Prevention and reversal of delta-9-tetrahydrocannabinol induced depression of natural killer cell activity by interleukin-2

The effects of delta-9-tetrahydrocannabinol (THC) on NK cell activity were studied. Previously, we reported that incubation of human peripheral blood mononuclear cells in THC resulted in an inhibition of natural killer (NK) cell activity. The present study examined the mechanism(s) of the decrease in NK cell activity. The inhibition of killing by NK cells was not due to a failure of NK cells to bind to K562 target cells. Furthermore, indomethacin did not abrogate the THC-mediated effect, suggesting that prostaglandins are not involved in the process leading to suppression of NK cell activity. However, NK activity was partially restored if cells, pretreated with THC, were washed to remove excess drug and then incubated overnight in fresh medium before assay. Addition of 1-100 U IL-2, either during pretreatment with THC or during overnight incubation, precluded or promoted the reversal of the inhibition of NK cell cytotoxicity. We conclude that the regulatory mechanism(s) involved in depression of NK cell cytotoxicity by THC is significantly influenced by IL-2.     

Marijuana effects on immunity: suppression of human natural killer cell activity of delta-9-tetrahydrocannabinol

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, was tested for its ability to modulate human natural killer (NK) cell function. THC was toxic for peripheral blood lymphocytes at 20 micrograms/ml but not at 10 micrograms/ml or less. This component of marijuana also was inhibitory for NK activity against K562, a human tumor cell line at concentrations down to 5 micrograms/ml when pre-incubated with the effector cells. Suppression of NK function was dependent upon the concentration of THC and the length of time of pre-incubation but was independent of the ratio of effector to target cells. Prostaglandins were not involved in suppression of NK activity.     

Suppression of lymphokine-activated killer cell generation by tumor-infiltrating lymphocytes

A functional analysis of tumor-infiltrating lymphocytes (TILs) from renal cell carcinoma (RCC) and malignant melanoma was performed. TILs were expanded in recombinant interleukin-2 (50 U/ml) in Iscoves medium. Phenotypic and functional (cytolytic vs regulatory) analyses were carried out with the fresh and expanded TIL populations after 4 weeks in culture. Only one TIL population from an RCC case (out of six cases studied) was CD8+ and demonstrated MHC class I-restricted tumor-specific cytotoxicity against the autologous RCC target. TIL populations from the other five cases became predominantly CD4+ and they neither killed the respective autologous tumor cells nor killed the NK-sensitive target K-562 cells. When studied for other functions, two CD4+ TIL populations were found to suppress the lymphokine-activated killer cell response by peripheral blood lymphocytes (PBL) in coculture. Of these two, a TIL population from an RCC case (MJ TIL) was used to study the cellular and molecular mechanisms of suppression. The MJ TIL synthesized a supernatant factor that blocked activation of resting PBL as measured by the induction of high-affinity IL-2 receptor (IL-2R) when stimulated by phytohemagglutinin but did not down-regulate the fully expressed IL-2R on activated T cells. The suppression of high-affinity IL-2R induction on T cells did not result from tumor necrosis factor-alpha and beta or from transforming growth factor-beta as these cytokines were not detected in the cell-free supernatant from the MJ TIL culture. The supernatant factor also suppressed IL-2-mediated enhancement of cytotoxicity by natural killer (NK) cells without demonstrating direct toxic effect on the NK cells. Thus, when TIL are used for adoptive immunocytotherapy, it may be useful to fully characterize them functionally, in vitro.     

Selective inhibition of natural killer but not natural cytotoxic activity in a cloned cell line by delta-9-tetrahydrocannabinol.

Natural killer (NK) and natural cytotoxic (NC) activities are spontaneously generated against certain tumors in vitro and their contribution to tumor immunity is being extensively investigated. We report here that the interleukin-2 (IL-2)-dependent murine cell line, NKB61A2, which we recently found to express both NK and NC functions, can be modulated selectively by 9 delta-tetrahydrocannabinol (THC). THC, a major psychoactive metabolite of marijuana, could significantly inhibit NK activity without altering NC activity in NKB61A2 cells. Inhibition of NK function occurred at a post-binding stage because effector/target conjugation was unaffected by THC. With regard to NC function, neither the cytotoxic activity of the cells nor release of tumor necrosis factor was interrupted by THC. Therefore, THC may provide a useful tool for dissociating the mechanism of NK and NC activities within a single population of cells.     

