Age-related suppression of murine lymphoid cell blastogenesis by marijuana components

Marijuana components modulate a variety of immune response parameters. The cannabinoids delta 9-tetrahydrocannabinol (THC) and 11-hydroxy-tetrahydrocannabinol (11 OH-THC) are known to depress the in vitro proliferative response of murine lymphoid cells to the mitogens concanavalin A (Con A) and phytohemagglutinin (PHA). In the present report the effects of THC and 11 OH-THC on adult thymus and spleen cells were compared to effects on lymphoid cells of those organs from juvenile mice at various ages. The results demonstrate differences in susceptibility to cannabinoid-induced suppression by lymphoid cells from different organs and different age mice. In adults, thymus cells were suppressed more readily than spleen cells. Splenocytes from mice under 2 weeks old were suppressed much more readily than those from older mice. Cell populations from organs with higher proportions of L3T4+/Lyt2- cells were more difficult to suppress. The possible mechanisms involved and directions for future work are discussed.     

Contrasting Effects of thc on Adult Murine Lymph Node and Spleen Cell Populations Stimulated with Mitogen or Anti-CD3 Antibody

Marijuana, and specifically its psychoactive component, THC, can up or down regulate lymphocyte proliferation in murine spleen cells depending in part on the method used to stimulate the cells. This study identifies a difference in THC induced disregulation using cells derived from two different secondary lymphoid organs, the spleen and the lymph node. It was found that THC treatment of mitogen (concanavalin A or phytohemagglutinin) stimulated cells derived from either organ resulted in suppression of the proliferative response. In contrast, spleen cells stimulated with anti-CD3 antibody and treated with low doses of THC displayed an enhanced proliferation whereas the response in lymph nodes did not change. The cell type involved with this THC immunoenhancement in spleen cells was found to be the Ly2 cell. Further differences in the THC modulation of Ly2 spleen cells as compared to lymph node cells were noted following stimulation with PHA. Proliferation of Ly2 cells of splenic origin was inhibited with low doses of THC whereas the Ly2 cells of lymph node origin were more resistant to this drug induced suppression. This study, therefore, demonstrates differences in the immunomodulatory capability of THC dependent upon the organ source of the lymphocytes.     

Contrasting Effects of thc on Adult Murine Lymph Node and Spleen Cell Populations Stimulated with Mitogen or Anti-CD3 Antibody

Abstract

Marijuana, and specifically its psychoactive component, THC, can up or down regulate lymphocyte proliferation in murine spleen cells depending in part on the method used to stimulate the cells. This study identifies a difference in THC induced disregulation using cells derived from two different secondary lymphoid organs, the spleen and the lymph node. It was found that THC treatment of mitogen (concanavalin A or phytohemagglutinin) stimulated cells derived from either organ resulted in suppression of the proliferative response. In contrast, spleen cells stimulated with anti-CD3 antibody and treated with low doses of THC displayed an enhanced proliferation whereas the response in lymph nodes did not change. The cell type involved with this THC immunoenhancement in spleen cells was found to be the Ly2 cell. Further differences in the THC modulation of Ly2 spleen cells as compared to lymph node cells were noted following stimulation with PHA. Proliferation of Ly2 cells of splenic origin was inhibited with low doses of THC whereas the Ly2 cells of lymph node origin were more resistant to this drug induced suppression. This study, therefore, demonstrates differences in the immunomodulatory capability of THC dependent upon the organ source of the lymphocytes.     

In vivo effects of an acute nonimmunological inflammation in rats on the lymphoproliferative response to mitogens

Lymphoid cells (spleen, lymph node and thymus) derived from rats after induction of an acute nonimmunological inflammatory reaction responded to various mitogens (Phytohemagglutinin, PHA; Concanavalin A, Con A; Lipopolysaccharide, LPS) with increased proliferation when compared with cells derived from normal animals. In the absence of mitogens, lymphoid cells from animals undergoing an acute nonimmunological inflammation demonstrated enhanced proliferation compared with cells from normal animals. These results clearly demonstrated that during acute nonimmunological inflammation the reactivity of lymphoid cells was increased.     

