Susan Pross

Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species.

Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene super-family in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems.

Tetrahydrocannabinol inhibition of macrophage nitric oxide production.

delta 9-Tetrahydrocannabinol (THC) inhibited nitric oxide (NO) production by mouse peritoneal macrophages activated by bacterial endotoxin lipopolysaccharide (LPS) and interferon-gamma (IFN)-gamma). Inhibition of NO production was noted at THC concentrations as low as 0.5 microgram/mL, and was nearly total at 7 micrograms/mL. Inhibition was greatest if THC was added 1-4 hr before induction of nitric oxide synthase (NOS) by LPS and IFN-gamma, and declined with time after addition of the inducing agents. This suggested that an early step such as NOS gene transcription or NOS synthesis, rather than NOS activity, was affected by THC. Steady-state levels of mRNA for NOS were not affected by THC. In contrast, protein synthesis was inhibited as indicated by immunoblotting. NOS activity was also decreased in the cytosol of cells pretreated with THC. Addition of excess cofactors did not restore activity. Inhibition of NO production was greater at low levels of IFN-gamma, indicating the ability of the cytokine to overcome inhibition. The effectiveness of various THC analogues, in decreasing order of potency, was delta 8-THC > delta 9-THC > cannabidiol > or = 11-OH-THC > cannabinol. The presumably inactive stereoisomer, (+)delta 9-THC, and the endogenous ligand for cannabinoid receptors, anandamide, were weakly inhibitory. Inhibition may be mediated by a process that depends partly on stereoselective receptors and partly on a nonselective process. LPS, IFN-gamma, hormone receptor agonists, and forskolin increased macrophage cyclic AMP levels. THC inhibited this increase, indicating functional cannabinoid receptors. Addition of 8-bromocyclic AMP increased NO 2-fold, and partially restored NO production that had been inhibited by THC. This occurred only under conditions of limited NOS induction, suggesting that the effect of THC on cyclic AMP was responsible for only a small portion of the inhibition of NO.

Inhibition of macrophage nitric oxide production by tetrahydrocannabinol in vivo and in vitro

delta 9-Tetrahydrocannabinol (THC, 10 micrograms) was administered intraperitoneally to thioglycollate-treated mice. After 18 h, peritoneal macrophages were harvested and nitric oxide (NO.) production was induced by lipopolysaccharide (LPS, 1 microgram/ml) and interferon-gamma (IFN-gamma, 0.1-10 U/ml). Macrophages from THC-treated mice produced about half as much NO. as controls. THC (1 microgram/ml) added in vitro caused further inhibition. Greater inhibition was observed at the lower (0.1-0.3 U/ml) IFN-gamma concentrations. The results suggest that the use of THC can reduce NO. production and thereby affect host defense mechanisms, inflammation and autoimmune responses.

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)

Nicotine modulation of apoptosis in human coronary artery endothelial cells.

It has been recently reported that nicotine, the addictive component of tobacco, is an important modulator at the level of immune cell apoptosis or programmed cell death. Apoptosis is a process that helps maintain the homeostasis of the vascular endothelium and vascular smooth muscle cells, and alteration of the apoptotic process has been associated with cardiovascular diseases. The present study examined the effects and the mechanisms of action of nicotine on apoptosis in human coronary artery endothelial cells (HCAECs). Cultured HCAECs were treated with nicotine at a concentration that correlates with the tissue level of smokers (1 microg/ml), concurrently with tumor necrosis factor-alpha (TNF-alpha) and dexamethasone to induce apoptosis. The data showed that nicotine significantly inhibited apoptosis in HCAECs, as verified by the decreased expression level of active caspases compared to cells treated with the apoptosis inducers alone. This decrease was blocked by the addition of d-tubocurarine chloride (d-TC), a general nicotinic receptor antagonist, providing evidence that this action of nicotine was receptor-mediated. The findings were further confirmed by TUNEL assay for DNA fragmentation, a biochemical marker of apoptosis. This action of nicotine on apoptosis in human coronary artery endothelial cells suggests that nicotine may have an impact on cardiovascular pathology and atherogenesis.

Differing effects of delta-9-tetrahydrocannabinol (THC) on murine spleen cell populations dependent upon stimulators

Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, can suppress the immune response, both in vitro and in vivo. In the present study, THC was found to either up-regulate or down-regulate lymphocytes depending on the method of stimulation. When the mitogens concanavalin A (Con A) or phytohemagglutinin (PHA) were used to stimulate THC-treated splenocytes, a down-regulation of lymphocyte proliferation occurred, which reflected lower T-cell numbers in general and Ly2 positive cells specifically. When splenocytes were stimulated directly by using anti-CD3 antibody it was found that low concentrations of THC enhanced lymphocyte proliferation, T-cell numbers in general, and Ly2 cells specifically. These results emphasize that THC can either enhance or suppress aspects of the immune response, depending on the specific immune stimulants used and the specific parameter of immunity measured.