Induction of endogenous lymphokine-activated killer activity by combined administration of lentinan and interleukin 2

Lymphokine-activated killer activity in vivo (endogenous LAK activity) was found to be augmented by combined administration of lentinan, a beta (1-3) glucan with beta-1,6 branches, and interleukin 2 (IL-2). In contrast, addition of lentinan during culture in vitro did not augment LAK activity induced by IL-2. Surface marker analysis of endogenous LAK cells revealed that endogenous LAK cells induced by a combined administration of lentinan and IL-2 were all NK-type LAK cells, which express asialo-GM1 and lack T3, Thy-1 and Lyt2, whereas LAK cells generated in vitro were composed of both NK-type LAK and T-type LAK cells, which express T3 and Thy-1, and lack asialo-GM1. Furthermore, combined administration of lentinan and IL-2 was found to augment the endogenous LAK activity even in the tumor bearer, and show a substantial inhibition of tumor growth and a significant increase in survival rate in the C3H/HeN/MM46 system. Results of the present investigation offer a possible clinical application of a combination of lentinan and IL-2 for immunotherapy against cancer without detrimental side effects.     

Effects of delta-9-tetrahydrocannabinol on cultured HeLa cell growth and development

Monolayer cultures of HeLa cells were used to monitor the effects of non-lethal concentrations of delta-9-tetrahydrocannabinol (delta-9-THC) on the pool sizes of the acid-soluble and acid-insoluble DNA moieties and cytoplasmic RNA pool sizes. The DNA fractions were separated using acid precipitation and low speed centrifugation, while the RNA was examined through the use of sucrose gradients and high speed ultracentrifugation. The ratio of acid-insoluble to acid-soluble DNA per cell in untreated HeLa cells is 16:1, which did not change appreciably following delta-9-THC treatment. However, cell division was retarded as much as 25% in the 24 hours treatment period indicating that nucleic acid synthesis, while not inhibited, is depressed by delta-9-THC. This is not related to cell death as indicated by cell viability (> 95%). At both 1.0 x 10(-5)M and 3.2 x 10(-7)M, delta-9-THC caused a marked change in the free ribosomal RNA (an increase with 3.2 x 10(-7)M and a decrease with the 10(-5)M), total ribosomal RNA (a decrease with both observed delta-9-THC concentrations) and non-sedimental RNA (an increase with both observed delta-9-THC concentrations).     

Augmentation of lymphokine-activated killer cell activity in vitro by aloctin A

Culturing of mouse spleen cells with IL2-containing MLA144 conditioned medium (MLA144 CM) resulted in the induction of cells cytotoxic to syngeneic and allogeneic tumor cells in vitro. The cytotoxicity was markedly augmented by the presence of aloctin A (Alo A), a lectin having anti-tumor activity, during the assay for the cytotoxicity. The results of cytotoxicity assay after treatment with antisera and complement indicated that the killer cells mainly consisted of Thy 1+, Lyt 1-2-, asialo GM1-T cells.     

Convulsant-anticonvulsant properties of delta-9-tetrahydrocannabinol in rabbits

Sound stimulation causes audiogenic seizures (AGS) in ACEP/J, but not Uaz:NZW-thc, rabbits. Delta-9-tetrahydrocannabinol (THC) causes behavioral seizures in the latter, but not in the former, rabbits. Also, delta-9-THC blocks AGS in ACEP/J rabbits. These results support the concept that the genes conferring seizure susceptibility to these two rabbit populations are different.     

