A Randomized Controlled Trial of the Effects of Mindfulness-Based Stress Reduction (MBSR[BC]) on Levels of Inflammatory Biomarkers Among Recovering Breast Cancer Survivors.

PURPOSE: The purpose of this substudy of a large randomized controlled trial was to evaluate the efficacy of the Mindfulness-Based Stress Reduction (Breast Cancer) (MBSR[BC]) program compared to usual care (UC) in normalizing blood levels of pro-inflammatory cytokines among breast cancer survivors (BCS). METHOD: A total of 322 BCS were randomized to either a 6-week MBSR(BC) program or a UC. At baseline and 6 and 12 weeks, 10 ml of venous blood and demographic and clinical data were collected and/or updated. Plasma cytokines (interleukin [IL]-1ß, IL-6, IL-10, tumor necrosis factor [TNF] α, transforming growth factor [TGF] ß1, soluble tumor necrosis factor receptor [sTNFR] 1) were assayed. Linear mixed models were used to assess cytokine levels across three time points (baseline and 6 and 12 weeks) by group (MBSR[BC] vs. UC). RESULTS: Of the six measured cytokines, three were nondetectable at rates greater than 50% (IL-10, IL-1ß, TGF-ß1) and, because of overall low prevalence, were not analyzed further. For the remaining cytokines (TNFα, IL-6, sTNFR1), results showed that TNFα and IL-6 increased during the follow-up period (between 6 and 12 weeks) rather than during the MBSR(BC) training period (between baseline and 6 weeks), while sTNFR1 levels did not change significantly across the 12-week period. CONCLUSIONS: Study results suggest that MBSR(BC) affects cytokine levels in BCS, mainly with increases in TNFα and IL-6. The data further suggest that B-cell modulation may be a part of immune recovery during breast cancer management and that increases in TNFα and IL-6 may be markers for MBSR(BC)-related recovery.

Lymphocyte recovery after breast cancer treatment and mindfulness-based stress reduction (MBSR) therapy.

OBJECTIVES: This randomized controlled trial was conducted to examine immune recovery following breast cancer (BC) therapy and evaluate the effect of mindfulness-based stress reduction therapy (MBSR) on immune recovery with emphasis on lymphocyte subsets, T cell activation, and production of T-helper 1 (Th1; interferon [IFN]-γ) and T-helper 2 (Th2; interleukin-4 [IL-4]) cytokines. METHOD: Participants who completed the study consisted of 82 patients diagnosed with Stage 0-III BC, who received lumpectomy and adjuvant radiation ± chemotherapy. Patients were randomized into an MBSR(BC) intervention program or a control (usual care) group. Immune cell measures were assessed at baseline and within 2 weeks after the 6-week intervention. The numbers and percentages of lymphocyte subsets, activated T cells, and Th1 and Th2 cells in peripheral blood samples were determined by immunostaining and flow cytometry. RESULTS: Immune subset recovery after cancer treatment showed positive associations with time since treatment completion. The B and natural killer (NK) cells were more susceptible than T cells in being suppressed by cancer treatment. Women who received MBSR(BC) had T cells more readily activated by the mitogen phytohemagglutinin (PHA) and an increase in the Th1/Th2 ratio. Activation was also higher for the MBSR(BC) group if <12 weeks from the end of treatment and women in MBSR(BC) <12 weeks had higher T cell count for CD4(+). CONCLUSION: MBSR(BC) promotes a more rapid recovery of functional T cells capable of being activated by a mitogen with the Th1 phenotype, whereas substantial recovery of B and NK cells after completion of cancer treatment appears to occur independent of stress-reducing interventions.

Identification of transcription start sites and preferential expression of select CB2 transcripts in mouse and human B lymphocytes.

Marijuana cannabinoids, the endocannabinoids, and cannabinoid cell receptors have been shown to play important roles in immune regulation particularly as potent modulators of anti-inflammatory cytokines. The predominant cannabinoid receptor involved in this immune regulation is cannabinoid receptor 2 (CB(2)), which is predominantly expressed in B lymphocytes. However, the promoter region and mechanisms of CB(2) gene regulation are unknown in this immune cell type. Utilizing a combination of bioinformatics, 5' rapid amplification of cDNA ends (5' RACE), real-time reverse transcription-polymerase chain reaction, DNA sequencing, and luciferase reporter assays, we show that human B cells express one CB(2) transcript while mouse B cells express three CB(2) transcripts, with specific transcript selection occurring during B cell activation by lipopolysaccharide. Alignment of our sequenced RACE products to either the mouse or human genome, along with the GenBank submitted mRNA sequences, revealed that the transcripts we isolated contained previously unidentified transcriptional start sites (TSS). In addition, expression construct testing of the genomic region containing the TSSs of the mouse CB(2) exon 1 transcripts showed an eightfold increase of promoter activity over baseline. These data show for the first time that human B cells use only one TSS for CB(2) while mouse B cells use multiple TSSs and that the mouse TSSs are in a genomic area with promoter activity, thus suggesting the location of the gene promoter region. Defining these TSSs also provides clues to the various gene regulatory factors involved in the expression of CB(2) during B cell activation.

