The Effect of Delta-9-Tetrahydrocannabinol and 11-Hydroxy-Delta-9-Tetrahydrocannabinol on T-Lymphocyte and B-Lymphocyte Mitogen Responses

Abstract

Previous studies have shown that delta-9-tetrahydrocanna-binol (THC) suppresses T-lymphocyte proliferation when added to human cell cultures. We report that THC when added to mouse splenocyte cultures suppressed T-lymphocyte (Con A, PHA) and B-lymphocyte (LPS) mitogen-induced proliferation. Although the ED₅₀ concentrations (5ug/ml; 1.6×10^?5 M) of THC were similar for suppressing all three mitogen responses, higher threshold concentrations of drug were required to effect suppression of the T-lymphocyte mitogen responses. Complete suppression of T- and B-lymphocyte responses was achieved with THC concentrations (8ug/ml or 2.6×10^?5 M) which were not directly toxic as judged by vital dye exclusion. The hydroxylated metabolite of THC, 11-hydroxy-THC, was observed to be much less potent in the inhibition of lymphocyte proliferation. However, as with the parent compound, B-lymphocyte responses appeared to be the most affected by the drug. Additional studies demonstrated that both T- and B-lymphocyte proliferation is rapidly suppressed following THC treatment, not affected by a 24 hr. pretreatment with THC, and not as readily suppressed by THC in cultures containing 20% serum. Thus, THC appears to inhibit both T- and B-lymphocyte proliferation with B-lymphocyte responses displaying greater inhibition at lower drug concentration. The 11-hydroxy metabolite is much less suppressive in this system than the parent compound.     

Enhancement of Human B Lymphocyte Differentiation in Vitro by Thyroid Hormone

A study was made of the regulatory influence of a thyroid hormone, triiodothyronine (T₃), on human blood T- and B-lymphocyte responses in vitro. Blood lymphocytes were stimulated with the T-cell mitogens concanavalin A (Con A) and phytohaemagglutinin (PHA), with the T-dependent B-cell activator pokeweed mitogen (PWM), and with the T-independ-ent B-cell activator Staphylococcus aureus Cowan I bacteria in the presence of various hormone concentrations. T₃ did not stimulate T-cell proliferation. The number of immuno-globulin-containing and -secreting cells (plasmablasts) was increased in PWM and Staph. aureus cultures treated with T₃. The maximal enhancement was reached at a concentration of 10⁻⁹-10⁻⁷ M T₃. Cell fractionation techniques revealed that T₃ apparently had a direct stimulatory effect on B-cell differentiation.     

Expression of heat shock proteins 65, 72 and 90 in mercuric chloride-treated human peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) were challenged with different concentrations of mercuric chloride and the expression of heat shock protein (hsp) 65, 72 and 90, were investigated by an indirect peroxidase method as well as the proliferation was measured by uptake of tritiated thymidine into DNA. Hsp 65 immunoreactivity was peripherally located in the cells at a mercuric chloride concentration of 5.5-2.8×10^-5 mol/L, which also gave a decreased DNA synthesis. Hsp 72 positive cells, with a nuclear and cytoplasmic immunoreactivity, were found at a concentration of 5.5-2.2×10^-5 mol/L. Hsp 90 positive cells also with an immunoreactivity at a nuclear and cytoplasmic level, were found at 1.1×10^-5-5.5×10^-6 mol/L. A stimulated DNA synthesis was obtained already at 2.2×10^-5 mol/L and was found down to 5.5×10^-6 mol/L. High concentrations of mercuric chloride might induce expression of hsp 65 and 72 as a protective mechanism. Hsp 90 as well as hsp 72 might be involved in the proliferation of human PBMC to mercuric chloride. Thus, this expression of hsp 72 may have a dual role.     

Effects of Dynorphin on the Pha-Induced Lymphocyte Proliferation in Vitro

The effect of the opioid peptide dynorphin (DYN) on PHA-induced lymphocyte proliferation has been evaluated in the present study.

A significant increase in PHA-induced lymphocyte activation was observed when DYN was added to cultures 48 hr after the mitogenic stimulation. This effect occurred at suboptimal (3.12 and 6.25 ug/ml) PHA concentrations and at DYN doses ranging from 10^-9 to 10^-12 M. Conversely, DYN did not affect the lymphocyte blastogenic process either when added before, or simultaneously or after intense PHA (12.5 ug/ml) stimulation.