Suppression of antigen-specific lymphocyte proliferation by Gram-positive bacterial cell walls

It is largely unknown how bacterial cell walls (BCW) modulate human immune responses. In the present work the effect of Gram-positive BCW on lymphocyte proliferation responses towards several microbial antigens (Ag) or mitogens was studied. Gram-positive BCW were derived from four indigenous bacterial strains and from one pathogen (Streptococcus pyogenes). All BCW preparations used non-specifically suppressed the proliferation responses of peripheral blood mononuclear cells (PBMC) against bacterial and viral Ag, but not against mitogens. Both lymphocytes and macrophages or their secreted products mediated the suppressive effects of BCW, which were not IL-10 dependent. Furthermore, the expression of HLA-DR and CD86 on monocytes/macrophages was downregulated by BCW. Unlike in LPS-induced suppression, the CD14 pathway was not used by BCW of Lactobacillus casei (L.c.). The observed results indicate that Gram-positive BCW suppress antigen-specific lymphocyte proliferation through several mechanisms. This non-specific immunosuppression might be a general function of BCW in the bacteria-host interaction, being of importance for bacterial survival and pathogenicity.     

Secondary immunity to Legionella pneumophila and Th1 activity are suppressed by delta-9-tetrahydrocannabinol injection.

Resistance to infection with Legionella pneumophila is primarily dependent upon cell-mediated immunity rather than humoral immunity. Recent evidence suggests that activation of cell-mediated immunity depends on Th1 cells and activation of humoral immunity depends on Th2 cells. In this report, delta 9-tetrahydrocannabinol (THC), the major psychoactive cannabinoid of marijuana and an immunomodulator, suppressed development of secondary immunity to L. pneumophila, which correlated with a reduction in Th1 activity. BALB/c mice, infected with a primary sublethal dose of L. pneumophila, developed resistance to a larger challenge infection 3 to 4 weeks later. However, intravenous injection of THC (4 mg/kg of body weight) 1 day prior to primary infection resulted in increased mortality after the challenge infection. The level of anti-L. pneumophila antibodies in serum increased in both THC-treated and control mice; however, in the THC group IgG1 antibodies which are stimulated by Th2 cells were elevated while Th1-regulated, IgG2a antibodies were depressed. Furthermore, cultured splenocytes from THC-treated mice had less L. pneumophila-specific lymphoproliferation, indicating a deficiency in cell-mediated immunity. Normal mouse splenocytes treated in vitro with THC and pokeweed mitogen showed suppressed production of gamma interferon, a cytokine associated with Th1 cells, but increased production of interleukin 4, a cytokine produced by Th2 cells. Splenocytes from THC-treated mice, stimulated in vitro with either pokeweed mitogen or anti-CD3 antibodies, also produced less gamma interferon, indicating less Th1 activity in these mice. These results suggest that THC decreases the development of anti-L. pneumophila immunity by causing a change in the balance of Th1 and Th2 activities.     

Modulation of natural killer and lymphokine-activated killer cell cytotoxicity by lactoferrin.

Natural killer (NK) and lymphokine-activated killer (LAK) cell cytotoxic functions can be strongly augmented by the iron-carrier protein lactoferrin (LF). LF significantly enhances NK and LAK activities when added at the beginning of NK or LAK cytotoxicity assays. LF is effective in augmenting cytotoxic activities at concentrations as low as 0.75 microgram/ml, and higher concentrations of LF induce greater augmentation of NK and LAK. Iron does not appear to be essential for LF to increase NK and LAK, as depleting iron from LF with the chelator deferoxamine does not affect the capacity of LF to increase cytotoxicity. LF is known to have RNase enzymatic activity, and LF enhancement of NK and LAK can be blocked by RNA. However, LFs from two different sources with over 100-fold difference in RNase activity are equally effective in enhancing NK and LAK. Furthermore, purified non-LF RNase does not modulate NK or LAK activity and DNA is as effective as RNA in blocking LF augmentation of NK or LAK cytotoxicity. Therefore, the RNase activity is unlikely to be responsible for LF enhancement of the cytotoxicities. Newborn infants are known to have low NK activity and NK and LAK cells have been implicated in host defense against microbial infections. Thus, maternal milk-derived LF may have a role in boosting antimicrobial immunity in the early stages of life. In adults, LF released from neutrophils may enhance NK and LAK functions in the inflammatory process induced by microbial infections.     