Cannabinoid receptor 2 (CB2) mediates immunoglobulin class switching from IgM to IgE in cultures of murine-purified B lymphocytes.

Marijuana cannabinoid treatment increases Th2 activity, and previous reports showed that B cells express the highest level of CB(2) mRNA relative to other immune cells, suggesting that cannabinoids play a critical role in B cell activation and maturation. We previously reported evidence of Th2 biasing and class switching in cannabinoid-treated and antigen-challenged mice. We now explore the possibility that cannabinoids directly influence B cell antibody class switching. Mouse splenic B cells were purified by negative selection and cultured with IL4 and anti-CD40 in the presence or absence of the nonselective cannabinoid agonist, CP55940, or the CB(1) selective cannabinoid agonist, methanandamide, and analyzed at different days by flow cytometry for surface expression of either IgM or IgE. Cells treated with CP55940 showed an increase in expression of IgE by day 5 in culture; methanandamide had no effect. CP55940 also induced an increase in secreted IgE in culture supernatants as analyzed by ELISA. In addition, CB(2) receptors were increased on B cells after stimulation with IL-4 and anti-CD40, and the class switching effect of CP55940 was attenuated by the CB(2) antagonist, SR144528. These results suggest that cannabinoids bias toward Th2-type immunity by directly inducing B cell class switching from IgM to IgE through a mechanism involving CB(2) receptors.

Role of Toll-like receptor 9 in Legionella pneumophila-induced interleukin-12 p40 production in bone marrow-derived dendritic cells and macrophages from permissive and nonpermissive mice.

The progression of Legionella pneumophila infection in macrophages is controlled by the Lgn1 gene locus, which expresses the nonpermissive phenotype in cells from BALB/c mice but the permissive phenotype in cells from A/J mice. Activation of dendritic cells and macrophages by L. pneumophila is mediated by the pathogen recognition receptor Toll-like receptor 2 (TLR2); furthermore, Legionella induces innate and adaptive immune cytokines by the MyD88-dependent pathway. TLR9 is coupled to MyD88 and mediates the production of interleukin-12 (IL-12) in dendritic cells infected with other facultatively intracellular pathogens. In the current study, L. pneumophila growth in dendritic cells from BALB/c and A/J mice was examined along with the role of TLR9 in the induction of IL-12 in these cells. Dendritic cells from both strains were nonpermissive for L. pneumophila intracellular growth, suggesting that the products of the Lgn1 gene locus that control intracellular growth in macrophages do not control the growth of Legionella in dendritic cells. In addition, chloroquine treatment suppressed IL-12 p40 production in response to Legionella treatment in dendritic cells and macrophages from BALB/c and A/J mice. Furthermore, the TLR9 inhibitor ODN2088 suppressed the Legionella-induced IL-12 production in dendritic cells from both mouse strains. These results suggest that L. pneumophila is similar to other intracellular bacteria in that it stimulates the production of immune-transitioning cytokines, such as IL-12, through activation of TLR9 and that this receptor provides a common mechanism for sensing these types of microbes and inducing innate and adaptive immunity.

Cannabinoid treatment suppresses the T-helper cell-polarizing function of mouse dendritic cells stimulated with Legionella pneumophila infection.