Naloxone preincubation did not modify the above described effects.

Results suggest that DYN might play a role in neuroendocrine regulation of the lymphocyte blastogenic process.     

Chemiluminescence in Human Whole Blood: Modulation by the Cocarcinogens Phorbol Diester and Polychlorobiphenyls

A human whole blood chemiluminescence (CL) assay was established using zymosan as cell activator. Aroclor 1254 was found to inhibit this CL response in a direct linear relation to its concentration, (50% inhibitory dose, (ID₅₀) equal to 5 × 10^-4 M) in diluted blood samples of 10 normal human subjects. In comparison the ID₅₀ of other inhibitors was 1.3 × 10^-3 M for ethylenediamine tetraacetic acid, 3.3 × 10^-3 M for ascorbic acid, 4 × 10^-3 M for reduced glutathione, 1.2 × 10^-3M for ethanol, 2.5 × 10^-1 for methanol and 3.7 × 10^-1 M for dimethyl sulfoxide. Using 12-o-tetradecanoyl-phorbol-13-acetate (TPA) as cell activator the CL response was likewise inhibited by Aroclor 1254 with an ID₅₀ of 4.5 × 10^-4 M. However, it was found that Aroclor 1254 alone has a stimulatory CL effect on otherwise unactivated cells. To compare the mechanisms involved in the CL elicited by the three stimulants zymosan, TPA and Aroclor 1254, the CL signal was measured in the presence of cytochalasin B. Cytochalasin B inhibited zymosan-induced CL, had a smaller inhibitory effect on TPA-induced CL but it could augment the CL response initiated by Aroclor 1254. This pattern of responses implicates Aroclor 1254 in the activation of eicosanoid metabolism as it matches the differential responses reported for arachidonic acid.     

Ontogeny of T-cell mitogen response in Lewis rats: II. Early appearance and loss of suppressor activity

Spleen cells from rats 2 to 132 days old were cultured with 1 - 125 ug/ml Concanavalin A (Con A). At high doses of Con A, the high spontaneous thymidine uptake of spleen cells from rats 15 to 21 days old was suppressed, whereas spleen cells from younger rats showed no suppression of spontaneous mitogenesis at equally high Con A doses. Removal of either plastic⁻, nylon wool⁻, or carbonyl iron (cFe) adherent cells not only removed suppression of background by high Con A doses, but also allowed mitogenic responses at adult levels in normally unresponsive 15 to 21 day old pups. Low doses of X-irradiation did not cause a similar loss of suppression. We suggest that although there is an influx of ConA responsive cells into the rat spleens at 15 to 16 days, the mitogen responses of these cells are suppressed by an adherent cell population which is activated by high doses of Con A.     

Inhibitoty Effects of Local Anesthetics on Migration, Extracellular Release of Lysosomal Enzyme, and Superoxide Anion Production in Human Polymorphonuclear Leukocytes

This study examined the effects of four typical local anesthetics, lidocaine, prilocaine, procaine and tetracaine, on the functioning of human polymorphonuclear leukocytes (PMN). PMN were stimulated by fMet-Leu-Phe (FMLP) or phorbol myristate acetate (PMA) to elicit chemotaxis, extracellular release of beta-glucuronidase (BGL) and superoxide anion (SOA) production. the four agents inhibited chemotaxis efficiently and in a concentration-dependent manner but had only weak effects on the release of BGL. the effect of tetracaine was strongest, followed by lidocaine, then prilocaine, whereas the effect of procaine was blunt. the 50% inhibitory concentrations (IC₅₀ in molarity) of the four local aesthetics for chemetaxis were as follows: tetracaine=4.1×10^-4, lidocaine=3.2×10^-3, prilocaine=3.6×10, procaine=4.9×10^-3, those for SOA production induced by FMLP were : tetraaine=3.1×10^-4, lidocine=5.9×10^-3, prilocaine=1.9×10^-2, procaine=1.2×10^-2, those for SOA production indced by PMA were : tetracaine=1.1×10^-3, lidocine=1.2×10^-2, prilocaine=1.5×10-2, procaine=2.5×10^-2, and those for rlease of BGL were : tetracaine=1.6×10^-, lidocaine=5.3×10^-3, prilocaine=2.8×10^-2, procaine=1.2×10^-1. the IC₅₀ seemed to relate to the anesthetic's chemical structures and their inhibitory properties on PMN functions, as lidocaine and prilocaine, which are aminoamide type anesthetics, preferentially inhibited chemotaxis, whereas tetracaine and procaine, aminoester type anesthetics, inhibited SOA production induced by FMLP. the results suggest that the inhibitory effects of local anesthetics on human PMN functions are also correlated with local anesthetic potency and vary according to differences in their chemical structures.     