Alterations in the Mitogen Responsiveness of Lymphocytes from Mice Bearing Moloney Sarcoma Virus Induced Tumors

The effects of Moloney Sarcoma Virus (MSV) induced tumor growth dynamics on the blastogenic responsiveness of lymphocytes from BALB/c mice were investigated. Lymphocytes from spleen, thymus and lymph node pools were tested for responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A). Results showed a significant decrease in PHA-induced blastogenesis of all lymphocytes tested at the time of maximal tumor volume, with a return to normal responsiveness as the tumor regressed. In contrast, a differential dose dependent Con A response occurred in spleen and thymus lymphocytes. A decreased ³H-thymidine uptake occurred at optimal Con A dose, correlating with the PHA decrease. However, at a lower Con A dose an increased response was observed, beginning shortly before the PHA depression and continuing until regression of tumor began. This phenomena was not observed in lymph node lymphocytes.

Based upon these observations, we suggest that the cell or cells responsible for the transient suppression of PHA responsiveness may be Con A responsive T lymphocytes.     

Alterations in the mitogen responsiveness of lymphocytes from mice bearing Moloney sarcoma virus induced tumors.

The effects of Moloney Sarcoma Virus (MSV) induced tumor growth dynamics on the blastogenic responsiveness of lymphocytes from BALB/c mice were investigated. Lymphocytes from spleen, thymus and lymph node pools were tested for responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A). Results showed a significant decrease in PHA-induced blastogenesis of all lymphocytes tested at the time of maximal tumor volume, with a return to normal responsiveness as the tumor regressed. In contrast, a differential dose dependent Con A response occurred in spleen and thymus lymphocytes. A decreased 3H-thymidine uptake occurred at optimal Con A dose, correlating with the PHA decrease. However, at a lower Con A dose an increased response was observed, beginning shortly before the PHA depression and continuing until regression of tumor began. This phenomena was not observed in lymph node lymphocytes. Based upon these observations, we suggest that the cell or cells responsible for the transient suppression of PHA responsiveness may be Con A responsive T lymphocytes.     

Immunoregulation in experimental filariasis. IV. Induction of non-specific suppression following in vitro exposure of spleen cells from infected jirds to Brugia pahangi antigen.

Studies with inbred jirds chronically infected (greater than 5 months) with Brugia pahangi have demonstrated splenic suppressor cells which modulate in vitro responsiveness to mitogens and parasite antigens. The stimuli which induce suppression were characterized by analysing the effect of activated cells from inbred normal or B. pahangi-infected jirds on the PHA and PWM responsiveness of cultures on normal cells. Regulatory cells were stimulated in vitro with concanavalin A (Con A; 5 micrograms/ml) or an extract of adult B. pahangi (20 micrograms/ml) for 72 hr and irradiated (1500 rads) prior to cocultivation with normal cells. Addition of Con A-activated normal spleen cells to normal cells produced moderate suppression of PHA and enhancement of PWM responsiveness. However, Con A-stimulated spleen cells from infected animals consistently suppressed both the PHA and PWM responsiveness of normal cells by 80-90%. Spleen cells from chronically infected jirds were also induced by B. pahangi antigen to suppress both the PHA and PWM responsiveness of normal lymphocytes. In contrast, spleen cells from animals 3-15 weeks after infection and lymph node cells from all time points were capable of suppressing only PWM responses when stimulated by antigen. Normal spleen cells were not induced by B. pahangi antigens to exhibit immunoregulatory activity. The suppression mediated by antigen-induced spleen cells from chronically infected jirds was partially or totally alleviated by removal of non-specific suppressor cells which are plastic adherent and cyclophosphamide-sensitive, or by removal of antigen-specific suppressor cells which bear receptors for histamine. the results suggest the involvement of regulatory cell circuits in experimental filarial infections.     