Differential suppression of T-cell subpopulations by thc (delta-9-tetrahydrocannabinol)

THC (delta-9-tetrahydrocannabinol), the major psychoactive component of marijuana, has been shown to suppress various immune functions in vivo and in vitro. THC suppresses murine T-lymphocyte proliferation; however, the effects on T-cell subsets remain unclear. We have stimulated cultured murine splenocytes with the mitogens concanavalin A (Con A) or phytohemagglutinin (PHA) while exposing them to varying concentrations of THC. After three days, the cells were analyzed by the fluorescent activated cell sorter for the following T-cell markers--Thy1, L3T4 and Ly2. The Ly2 cells represent the suppressor/effector T-cells while L3T4 cells represent the helper T-cell subpopulations. The results show that the dose response suppressive effect of THC on T-cell proliferation reflects a preferential inhibition of Ly2 vs L3T4 cells. The effects of THC on other functional parameters are in the process of investigation.     

Differential effects of IL-4 and IL-10 on IL-2-induced IFN-gamma synthesis and lymphokine-activated killer activity.

Culture of human peripheral blood mononuclear cells (PBMC) with IL-2 stimulates synthesis of cytokines and generation of lymphokine-activated killer (LAK) activity. Both IL-4 and IL-10 [cytokine synthesis inhibitory factor (CSIF)] inhibit IL-2-induced synthesis of IFN-gamma and tumor necrosis factor (TNF)-alpha by human PBMC. However, unlike IL-4, IL-10 inhibits neither IL-2-induced proliferation of PBMC and fresh natural killer (NK) cells, nor IL-2-induced LAK activity. Moreover, IL-4 inhibits IL-2-induced IFN-gamma synthesis by purified fresh NK cells, while in contrast the inhibitory effect of IL-10 is mediated by CD14+ cells (monocytes/macrophages). IL-10 inhibits TNF-alpha synthesis by monocytes or monocytes plus NK cells, but not by NK cells alone. These results suggest that IL-4 and IL-10 act on NK cells via distinct pathways, and that IL-2-induced cytokine synthesis and LAK activity are regulated via different mechanisms.     

Bone marrow granulomas in acute myeloid leukaemia following interleukin 2 and lymphokine-activated killer cells

Granulomas are thought to represent an immune reaction to antigenic stimulation. Bone marrow granulomas are uncommon, but in the following case report we show their transient appearance after interleukin 2 (IL-2) and autologous lymphokine-activated killer cell therapy. This is a previously unreported association, and may implicate IL-2 in the pathogenesis of granulomas.     

The ontogeny of delta-9-tetrahydrocannabinol responsiveness in the rabbit

An autosomal recessive condition (thc/thc) in our closed colony of New Zealand White rabbits (Uaz:NZW-thc) results in nonfatal, behavioral convulsions following intravenous (i.v.) injections of delta-9-tetrahydrocannabinol (THC) and other psychoactive cannabinoids of marijuana. The ontogeny of the convulsive response was evaluated in potential THC-seizure-susceptible (SS) rabbits from postnatal Days (PN) 15-548. Ages of nonsusceptibility (PN 15-23), partial susceptibility (PN 24-38), and complete susceptibility (PN 39-548) were found. Also, open-field activity was determined in PN 14-25 THC-SS and THC-seizure-resistant (SR) rabbits. Administration of THC during the seizure-insensitive period resulted in genotype-dependent alternations in photocell activity and sprawling.     

In vivo boosting of lung natural killer and lymphokine-activated killer cell activity by interleukin-2: comparison of systemic, intrapleural and inhalation routes.

