to those from autoimmune donors ensured complete inhibition of the adoptive transfer of immune reactivity. Second, the phenotype of this host resistance mechanism in normal splenocytes involves dual regulatory T cell subsets. A helper/inducer subset (W3/25+) must be cotransferred with a cytotoxic/suppressor subset (OX8+) in a ratio that approximates the normal ratio in normal unfractionated splenocytes in order to affect inhibition of the transfer of SGVHD. Moreover the specific inducer regulatory activity resides in the OX22-, W3/25+ subset of Th cells.     

Murine lymphokine-activated killer (LAK) cells: phenotypic characterization of the precursor and effector cells

Murine and human lymphocytes incubated in recombinant interleukin 2 (RIL 2) generate a population of cytotoxic cells (lymphokine-activated killer cells [LAK]), which are able to lyse a wide array of fresh tumor cells but do not lyse fresh normal cells. Intravenous administration of these cells with the concomitant administration of RIL 2 can eliminate established pulmonary and hepatic metastases in mice. To characterize the cell that has in vitro LAK activity, we subdivided murine lymphocytes by lysing select subpopulations with the use of complement and antibodies against lymphocyte surface markers or by fluorescence-activated cell sorting. Thy-1.2-negative splenocytes were found to generate near normal amounts of LAK activity after RIL 2 incubation. Small and inconsistent LAK cell activity was generated from Thy-1.2-positive splenocytes. Ia-positive and surface immunoglobulin-positive splenocytes had little or no LAK precursor capability and did not appear to be necessary for LAK activation. Treatment of splenocytes with anti-asialo GM1 (anti-ASGM1) heterosera and complement markedly decreased their ability to generate LAK activity. At the effector stage, cytotoxic cells were of the Thy-1.2-positive, Ia-negative phenotype. Ia-depleted cells were separated into subpopulations bearing or not bearing the gamma Fc receptor (gamma FcR). The majority of cytotoxicity resided in gamma FcR-positive cells. Thus the precursors of murine LAK cells are "null" lymphocytes bearing neither T nor B cell surface markers but develop the Thy-1.2 cell surface marker in vitro, in association with the development of lytic activity for fresh tumor cells after stimulation by RIL 2.     

Different subsets of T cells in the adult mouse bone marrow and spleen induce or suppress acute graft-versus-host disease.

Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated spleen cells, all fractions protected the BALB/c recipients. Unfractionated bone marrow cells showed modest protection. Evaluation of the dose response characteristics of the suppressive activity of the low and middle density (1.060-1.068 g/ml) bone marrow cell fraction showed that reproducible protection could be achieved at a 5:1 ratio of inducing to suppressing cells. The low density fractions of both bone marrow and spleen cells had a marked depletion of typical TCR(+)-alpha beta CD4+ or CD8+ T cells, and a predominant population of TCR(+)-alpha beta CD4- CD8- T cells. Purified populations of the latter cells suppressed GVHD. Recipients given unfractionated C57BL/Ka spleen cells and protected with low-density bone marrow or spleen cells were chimeras.     

Immunosuppressor activity of rat endometrial granulated cells and their differentiation