Gastrointestinal Regulatory Peptides Modulate Mouse Lymphocyte Functions Under Serum-Free Conditions In Vitro

Conditions are described for performing mitogen (Concanavalin A, Con A; lipopolysaccharide, LPS) and mixed lymphocyte reaction (MLR) cultures using serum-free medium. The effects of exogenously adding several gastrointestinal regulatory peptides (β-endorphin, substance P, met-enkephalin, vasoactive intestinal peptide, bombesin and somatostatin) on the incorporation of ³H-methyl-thymidine was determined. It was observed that mitogen stimulation of lymph node cells with Con A was inhibited (70% of control) by vasoactive intestinal peptide (VIP) but spleen cells stimulated by LPS were insensitive to immunomodulation (98% of control). The ability of VIP to inhibit Con A induced thymidine incorporation was concentration dependent (10^-6 to 10^-18 M) and was not attributable to kinetic shifts or cell toxicity. None of the other tested neuropeptides affected Con A or LPS induced blastogenesis. MLR cultures were inhibited by VIP, β-endorphin and somatostatin in a biphasic manner with maximal inhibition observed at 10^-8 to 10^-12 M. Both substance P and bombesin exhibited slight immunoenhancing properties at 10^-14 to 10^-18 M. Met-enkephalin was ineffective as an immunomodulator of MLR cultures. The utility of using serum-free medium in identifying neuropeptides with immunomodulatory properties are discussed.     

Regulation of Human Cytotoxic Responses by Complement: C3, C3b and C3d Preparations Enhance Human Allogeneic Cytotoxic Responses

Complement components and complement. breakdown products have been found to participate in the regulation of the immune response. In the present study we investigated the effect of C3 and its fragments, C3b, C3c and C3d on human allogeneic cell mediated lympholysis (CML). C3 and C3b at a concentration of 275 M × 10^-9 and C3d at a concentration of 330 M × 10^-9 enhanced human allogeneic CML by at least two fold. In contrast C3C did not affect CML responses. Both C3b and C3d had to be present at the initiation of the cultures in order to exert their effect. Similar doses of C3b and C3d did not affect the mixed lymphocyte responses (3_H-thymidine uptake) while higher doses were clearly inhibitory. None of the preparations induced proliferative or cytotoxic responses in the absence of allogeneic stimulating cells. C3b and C3d added to the mixed lymphocyte cultures caused increased production of interleukin 2. We conclude that C3b and C3d facilitate allogeneic cytotoxic responses through increased production of interleukin 2.     

Marijuana and immunity: tetrahydrocannabinol mediated inhibition of lymphocyte blastogenesis

The effects of delta-9-tetrahydrocannabinol (THC) and its major metabolite, 11-OH delta-9-tetrahydrocannabinol (11-OH THC) on mitogen driven lymphocyte blastogenic transformation (LBT) were studied. THC inhibited LBT responses to the T lymphocyte mitogens phytohemagglutinin and concanavalin A but not the B lymphocyte stimulant pokeweed mitogen. The metabolite 11-OH THC caused a comparable, but less significant, inhibition of LBT responses to the T cell mitogens. These inhibitions were dependent upon the drug doses and the time of incubation with the lymphocytes. There was no significant inhibitory activity of THC to the LBT when it was added 24 or 48 h after mitogen. In addition, exposure of lymphocytes to THC for 3 h, followed by removal of the drug prior to addition of mitogen had no effect on the cells' ability to respond to the mitogen. Thus, there appears to be a specified temporal period during which exposure of lymphocytes to THC results in an inhibition of blastogenesis. Interleukin 2 (30 units/ml) could not preclude the THC induced inhibition of lymphocyte blastogenesis. We conclude that THC and 11-OH THC inhibit T lymphocyte blastogenesis. However, unlike the THC mediated inhibition of natural killer cell activity (as shown previously), the process is not responsive to the cytokine interleukin 2.     