In vitro and in vivo suppressive effects of delta-9-tetrahydrocannabinol on interferon production by murine spleen cells

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, has been reported to be suppressive on some immune functions. Since interferons (IFNs) are important immunomodulatory proteins, the effect of in vivo or in vitro administration of THC on induction of IFN by various mitogens was examined. Splenocytes from normal mice in the presence of THC produced significantly less IFN when stimulated by phytohemagglutinin (PHA), concanavalin A (Con A), or Escherichia coli lipopolysaccharide (LPS). Induction of IFN by a bacterial antigen, Legionella pneumophila bacterial cells, was also suppressed by THC. Also, splenocytes which were incubated up to 24 h in the presence of THC partially recovered responses to mitogens when cells were washed before stimulation. This suggested that THC must be present in order to mitigate IFN induction. Splenocyte cultures from mice which were chronically injected with THC for 6-8 weeks were also less responsive to induction of IFN by the various mitogens. These results suggest that at least part of the immunosuppressive effects of THC may be related to depressed IFN production by stimulated lymphocytes. Since Con A and PHA are T cell mitogens and LPS is considered to be a macrophage and B cell stimulator, suppression of IFN production by these classes of cells indicate a wide range of effects of THC.     

Studies on the immunobiology of rnu/rnu "nude" rats with congenital aplasia of the thymus

A detailed study of the recently described “nude” rat, which suffers from congenital aplasia of the thymus, has shown that this animal is grossly deficient in functional mature T cells. Histological investigation revealed that those areas of lymph node, spleen and Peyer's patches, which are considered to be thymus-dependent, were markedly depleted of lymphocytes. The areas of lymph node and Peyer's patches, which are normally considered to be thymus-independent, were also partially depleted of lymphocytes, whereas the thymus-independent areas of splenic white pulp showed greater activity than in control animals. Indirect immunofluorescence with a monoclonal anti-T cell antibody showed that lymph node and spleen cells were severely depleted of mature T cells, whereas staining with anti-immunoglobulin antisera revealed an increased percentage of B cells in these organs. The nature of the residual anti-T⁺ cells in rnu/rnu lymph node and spleen is not clear, but at least some of these may be precursor T cells. Spleen cells from rnu/rnu rats gave no response to phytohemagglutinin (PHA), and only occasional small responses to concanavalin A (Con A), but showed normal responses to Staphylococcus aureus bacteria. The absence of responses to PHA and Con A was not caused by suppressor cells nor by any defect in helper cell activity. In so far as PHA and Con A are considered to be T cell-dependent mitogens and S. aureus a T cell-independent mitogen, these results indicate that rnu/rnu rats lack functional T cells.     

Ontogeny of T-cell mitogen response in Lewis rats: I. Culture conditions and developmental patterns

Spleen and thymus cells from female Lewis rats 2 to 220 days old were cultured with phytohemagglutinin (PHA) or Concanavalin A (Con A). The optimal dose of Con A for mitogenesis ranged from 1 ug/ml for suckling rats to 125 ug/ml for adult rats. Thymus cells differed from spleen cells in that they required 72 hours for maximum thymidine incorporation; whereas spleen cells attained maximum incorporation within 48 hours. Thymidine uptake in unstimulated spleen cell cultures was high in the neonate and decreased with age. Maximum splenic cell responses to Con A increased from very low levels in the 2-day old to adult levels by 3 weeks coincident with weaning. Maximum PHA responses of splenocytes matured more slowly, reaching adult levels by 8 weeks. Thymus cell responses to Con A and PHA were mature from birth     

Cell-mediated immunity in Treponema pallidum infected rabbits: in vitro response of splenic and lymph node lymphocytes to mitogens and specific antigens.

Peripheral blood lymphocytes from Treponema pallidum infected rabbits respond poorly to mitogen and specific antigens when cultured in the presence of autologous serum. Reactivity of lymphocytes from the spleen and popliteal lymph nodes of T. pallidum infected rabbits have therefore been examined by lymphocyte transformation using the mitogens phytohaemagglutinin (PHA) and concanavalin A (Con A) and extracts of T. pallidum. Spleen cell populations, both T cell enriched (by nylon wool elution) and non-nylon wool treated, which respond to T. pallidum as early as ten days post infection in normal serum, were suppressed in responses to T. pallidum when cultured in autologous serum. The same lymphocytes responded normally to PHA and Con A. Lymph node cells from infected rabbits responded normally to both T. pallidum antigen and mitogens in either autologous or normal rabbit serum. These data indicate that splenic lymphocytes are sensitive to regulatory factors in autologous serum during the early stages of T. pallidum infection whereas lymph node cells are not.     