Nicotine inhibition of apoptosis in murine immune cells.

Nicotine, the addictive component of tobacco, is thought to be at least partially responsible for the deleterious effects of smoking such as heart disease and cancer. Evidence shows that nicotine is an immunomodulator and that one of its possible mechanisms is regulation of apoptosis, or programmed cell death, in immune cells. This study examined the effects and the mechanisms of action of nicotine on dexamethasone (DEX)-induced apoptosis in murine immune cells by examining the expression of levels of the 17-kDa active caspase-3, a marker of apoptosis. Thymocytes and splenocytes from adult BALB/c female mice were Incubated with concentrations of nicotine correlating to those found in the blood and tissue of smokers (0.01 μg/ml [0.022 μM] and 1 μg/ml [2.2 μM]), concurrently with 100 nM DEX, to induce apoptosis. Cytosolic protein fractions were analyzed by Western blotting with polyclonal antibodies that recognize the active form of caspase-3. The data showed that nicotine significantly blocked the formation of the DEX-induced 17-kDa caspase-3 subunit expression. This downregulation ranged from 65% to 100% of the active caspase-3 expressed in cultures treated with DEX alone. Addition of d-tubocurarine chloride (dTC), a general nicotinic receptor antagonist, inhibited nicotine downregulation of the DEX-lnduced active caspase-3 expression, providing evidence that this action of nicotine was receptor-mediated. These data support that nicotine is an important immunomodulator at the level of immune cell apoptosis, a process thought to be a contributory mechanism of autoimmunity, cardiovascular disease, and carcinogenesis.

Differential impact of nicotine on cellular proliferation and cytokine production by LPS-stimulated murine splenocytes.

The immunoregulatory effects of nicotine have not been fully clarified and the reported data are often conflicting. The present study investigated the role of nicotine as an immunomodulator of murine splenocytes stimulated by lipopolysaccharide (LPS), the endotoxin component of gram-negative bacteria. BALB/c female mice of two different ages, young (2-3 months) and old (18-22 months), were used. The cells were incubated with nicotine at two different time points, 3 h pre-incubation and concurrent incubation relevant to LPS stimulation, before further incubation for 48 or 72 h. Treatment of murine splenocytes with nicotine showed an impact on cellular proliferation as well as on the production of the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). The results indicated that nicotine significantly inhibited cellular proliferation of murine splenocytes in a concentration-related manner (32, 64 and 128 microg/ml). Timing of nicotine exposure prior to LPS stimulation was critical in terms of immunological impact on cytokine production. TNF-alpha and IL-6 production were significantly enhanced by 1 microg/ml of nicotine when cells were pre-incubated with nicotine for 3 h compared to concurrent incubation relative to LPS stimulation. The alteration in cytokine production varied with the age of the mouse. TNF-alpha production was significantly inhibited by nicotine in young mice, while IL-6 production was significantly inhibited by nicotine in old mice. Since any immunomodulation that alters the profile of these cytokines may cause an imbalance in the immune system impinging on health status, these findings may be important when dealing with the concept of nicotine as a therapeutic agent.

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.

Modulation of interleukin 2 activity by Δ9-tetrahydrocannabinol after stimulation with concanavalin A, phytohemagglutinin, or Anti-CD3 antibody

Abstract The effects of Δ9-tetrahydrocannabinol (THC) on lymphocyte proliferation and interleukin (IL) 2 activity was investigated using adult murine spleen cells stimulated with either the mitogens concanavalin A, phytohemagglutinin, or anti-CD3 antibody. THC was found to suppress mitogen-induced proliferation, but to enhance anti-CD3-antibody-induced proliferation. These results reflected THC-induced suppression of Ly2 cells following concanavalin A or phytohemagglutinin stimulation and THC-induced enhancement of Ly2 cells following CD3 stimulation. The combination of THC and concanavalin A or phytohemagglutinin resulted in suppressed IL-2 activity, whereas the combination of THC and anti-CD3 antibody resulted in enhanced IL-2 activity. This drug-related modulation of IL-2 activity corresponded to the changes in blastogenic activity as well as to variations in numbers of Tac positive cells. These results suggest that the dysregulation in immune responses following THC treatment, either suppression or enhancement, may relate to the effects of THC on IL-2 production.