The Modulatory Effect of Exogenously Added Prostaglandin E1 Or E2 On the Delayed-Type Hypersensitivity Reaction of Normal or Tumored Mice

The effect of exogenously added prostaglandin (PG) E₁ or E₂ over concentration ranges of from 1 × 10^-4 to 1 × 10^-9 M were studied in order to determine their effect on the delayed-type hypersensitivity (DtH) reaction of normal or tumored mice. PGE or PGE₂. generally caused a stimulation over the control values of normal mice as detected by the footpad swelling assay. However, PGE¹ or PGE₂ at all concentrations tested were found to significantly inhibit the DtH reaction of CD₂F₁ tumored mice.     

Synergy between human T and B lymphocytes in their response to phythaemagglutinin and pokeweed mitogen.

Human B- and T-lymphocyte preparations were isolated by separating T lymphocytes that formed rosettes with sheep erythrocytes from unrosetted B lymphocytes. Pokeweed mitogen stimulates the proliferation of both B- and T-lymphocyte preparations. In contrast, phytohaemagglutinin stimulates little or no proliferation of purified B lymphocytes although it stimulates the proliferation of T lymphocytes. Lymphoid preparations containing both T and B lymphocytes are more responsive to both mitogens than are either T- or B-lymphocyte preparations. This observation suggested synergy between T and B lymphocytes in the response of unfractionated lymphocytes to mitogens. The basis for this synergy was shown to be the capacity of T lymphocytes to facilitate the proliferation of B lymphocytes cultured with pokeweed mitogen or phytohaemagglutinin. The activity of T lymphocytes is not dependent upon their proliferation or attributable to their release of mitogenic factors. With regard to the clinical evaluation of immune function, our results indicate that the proliferative response of human lymphocytes to phytohaemagglutinin or pokeweed mitogen cannot be directly related to the percentage of T lymphocytes in the lymphoid preparation.     

The Impact of Acemannan on the Generation and Function of Cytotoxic T-Lymphocytes

Acemannan, an antiviral agent with immune enhancement capabilities, was studied for its impact on cytotoxic T-lymphocyte (Tc) function generated in response to alloantigen. To investigate whether acemannan directly stimulated the generation of Tc from primary mixed lymphocyte cultures (MLC), the drug was added at the initiation of the MLC. There was a dose-related, statistical increase in killer T-cell generation produced by acemannan in the clinically relevant dose range. The lowest test dose of the drug (2.6 × 10^-9M) increased chromium release nearly two-fold; the 2.6 × 10^-8 M dose gave a maximal 3.5 fold increase in cytotoxic T-cells. To study whether acemannan enhanced the capacity of Tc once generated to alloantigen to destroy targets bearing the sensitizing antigens, MLR were established in the absence of any drug. Acemannan at the two highest doses increased the functional capacity of Tc to destroy target cells to which they had been sensitized in the MLR. To control for the possibility that acemannan was directly cytotoxic to target cells, targets were incubated alone with drug and without sensitized killer T-cells. No dose of acemannan was found to be cytotoxic to these cells. In conclusion, acemannan did enhance the generation of cytotoxic T-cells when added at the initiation of the MLR. When acemannan was added at the completion of allostimulation, an increase of almost 50% killing by Tc was also observed. These effects can not be explained by direct drug related toxicity and suggest a functional correlate to the previously described immune enhancing properties of the agent. As this drug is being tested for the treatment of HIV infections, these data provide at least one immunologic mechanism by which acemannan may be clinically salutory.     

Diazepam Inhibits Phagocytosis and Killing Exerted by Polymorphonuclear Cells and Monocytes from Healthy Donors. In Vitro Studies

The effect of a benzodiazepine (BDZ), diazepam on human polymorphonuclear cell (PMN) and monocyte pha gocytosis and killing from healthy volunteers has been evaluated. Diazepam is able to inhibit in vitro both functions exerted by PMN and monocytes at 10^-5 and 10^-6 M concentrations/ 4 × 10⁶ phagocytes. 10^-7 M con centration was not effective in all the instances.