Natural killer (NK) cells are thought to play a role in host defence against malignancy and infection, in immunoregulation and as precursor cells in a generation of lymphokine-activated killer (LAK) cells which can lyse NK-resistant tumour cells. As the lung is a major site for malignancy and infection and as there are large numbers of lymphoid cells including NK cells in the interstitial compartment of the lung, we evaluated the capacity of interleukin-2 (IL-2), a lymphokine capable of augmenting NK activity in vitro, to augment lung NK cell activity in vivo, using different routes of IL-2 administration. We compared both systemic (i.v. and i.p.) and local (intrapleural and inhalation) routes of IL-2 administration (50,000 U/daily for 5 days) using CBA mice, assessing NK and LAK cell activity in the spleen (systemic) and in the lung. The target cells used for these studies were the YAC-1 (NK-sensitive) and P815, NO36 and HA56 (NK-resistant, LAK-sensitive) cell lines. Splenic NK activity was increased by 1.4-1.9-fold for i.v./i.p., respectively, compared with controls with both systemic routes of administration, and lung NK activity was increased 3.2-fold and 3.8-fold (i.v./i.p, respectively, P less than 0.05), to levels which were comparable to systemic (splenic) NK activity following the same therapy. Intrapleural IL-2 administration similarly enhanced lung NK activity (3.3-fold) and splenic NK activity (1.3-fold; P less than 0.05 versus controls for both). Surprisingly, inhaled IL-2 suppressed both splenic and lung NK cell activity (84 +/- 8% and 78 +/- 10% suppression, respectively, P less than 0.05). LAK cell activity was also enhanced in the lung by 1.8-8-fold in response to i.v., i.p. and intrapleural IL-2, whereas inhaled IL-2 was ineffective in generating LAK cell activity. These results suggest that the systemic and intrapleural administration of IL-2 effectively boost pulmonary NK and LAK activity whereas inhalation of IL-2 does not. Thus, in clinical situations where boosting of local lung NK or LAK cell activity is desired, these routes of IL-2 administration may be effective.     

Human B cell proliferation is stimulated by interleukin 2

The proliferation of human B cells was studied for response to interleukin 2 (IL-2) produced in Escherichia coli using recombinant DNA technology. The IL-2 was found to be an homogenous preparation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using the anti-IL-2 monoclonal antibody DMS-1. IL-2 was found to stimulate B cell proliferation. Activation of the B cells using anti-IgM antibodies increased this response. Resting T cells from the same donors were found to be less reactive to IL-2. The results suggest that human B cell proliferation can be stimulated by IL-2 alone.     

The combined effect of prostaglandin E2 and interleukin 1 on interleukin 2 production and lymphocyte proliferation

In this study the combined effect of prostaglandin E2 (PGE2) and interleukin-1 (IL-1) on the proliferation of concanavalin A (Con A) stimulated mouse spleen lymphocytes was studied. IL-1 in concentrations ranging from 0.5 to 5.0 U/ml consistently and significantly enhanced Con A activity. However, to be effective, IL-1 needed to be added at the time of initiation of the culture. If added 24 h later, IL-1 failed to enhance the proliferative response. PGE2 at concentrations ranging from 0.1 to 5.0 ng/ml effectively inhibited or antagonized this enhancing effect of IL-1, with the majority of this IL-1 augmentation abrogated by 5.0 ng/ml PGE2. Unlike IL-1, PGE2 was as effective if added 24 h after initiation of the culture as if added simultaneously with IL-1. PGE2 was also found to markedly suppress the enhanced production of IL-2 resulting from the addition of IL-1 to Con A stimulated lymphocytes, however, the amount of IL-2 produced in the cultures containing both IL-1 and PGE2 was always greater than that produced in the cultures which contained only PGE2. This finding indicates that IL-1 could partially reverse or antagonize the suppressive effect of PGE2 on IL-2 production. In addition, PGE2 at concentrations of 1.0 and 5.0 ng/ml was also found to inhibit the proliferation of IL-2 stimulated cultured T lymphocytes, but by only about 15-20%. The addition of IL-1 to these cultured T cells neither altered the response of the culture T cells to IL-2 nor altered the sensitivity of these cells to PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)