Genetic difference in the proliferative response to T mitogens between Hi/PHA and Lo/PHA lymphocytes is independent of accessory cell function

The role of the macrophage as accessory cell in the proliferative response of lymphocytes to phytohemagglutinin (PHA) was studied in two lines of mice genetically selected for high and low responsiveness to T mitogens. Adherent cell depletion of lymph node cells abrogated the low (Lo)/PHA response, but only partially inhibited the high (Hi)/PHA response. Addition of peritoneal cells provided either by Hi/PHA or by Lo/PHA mice equally restored Hi/PHA responsiveness but had only a slight reconstituting effect on the inhibited Lo/PHA response. Equivalent enhancement or suppression of proliferation of untreated lymph node cells was obtained by the addition of increasing percentages of each of the two peritoneal cell populations. However, the maximum level of the Lo/PHA response never reached that of Hi/PHA cells. These data indicate that the bidirectional selective breeding has not modified the potentialities of the macrophages as accessory cells but has resulted in an impaired response of Lo/PHA lymphocytes to the signals delivered either by accessory cells or by T mitogens.     

During frog ontogeny, PHA and Con A responsiveness of splenocytes precedes that of thymocytes

The in-vitro proliferation of splenocytes and thymocytes from Xenopus laevis-gilli (hybrid clone LG-15) to the T cell mitogens, concanavalin A (Con A) and phytohaemagglutinin-P (PHA), were examined at specific stages of larval development (stages 51-66 of Nieuwkoop & Faber, 1967) and at 2 months post-metamorphosis. The responses of splenic lymphocytes to each mitogen were significant at all stages with stimulation indices ranging from 1.9 to 50.5 and 2.6 to 45.5 for PHA and Con A, respectively. Stage-related differences in responses of splenocytes to both mitogens suggest two waves of emergence of proliferative activity during development, divided by periods of diminished responsiveness during the metamorphic crisis. In contrast to the responses observed with splenocytes, proliferation of thymocytes cultured with either mitogen was barely detectable, with stimulation indices ranging from 1.2 to 6.9 and 1.4 to 2.9 for PHA and Con A, respectively. These minimal responses were observed only when thymocytes were cultured at relatively high cell density (5 X 10(5) cells/ml); they were not improved by increased or decreased concentrations of mitogen or by increased concentrations of fetal calf serum (5 or 10%) in the medium. Co-culture of larval thymocytes with autologous splenocytes and each mitogen did not consistently increase thymocyte responses suggesting that the defect in thymocyte responsiveness is not due to lack of accessory cells. These findings suggest that if PHA- and Con A-reactive cells are present in the thymus, they are present in relatively low numbers at all stages of larval development. The pattern of early mitogen responsiveness in the spleen at a time when the thymus is unresponsive contrasts with that observed in mammalian development in which thymocytes become responsive to mitogens in fetal stages and mitogen responsiveness appears in the spleen only around the time of birth. The apparent inactivity of larval thymocytes may reflect a population of cells that can become tolerant to those neo-self-antigens that arise during and after metamorphosis. If so, the larval amphibian thymus may provide a model to study the early events of thymocyte 'education' and differentiation in a broader time framework than is possible with fetal mammals.     

Maturation of mitogenic response in foetal guinea-pig.

Lymphocytes obtained from thymus, spleen and heart blood of 33-day-old guinea-pig foetuses and from lymph nodes of 48-day-old foetuses onwards were stimulated by phytohaemagglutinin (PHA). concanavalin A (Con A) and dextran sulphate (DxS). The results, based on the analysis of ninety-five foetuses, indicated that the mitogenic responses of the guinea-pig T cells matured first in the thymus and then in the spleen and lymph nodes, in that order. The PHA and Con A responses in the thymus, spleen and lymph nodes and the DxS response in the spleen improved with the increasing age of the foetus. A clear improvement of the mitogenic responses did not take place in the blood. Thus, at birth the guinea-pig is immunologically mature as regards the PHA and Con A responses in the thymus, spleen and lymph nodes and the DxS response in the spleen. Regarding the maturation of PHA and Con A responses by lymphocytes from the blood, further studies are needed, since at birth both of these responses are clearly below the adult level.     