These results are discussed for their possible clinical implications, since previous studies have shown that in patients with phobic disorder there is evidence for reduced phagocytosis and killing capacities.     

Control of the Production of Oxygen Intermediates of Human Polymorphonuclear Leukocytes and Monocytes by B-Adrenergic Receptors

The control by R-adrenergic receptors of the production of oxygen radicals by zymosan-stimulated human polymorphonuclear leukocytes (PMN) and monocytes (Mψ) was studied in vitro by means of chemiluminescence. In addition we asked whether PMN Mpsi; exhibit differential sensitivity to β-adrenergic stimulation. Eor β-adrenergic stimulation we applied fenoterol ranging from 10^-9 to 10 M × 2.7. We found a dose-dependent suppression of the production of oxygen radicals, the ID50 being approximately 10^-6 M both for PMN and Mpsi;. By assessment of lactic dehydrogenase release a cytotoxic effect of the drug could be ruled out. When incubated together with the β-adrenergic antagonist propranolol at 10^-6 and 10^-7 M the suppressive effect of fenoterol could be reversed in dose-dependency. Preincubation with fenoterol revealed that the inhibitory action on Mpsi; persisted, in contrast, no such suppression could be verified with PMN. Our findings indicate the control of the production of oxygen intermediates of human PMN and Mpsi; by β-adrenergic stimulation. Furthermore, selective functional modulation of resting PMN and Mpsi; by β-adrenoceptors is suggested. These effects may be of importance in vivo, in particular since fenoterol was applied in pharmacological doses.     

Effect of ASTA-Z 7575 (INN Maphosphamide) on Human Lymphokine-Activated Killer Cell Induction

Recent studies combining chemotherapeutic agents with various biological response modifiers for the treatment of cancer have shown promising results. Cyclophosphamide (Cy) is the most widely used alkylating agent and a major constituent of combination chemotherapy regimens for many neoplastic diseases. It has been reported that Cy is a cytotoxic drug, which becomes immunosuppressive at higher doses. A synthetic metabolite of Cy, ASTA-Z, has recently been produced. ASTA-Z is more active and stable by itself and does not need to be metabolically converted to an active compound. the combined effect of Cy and interleukin-2 (IL-2) on the induction of lymphokine-activated killer (LAK) cells is not known. Therefore, we decided to investigate the effect of ASTA-Z on the induction and function of LAK. the coculture of peripheral blood mononuclear cells (PBMC) with various concentrations of ASTA-Z (0, 10^-6 10^-5, 10^-4 and 10^-3 dilution) and IL-2 (50 U/ml) for 4 days produced significant suppression of cytotoxicity and lytic ability of the LAK cells against NK-sensitive (K562) and NK-resistant (M14) tumor cell lines. the lower doses of ASTA-Z did not affect the generation of LAK cells, its cytotoxicity and lytic ability of ASTA-Z against both NK-sensitive and NK-resistant tumor cell lines. Furthermore, the ASTA-Z produced dose-dependent suppression of the proliferative response of LAK cells. the significant therapeutic benefit in the cancer patient may be achieved by the low dose regimen of Cy and IL-2 because it has no deleterious effect on the induction and function of LAK cells.     

Influence of Salbutamol and Isoproterenol on the Production of TNF and Reactive Oxygen Species by Bovine Alveolar Macrophs and Calcitriol Differentiated HL-60 Cells

The influence of the β-adrenergic agonists Salbutamol and Isoproterenol on the release of reactive oxyen species and Tumor Necrosis Factor (TNF) was tested with bovine alveolar macrophages and HL-60 cells differentiated to macrophages by calcitriol. The production of reactive oxygen species was analyzed by a microplate assay using dichlorofluorescein-diacetate. It could be shown that this method almost exclusively measures superoxide anions. TNF was determined by a bioassay with WEHI cells. The superoxide anion production was stimulated by Zymosan, the TNF release by LPS. By incubation with 5×10^-6 and 5×10^-7 M Salbutamol or 5×10^-7 and 5×10^-8 m lsoproterenol prior to the stimulation, the production of superoxide anions as well as of TNF was inhibited to a significant deree. The inhibitory effects of the adrenergic agonists were completely or at least partially inhibited by the respective antagonists, ICI 118.551 and Propanolol, respectively.     