Natural killer and natural suppressor activities of the rat endometrial granulated cells were assayed on day 13 of pregnancy or pseudopregnancy. Metrial gland granulated cells were used as endometrial granulated cells. The natural killer activities of metrial gland granulated cells and other cells were determined by means of Hashimoto-Sudo test with K562 cells as targets. The estimation of natural killer activity included removal of the cells sticking to glass from a suspension of material gland granulated cells. Cytochemically, metrial gland granulated cells were identified by the presence of PAS-positive granules in the cytoplasm after treatment of the cells with diastase and identification of a specific antigen with the help of specific antisera. The natural killer activity of metrial gland granulated cells was twice weaker than that of splenocytes from the same pregnant or pseudopregnant females. The level of natural killer activity was proportional to the content of metrial gland granulated cells in a cell system. These data suggest that the natural killer activity of metrial gland granulated cells is realized via their contact with cell targets. Natural killer and suppressor activities were determined simultaneously for metrial gland granulated cells and splenocytes of the same rat with common cell targets. When estimating the nuclear suppressor activity of metrial gland granulated cells, the splenocytes of the same rat were used as an effector in a natural killer test. Various amounts of metrial gland granulated cells were added to the effector : target system at a ratio of 50:1. The natural suppressor activity of metrial gland granulated cells did not depend on the amount of metrial gland granulated cells present in a natural killer system. After fractionation in a Percoll gradient, the highest natural killer activity was recorded in a 30% Percoll fraction. The highest and lowest natural suppressor activities were recorded in 30% and 60% Percoll fractions, respectively. The culture medium was characterized by natural suppressor activity as well. The differences in mean areas of metrial gland granulated cells in 30 and 60% Percoll fractions between the pregnant (144.7 +/- 13.4 and 75.0 +/- 12.5 microm2, respectively) and pseudopregnant (97.5 +/- 4.9 and 69.2 +/- 3.5 microm2) females were reliable. The natural killer activity was estimated in all studied 23 samples of metrial gland granulated cells, among which 18 (79.6 +/- 7.8%) displayed the natural suppressor activity as well. The absence of natural suppressor activity in five samples was combined with the absence of this activity in their culture medium and with a reduction in the mean area of metrial gland granulated cells in 30% Percoll fraction to 109.1 +/- 5.2 microm2. The data obtained confirm the known data on a low activity of metrial gland granulated cells and demonstrated for the first time the natural suppressor activity of these cells. It was concluded that the natural suppressor activity of metrial gland granulated cells is due to their differentiation from metrial gland granulated cells with natural killer activity.     

Interleukin-3 induces proliferation but not lymphokine activated killer activity from human and murine mononuclear cells

Recombinant IL-3 (rIL-3) is a potent colony stimulating factor capable of stimulating early hematopoietic pluripotential progenitor cells and of supporting the differentiation of multiple cells. IL-3 has also been shown to have effects on mature, differentiated circulating cells including eosinophils and T cells. We evaluated the role of exogenous rIL-3 in the generation of cells with LAK activity from murine splenocytes and human bone marrow, spleen, unseparated PBMC and purified null cell preparations. rIL-3 was unable to generate lytic activity from any of these populations by itself and appeared to decrease LAK activity in bone marrow cultures containing high dose IL-2, (bone marrow derived cells (n = 3) with LAK activity for fresh tumor, mean lytic units(LU) 94.6 +/- 63.5 vs 32.8 +/- 44.8 for IL-2 and IL-2 plus IL-3 cultures, respectively p2 less than 0.05). Unlike previous reports testing murine cells, IL-3 priming and subsequent culture in IL-2 of human unseparated bone marrow cells or human or murine splenocytes, failed to generate long-term cultures with lytic activity. IL-3 did, however, induce a dose dependent stimulation of bone marrow and null cell preparations (mean null cell stimulation (3H Thymidine incorporation) with IL-3, 436 +/- 168 cpm vs 9802 +/- 9799 cpm, for 0 vs 10(3) units of IL-3, respectively n = 4, p2 less than 0.05). Furthermore, in bone marrow, unseparated PBMC and null cell cultures, the addition of rIL-3 generated characteristic large blastic appearing cells with prominent basophilic granules.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphologic, phenotypic, and cytotoxic analyses of C57BL/6 murine non-parenchymal liver cells: new evidence associating murine liver large granular lymphocytes with monocyte precursors and implications for tumor immunotherapy.