Reactivity differences among human T cells from blood and lymphoid organs, analysed by limiting dilution: correlation with specific gravity and binding of peanut lectin.

Human T cells from peripheral blood, cord blood, thymus, spleen and lymph node were analysed for their proliferative response capacity to mitogens, for their specific gravity and size, and for their binding capacity of peanut agglutinin. A close correlation was found between these parameters: thymocytes and T cells from spleen were consistently heavier and smaller, and showed a lower proliferative response capacity, than T cells from blood or lymph node. Similarly, within each cell population, the small, heavy cells were least reactive. The limiting-dilution analysis revealed that heavy T cells from peripheral blood contain a lower number of reactive cells than the light peripheral T lymphocytes. Because heavy T cells from the thymus bound more peanut lectin than did light cells, it is speculated that the differences between T cells of high and low specific gravity might reflect differences in maturation level.     

Alteration of lymphocyte reactivities by thyroid hormones

The effects of thyroid hormones (L-T4, L-T3 and rT3) on the proliferative response of rabbit peripheral blood lymphocytes to T-cell mitogens, PHA and Con A, and B cell specific goat anti-rabbit light chain antibodies (Anti-L) were investigated. It was observed that L-T4 potentiated the lymphocyte response to mitogens and Anti-L in a dose-dependent manner: 10(-9) M and 10(-8) M had no effect while 10(-7)-10(-5) M significantly enhanced the lymphocyte response. L-T3 (10(-11)-10(-8) M) had no effect on the lymphocyte response to PHA and Con A. At 10(-7) M, L-T3 inhibited the response to PHA but not Con A. L-T3 (10(-11)-10(-7) M) suppressed the lymphocyte response to Anti-L. The suppression was directly proportional to the L-T3 concentration. rT3 (10(-11)-10(-7) M) inhibited the proliferative response to PHA and Anti-L in a dose-related manner. Its effect on the lymphocyte response to Con A was stimulatory at 10(-11) M but inhibitory at higher concentrations (10(-8) and 10(-7) M). rT3 suppressed the enhancement by L-T4 of the lymphocyte response to the mitogens and Anti-L. The degree of suppression was proportional to its concentration. This data indicated that thyroid hormones can alter the reactivities of lymphocytes. The direction and magnitude of the alteration appear to depend on the concentration of a specific thyroid hormone encountered by the responding cells.     

Age-related changes of mitogen responsiveness in different lymphoid organs from outbred NMRI mice

Lymphocytes from spleen, thymus and lymph nodes from individual young adult (3--4 months) and aged (26--30 months) NMRI mice were stimulated with the mitogens Con A, PHA and LPS. 24 hours later, the number of cell with increased RNA-content (G1 cells) was determined by cytofluorometry. In parallel the 3H-thymidine incorporation after 48 hours was measured for the same cell samples. Aged animals in average produced less G1 cells and incorporated less 3H-thymidine as compared to young adults. By calculating the 3H-thymidine incorporation per G1 cell, the proliferative capacity of mitogen-induced G1 cells can be estimated. These ratios are lower in aged mice as compared to young adult, suggesting that in these animals not only less cells can be activated as measured by cytofluorometry, but also from these activated cells again fewer continue the cell cycle by initiating DNA-synthesis. In response to Con A and PHA, aged mice in average produce less G1 cells in all of the three lymphoid organs tested. In response to LPS, however, the young adult produced only few G1 cells in lymph nodes and practically none in thymus, whereas in aged animals a considerable number of G1 cells was found in both organs. Corresponding results were found for the 3H-thymidine incorporation. These results indicate that in addition to the reduction of the mitogen-response an age-related change in the distribution of mitogen-responsive cells in the different lymphoid organs takes place.     