Modulating Effects of Sensory and Autonomic Neuropeptides on Murine Splenocyte Proliferation and Cytokine Secretion Indd by Leishmania Major

The intimate, bidirectional link between neuroendocrine and immune systems is now accepted. A modulating effect of the nervous system on immune and inflammatory responses has been corroborated by identification of neuropeptide receptors on immunocompetent cells and the finding that neuropeptides can regulate leukocyte functions. The present study was undertaken to investigate the possible immunomodulatory role of sensory (SOM, CGRP and SP) and autonomic (VIP and NPY) neuropeptides in a murine model of cutaneous leishmaniasis, using two genetically different inbred mouse strains, BALB/c and C57BL/6, respectively susceptible and resistant to Leishmania (L.) major infection. The parameters studied were extent of splenocyte proliferation, as measured by thymidine uptake, and the ability of these cells to secrete IFN-γ and IL-4 by using a two-site ELISA, upon in vitro challenge with L. major parasites and addition of the neuropeptides. The resistant mouse splenocyte proliferation was enhanced by SOM, CGRP, and VIP at 10^-5', 10^-6 and 10^-9 M concentration, respectively, but was inhibited by NPY at 10^-5M. Proliferation of the splenocytes from the susceptible strain was inhibited by SOM (10^-11 M) and CGRP(10^-5 M). Somatostatin, at various concentrations, stimulated IFN-γ secretion in both mouse strain splenocytes, and IL-4 production in the susceptible mouse. Calcitonin gene-related peptide enhanced IFN-γ secretion in susceptible mouse splenocytes at 10^-6 10^-7 and 10^-9 M, as did VIP at 10^-10 M and NPY at 10^-7 M. Vasuactive intestinal peptide also stimulated IL-4 production in BALB/c splenocytes at all concentrations used Substance P had no effect on either cell proliferation or cytokine sccrction in eithcr of the two mouse strains.

These findings indicatc that the nervous system, represented by sensory and autonomic nerve terminals and their content of neuroinediators, may be involved in the pathophysiology of cutaneous leishmaniasis.     

Inhibition of T and B lymphocyte proliferation by rapamycin.

The immunosuppressive macrolide rapamycin shows marked structural similarity to FK-506, and like FK-506 inhibits the activation of cultured T and B lymphocytes at concentrations as low as 10(-10) M. However, rapamycin blocks T-lymphocyte proliferation at a much later stage than FK-506. It also inhibits human, porcine and murine T- and B-lymphocyte activation by all pathways tested, including pathways which are insensitive to FK-506, such as interleukin-2 (IL-2)-mediated proliferation of IL-2-dependent T-cell lines, activation of human peripheral blood T lymphocytes by phorbol ester and anti-CD28 and activation of murine B lymphocytes by bacterial lipopolysaccharide. Thus these two macrolides that bind competitively to the same major intracellular receptor protein inhibit T- and B-lymphocyte activation by quite distinct mechanisms.     

Differential effects of marijuana components on proliferation of spleen, lymph node and thymus cells in vitro

The effects of delta 9 THC and 11-OH THC on the proliferative response of murine spleen cells stimulated in vitro with the T cell mitogens Con A or PHA were compared with the effects of these drugs on the mitogen-induced proliferation of murine thymus and lymph node cells. Thymus cells were found to be suppressed at lower cannabinoid concentration than either spleen or lymph node cells. However, splenic cells were more easily suppressed than were the lymph node cells. Lymphoid cell numbers were varied from 1 X 10(6) to 8 X 10(6) cells and treated with a constant dose of either THC or 11-OH THC. When suppression was noted with spleen and lymph node cells, the smallest number of cells in the assay resulted in the greatest level of suppression of cell proliferation. No significant suppression to PHA induced proliferation was found for lymph node cells at any cell number tested. Thymus cells were always more readily suppressed than spleen or lymph node cells regardless of the number of cells in culture. Furthermore, 11-OH THC suppressed the responsiveness of the thymus cells to PHA more than to Con A under the experimental conditions used. Thus, the ability of cannabinoids to induce suppression of the proliferative response of lymphoid cells to mitogens depends on the organ source of the cells, nature of the cannabinoid (THC or 11-OH THC), dose of the cannabinoid, mitogen used (PHA or Con A), and number of cells in culture.(ABSTRACT TRUNCATED AT 250 WORDS)