Non-parenchymal liver cells (NPCs) have been implicated in murine host resistance to hepatic metastases. We have examined the relative cell number, morphology, phenotype, and cytotoxic potential of Percoll fractionated C57BL/6 murine liver NPCs. Low density (Percoll fractions 2 and 3) cells showed a large granular lymphocyte morphology and made up 76% of all NPCs recoverable, while high density (fractions 5 and 6) showed a small lymphocyte morphology and made up 10% of all NPCs. Low density cells demonstrated the following phenotype: 14% of the cells demonstrated the Thy 1.2 marker; 12%, the Lyt-2 marker; 67%, the L3T4 marker; 74%, the asialo GM1 marker; 30%, the 49H.8 marker; and 65%, the F4/80 marker. The high density cells expressed the same markers on 71%, 21%, 33%, 68%, 37%, and 19% of their cell surface, respectively. There were no differences phenotypically between high density NPCs and splenocytes except for the F4/80 expression (fractions 5 and 6 NPCs, F4/80 expression 19%, fresh splenocytes 60%). Dual color analysis of L3T4+ NPCs documented that fractions 2 and 3 cells also expressed the F4/80 marker on 85% of their cell surface and the Thy 1.2 marker on 11% of their cell surface. The high density fractions 5 and 6 L3T4+ cells expressed the F4/80 marker on 16% of their cell surface, and the Thy 1.2 marker on 89% of their cell surface. Cytotoxicity against YAC-1 [a natural killer (NK) sensitive target], MCA-102 (a NK resistant target), and WEHI-164 (a natural cytotoxicity target) were similar for fractions 2 and 3, and 5 and 6 cells. Based upon the expression of the F4/80 marker on L3T4+ cells that are Thy 1.2 negative and appear to be similar to LGLs morphologically (fractions 2 and 3 NPCs), we propose that these cells are monocyte precursors while fractions 5 and 6 cells are small lymphocytes. These findings with liver LGLs support the need for the evaluation of monocyte directed biological response modifiers in therapeutic models of murine hepatic metastases.     

Isolation of viable type II alveolar epithelial cells by flow cytometry

We developed a new method for isolating viable type II cells from fractionated and unfractionated lung cell suspensions by flow cytometry using acridine orange (AO). Fischer-344 rat lungs were dispersed into single-cell suspensions by a technique that yields a high number of cells (4-5 X 10(8) cells/lung, congruent to 85% viable), congruent to 11% of which are type II cells. Elutriated fractions from the lung cell preparation and parent, unfractionated cell suspensions were incubated with 1.0-0.02 micrograms/ml AO and analyzed by flow cytometry. Parameters analyzed included axial light loss (ALL) and red fluorescence (RF). Based on their unique RF, attributable to AO staining of type II cell lamellar bodies, and their ALL characteristics, type II pneumocytes were sorted from elutriated fractions to greater than 95% purity. Using the same approach, type II pneumocytes were sorted from unfractionated lung cell suspensions at greater than or equal to 85% purity. The viabilities of the type II alveolar epithelial cells isolated by this method range from 85% to 95%, and the ultrastructural features of the sorted cells were unaltered by AO labeling or sorting.     

Immunosuppressor activity of rat endometrial granulated cells and their differentiation

Natural killer and natural suppressor activities of the rat endometrial granulated cells were assayed on days 13 and 14 of pregnancy or pseudopregnancy. Metrial gland granulated cells were used as endometrial granulated cells. The natural killer activities of metrial gland granulated cells and other cells were determined by means of Hashimoto-Sudo test with K562 cells as targets. The estimation of natural killer activity included removal of the cells sticking to glass from a suspension of material gland granulated cells. Cytochemically, metrial gland granulated cells were identified by the presence of PAS-positive granules in the cytoplasm after treatment of the cells with diastase and identification of a specific antigen with the help of specific antisera. The natural killer activity of metrial gland granulated cells was twice weaker than that of splenocytes from the same pregnant or pseudopregnant females. The level of natural killer activity was proportional to the content of metrial gland granulated cells in a cell system. These data suggest that the natural killer activity of metrial gland granulated cells is realized via their contact with cell targets. Natural killer and suppressor activities were determined simultaneously for metrial gland granulated cells and splenocytes of the same rat with common cell targets. When estimating the natural suppressor activity of metrial gland granulated cells, the splenocytes of the same rat were used as an effector in a natural killer test. Various amounts of metrial gland granulated cells were added to the effector: target system at a ratio of 50 : 1. The natural suppressor activity of metrial gland granulated cells did not depend on the amount of metrial gland granulated cells present in a natural killer system. After fractionation in a Percoll gradient, the highest natural killer activity was recorded in a 60% Percoll fraction. The highest and lowest natural suppressor activities were recorded in 30% and 60% Percoll fractions, respectively. The culture medium was characterized by natural suppressor activity as well. The differences in mean areas of metrial gland granulated cells in 30 and 60% Percoll fractions between the pregnant (144.7 ± 13.4 and 75.0 ± 12.5 µm², respectively) and pseudopregnant (97.5 ± 4.9 and 69.2 ± 3.5 µm², respectively) females were reliable. The natural killer activity was estimated in all studied 23 samples of metrial gland granulated cells, among which 18 (79.6 ± 7.8%) displayed the natural suppressor activity as well. The absence of natural suppressor activity in five samples was combined with the absence of this activity in their culture medium and with a reduction in the mean area of metrial gland granulated cells in 30% Percoll fraction to 109 ± 5 µm². The data obtained confirm the known data on a low killer activity of metrial gland granulated cells and demonstrated for the first time the natural suppressor activity of these cells. It was concluded that the natural suppressor activity of metrial gland granulated cells is due to their differentiation from metrial gland granulated cells with natural killer activity.Translated from Ontogenez, Vol. 36, No. 1, 2005, pp. 26–34.Original Russian Text Copyright © 2005 by Podporina, Mikhailov.     