Nematospiroides dubius: Modulation of the host immunological responsive

Altered immunological reactivity occurred in mice infected with the larval stages ofNematospiroides dubius. This was shown by an increased responsiveness of mouse spleen lymphocytes and suppressed responsiveness of mesenteric lymph node (MLN) lymphocytes to phytohaemagglutinin (PHA) and concanavalin A (Con A) during the first 10–15 days after infection while at the same time spleen, MLN, and cervical lymph node (CLN) lymphocyte reactivity to lipopolysaccharide (LPS) was suppressed. Splenomegaly, with a marked increase of white pulp in the spleen, began 3 days after infection and there was a concomitant decrease in the numbers of cells in the MLN with depletion of the paracortical and medullary areas in particular.N. dubius larvae cultured in vitro produced a dialysable immunosuppressive factor which inhibited the proliferation of spleen lymphocytes from normal and immune mice and sheep in the presence of mitogens. Serum collected from infected mice also suppressed mitogen-induced transformation of normal mouse lymphocytes.     

The proliferative capacities of popliteal lymph node lymphocytes and of their functionally distinct T-cell subpopulations from young-adult and old mice

Young adult and old mice were immunized by footpad injection of dinitrophenyl-conjugated bovine gamma-globulin (DNP-BGG) in complete Freund's adjuvant. A comparison of lymph node weight and total number of nucleated cells per lymph node as a function of time after antigen injection demonstrated a significantly greater absolute increase in lymph node weight and peak number of nucleated cells per lymph node in young-adult than in old animals. However, as judged by this increase in total nucleated cells, other than being delayed in old mice, the magnitude of these in situ proliferative responses appeared comparable for young-adult and old mice. That is, the antigen-stimulated to non-stimulated cell ratio did not differ significantly between young-adult and old animals. This was because lymph nodes from old animals prior to antigen injection always weighed less and had fewer numbers of nucleated cells compared with young-adult animals. Therefore, the in vitro cellular proliferative response of three T-cell-enriched lymphocyte subpopulations from young-adult and old mice was further characterized. This was done by measuring [3H]thymidine incorporation following antigen- (DNP-BGG)- or mitogen-[phytohemagglutinin (PHA) or Concanavalin A (Con A)]-induced proliferation and assessing their quantitative and/or qualitative requirements for macrophages. In contrast to the markedly reduced proliferation of the two T-cell subpopulations from popliteal lymph nodes which respond to PHA and Con A in old animals primed 21-days earlier with DNP-BGG, antigen-induced in vitro cellular proliferation of the small T-cell subset in old mice specifically responsive to the immunizing antigen DNP-BGG always responded as well as, if not better than, cells from young-adult mice.     

Mitogenic response of human thymocytes: identification of functional Ca2+-dependent and independent signals.

In contrast to peripheral blood mononuclear cells (PBMC), human thymocytes do not exhibit a proliferative response to the T cell mitogens phytohaemagglutinin (PHA), concanavalin A (Con A), or Staphylococcal protein A (SPA). In thymocytes and PBMC, Con A and PHA induce increases in free cytosolic calcium concentrations [( Ca2+]i). Since both Con A and PHA induce similar increases in [Ca2+]i in thymocytes and PBMC, the absence of thymocyte proliferation was not due to an inability to induce an increase in [Ca2+]i. The lack of proliferative response was secondary to the failure of the mitogens to induce interleukin 2 (IL-2) production. Incubation of mitogen-treated thymocytes with phorbol esters reconstituted IL-2 production and the proliferative response indicating that the cells were indeed activated by the mitogens. Similarly, addition of exogenous recombinant IL-2 also induced mitogen-treated thymocytes to proliferate. This IL-2-dependent proliferation established that SPA, Con A, and PHA triggered the expression of biologically active IL-2 receptors. Since an increase in [Ca2+]i is a prerequisite, and possibly a trigger, for IL-2 production, the failure of PHA, Con A, or SPA to result in thymocyte proliferation may be due to an inability of thymocytes to respond to increases in [Ca2+]i with subsequent IL-2 production.