Marijuana cannabinoids, such as delta-9-tetrahydrocannabinoid (THC), suppress type 1 T-helper 1 (Th1) immunity in a variety of models, including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, bone marrow-derived dendritic cells (DCs) were purified from BALB/c mice and studied following infection and drug treatment. DCs infected in vitro with Lp were able to protect mice when injected prior to a lethal Lp infection; however, the immunization potential of the Lp-loaded cells along with Th1 cytokine production was attenuated by THC treatment at the time of in vitro infection. In addition, THC-treated and Lp-loaded DCs were poorly stimulated in culture-primed splenic CD4(+) T cells to produce interferon-gamma; however, this stimulating deficiency was reversed by adding recombinant interleukin (IL)-12p40 protein to the cultures. Moreover, THC treatment inhibited the expression of DC maturation markers, such as major histocompatibility complex class II and costimulatory molecules CD86 and CD40 as determined by flow cytometry and suppressed the Notch ligand, Del-ta4, as determined by reverse transcription-polymerase chain reaction. However, THC treatment did not affect other DC functions, such as intracellular killing of Lp, determined by colony-forming unit counts of bacteria, and Lp-induced apoptosis, determined by annexin V staining. In conclusion, the data suggest that THC inhibits Th1 activation by targeting essential DC functions, such as IL-12p40 secretion, maturation, and expression of costimulatory and polarizing molecules.

Role of cannabinoid receptors in Delta-9-tetrahydrocannabinol suppression of IL-12p40 in mouse bone marrow-derived dendritic cells infected with Legionella pneumophila.

Delta-9-tetrahydrocannabinol (THC) injection suppresses serum interleukin-12 (IL-12) levels in Legionella pneumophila-infected mice. Dendritic cells are a major producer of IL-12 and mouse, bone marrow-derived dendritic cell cultures produced high levels of the IL-12p40 following L. pneumophila infection. Treatment with THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoid, 2-arachidonoyolglycerol, less potently suppressed cytokine production. Dendritic cells expressed mRNA for cannabinoid receptor 1 (CB(1)), cannabinoid CB(2) receptor, and vanilloid receptor 1 (TRPV1) and the addition of the G(i) inhibitor, pertussis toxin, completely attenuated suppression induced by 3 and 6 muM THC but not by 10 muM THC. Furthermore, THC suppression was partially attenuated in dendritic cells from cannabinoid CB(1) receptor and CB(2) receptor knockout mice and in dendritic cells co-treated with THC and cannabinoid receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist, capsazepine. These results suggest that THC-induced suppression of serum IL-12 is partly due to a suppression of IL-12 production by dendritic cells and that G(i) signaling and cannabinoid receptors, but not TRPV1, are involved in this suppressive effect.

Cannabinoid receptors and T helper cells.

We have reported that injection of marijuana cannabinoids, such as Delta(9)-tetrahydrocannabinol (THC), into mice, followed by infection with Legionella pneumophila (Lp), suppresses the development of cell-mediated immunity T helper 1 (Th1) activity. These effects are accompanied by suppression of interleukin (IL)-12 and interferon (IFN) gamma production and enhancement of IL-4 production suggesting THC-induced T helper cell biasing. In the current report, other T helper cell biasing mechanisms were studied. Mice were injected with THC followed 18 h later by a challenge infection with Lp. Two-hour post-infection, spleens were removed and analyzed for mRNA to either IL-12Rbeta2 or GATA3 gene products. The results showed that THC suppressed IL-12Rbeta2 but increased GATA3. Receptor antagonists for CB1 (SR141716A, SR1) and CB2 (SR144528, SR2) were also injected to analyze the involvement of cannabinoid receptors. It was determined that SR1 attenuated the THC suppression of IL-12Rbeta2, while SR2 attenuated the increase in GATA3 mRNA. These results suggest that THC suppresses Th1 biasing activity such as IL-12Rbeta2 by a CB1 mediated mechanism and enhances the Th2 biasing activity, GATA3, by a CB2 mechanism. This dichotomy of receptor involvement might result from differential expression and/or signaling function of CB1 and CB2 on Th1 and Th2 cells.

The cannabinoid system and immune modulation.

Studies on the effects of marijuana smoking have evolved into the discovery and description of the endocannabinoid system. To date, this system is composed of two receptors, CB1 and CB2, and endogenous ligands including anandamide, 2-arachidonoyl glycerol, and others. CB1 receptors and ligands are found in the brain as well as immune and other peripheral tissues. Conversely, CB2 receptors and ligands are found primarily in the periphery, especially in immune cells. Cannabinoid receptors are G protein-coupled receptors, and they have been linked to signaling pathways and gene activities in common with this receptor family. In addition, cannabinoids have been shown to modulate a variety of immune cell functions in humans and animals and more recently, have been shown to modulate T helper cell development, chemotaxis, and tumor development. Many of these drug effects occur through cannabinoid receptor signaling mechanisms and the modulation of cytokines and other gene products. It appears the immunocannabinoid system is involved in regulating the brain-immune axis and might be exploited in future therapies for chronic diseases and immune deficiency.