SUBCELLULAR DISTRIBUTION OF RADIOACTIVITY IN NEURONAL AND GLIAL-ENRICHED FRACTIONS AFTER INCORPORATION OF [3H]LEUCINE IN VIVO AND IN VITRO

Abstract— The distribution of protein-bound radioactivity among subcellular organelles of cerebral cortex was followed after intravenous administration of [3H]leucine and after incubation of brain slices in the presence of [3H]leucine. Neuronal and glial cell-enriched fractions were prepared by discontinuous sucroseFicol1 gradient centrifugation of cerebral cortex cell suspensions. Subcellular fractions were obtained from each of the cell prepara- tions and the protein-bound radioactivity determined after in uiuo and in vitro incorporation of [3H]leucine. The unfractionated neuronal material had a considerably higher level of protein-bound radioactivity than the glial material. The most marked neuronal-glial dif- ferences were observed in microsomes and soluble proteins, while the radioactive labelling of the nuclear and mitochondria1 fractions was similar for the two cell types.     

Exercise stress and murine natural killer cell function

Male C3He mice were trained to run on a treadmill (final speed, slope, and duration of 30 m/min, 8 degrees, 30 min/day, 5 days/week, respectively) for 10 weeks or they remained sedentary. At the end of the training program, half of the mice were sacrificed and half were given a single bout of exercise to exhaustion (50% stepwise increases in final running speed for 2-min intervals). Splenic catecholamine concentrations, splenic natural killer cell cytolytic activity against YAC-1 tumor targets, and frequency of asialo GM1 (a murine natural killer cell surface glycolipid)-positive splenocytes were assessed. Exhaustive exercise in both trained and untrained mice reduced the in vitro killing of tumor targets by splenic natural killer cells relative to killing by splenocytes from mice which did not undergo the acute exercise bout (P less than 0.05). The frequency of asialo GM1-positive splenocytes was also reduced in the exhaustively exercised animals (P less than 0.05). Training alone, without the additional stress of exhaustive exercise, reduced the frequency of asialo GM1-positive splenocytes relative to a sedentary condition (P less than 0.05), but did not compromise natural killer cell cytolytic activity against the tumor targets. Splenic epinephrine concentrations in the exhaustively exercised animals were elevated 3- to 5-fold above the concentrations observed in trained and sedentary mice. These results suggest that a single, acute exercise bout reduces the capacity of splenic natural killer cells to kill tumor targets in vitro and that training enhances splenic natural killer cell cytolytic activity, on a per cell basis, against tumor targets.     

The role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice

Gastritis due to Helicobacter pylori in mice and humans is considered a Th1-mediated disease, but the specific cell subsets and cytokines involved are still not well understood. The goal of this study was to investigate the immunopathogenesis of H. pylori-induced gastritis and delayed-type hypersensitivity (DTH) in mice. C57BL/6-Prkdc(scid) mice were infected with H. pylori and reconstituted with CD4+, CD4-depleted, CD4+CD45RB(high), or CD4+CD45RB(low) splenocytes from wild-type C57BL/6 mice or with splenocytes from C57BL/6(IFN-gamma-/-) or C57BL/6(IL-10-/-) mice. Four or eight weeks after transfer, DTH to H. pylori Ags was determined by footpad injection; gastritis and bacterial colonization were quantified; and IFN-gamma secretion by splenocytes in response to H. pylori Ag was determined. Gastritis and DTH were present in recipients of unfractionated splenocytes, CD4+ splenocytes, and CD4+CD45RB(high) splenocytes, but absent in the other groups. IFN-gamma secretion in response to H. pylori Ags was correlated with gastritis, although splenocytes from all groups of mice secreted some IFN-gamma. Gastritis was most severe in recipients of splenocytes from IL-10-deficient mice, and least severe in those given IFN-gamma-deficient splenocytes. Bacterial colonization in all groups was inversely correlated with gastritis. These data indicate that 1) CD4+ T cells are both necessary and sufficient for gastritis and DTH due to H. pylori in mice; 2) high expression of CD45RB is a marker for gastritis-inducing CD4+ cells; and 3) IFN-gamma contributes to gastritis and IL-10 suppresses it, but IFN-gamma secretion alone is not sufficient to induce gastritis. The results support the assertion that H. pylori is mediated by a Th1-biased cellular immune response.     

Macrophages activated as suspension cultures with lymphocyte mediators devoid of antigen become cytotoxic for tumor cells.

Normal peritoneal exudate cells (PEC) were activated as suspension cultures either in mediator-rich supernatants from o-chlorobenzoyl-bovine gamma-globulin (OCB-BGG) stimulated lymphocytes or in antigen-free Sephadex fractions from these supernants. After 24 hr incubation thration. The adherent cell fractions of PEC, recovered by trypsinization from monolayers and activated by this technique, were as cytotoxic as unfractionated PEC. Lymphocyte supernatants and antigen-free fractions of the supernatants induced comparable macrophage-mediated tumor cytotoxicity. Treatment of activated macrophages with trypsin did not alter their cytotoxic capacity.     

Expression profiling of lymphocyte plasma membrane proteins

The physicochemical properties of plasma membrane proteins of mammalian cells render them refractory to systematic analysis by two-dimensional electrophoresis. We have therefore used in vivo cell surface labeling with a water-soluble biotinylation reagent, followed by cell lysis and membrane purification, prior to affinity capture of biotinylated proteins. Purified membrane proteins were then separated by solution-phase isoelectric focusing and SDS-PAGE and identified by high-pressure liquid chromatography electrospray/tandem mass spectrometry. Using this approach, we identified 42 plasma membrane proteins from a murine T cell hybridoma and 46 from unfractionated primary murine splenocytes. These included three unexpected proteins; nicastrin, osteoclast inhibitory lectin, and a transmembrane domain-containing hypothetical protein of 11.4 kDa. Following stimulation of murine splenocytes with phorbol ester and calcium ionophore, we observed differences in expression of CD69, major histocompatibility complex class II molecules, the glucocorticoid-induced TNF receptor family-related gene product, and surface immunoglobulin M and D that were subsequently confirmed by Western blot or flow cytometric analysis. This approach offers a generic and powerful strategy for investigating differential expression of surface proteins in many cell types under varying environmental and pathophysiological conditions.     

Antibiotic Production of Hyphal Fractions of Streptomyces griseus: II. Streptomycin Production of Different Fractions Obtained by Density Gradient Centrifugation

The streptomycin-producing activity (SPA) of hyphal fractions from washed mycelium of submerged cultures of Streptomyces griseus strain 52-1, as obtained by density gradient centrifugation, was investigated. Activity of the various fractions differed strongly in intensity. The highest SPA was evident in the unfractionated mycelium. A synergistic effect upon SPA was found in the interaction of cultures of different ages, and a 55% increase in yield was obtained by mixing the 48- and 72-hr cultures. A synergistic effect occurred in all combinations studied. By use of fractions obtained from 72-hr mycelium for inoculation, differences in streptomycin production were noted. Some inoculum fractions yielded a greater amount of streptomycin (36%) than the unfractionated mycelial inoculum.