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. 2016 Jun;46(6):1472-9.
doi: 10.1002/eji.201546181.

Production of endocannabinoids by activated T cells and B cells modulates inflammation associated with delayed-type hypersensitivity

Jessica M Sido  1 , Prakash S Nagarkatti  1 , Mitzi Nagarkatti  1   2
Affiliations

Production of endocannabinoids by activated T cells and B cells modulates inflammation associated with delayed-type hypersensitivity

Jessica M Sido et al. Eur J Immunol. 2016 Jun.

Abstract

Endocannabinoids are endogenous ligands for the cannabinoid (CB) receptors which include anandamide (AEA) and 2-arachidonyl glycerol (2-AG). 2-AG has been linked to inflammation due to its elevated expression in animal models of autoimmunity and hypersensitivity. However, administration of exogenous 2-AG has been shown to suppress inflammation making its precise role unclear. In the current study, we investigated the role of 2-AG following immunization of C57BL/6 (BL6) mice with methylated BSA (mBSA) antigen, which triggers both delayed-type hypersensitivity (DTH) and antibody response. We found that while naïve T cells and B cells expressed low levels of 2-AG, expression significantly increased upon activation. Furthermore, mBSA-immunized mice exhibited higher 2-AG concentration than naïve mice. Exogenous 2-AG treatment (40 mg/kg) in mBSA-immunized mice led to reduced DTH response, and decreased Th1 and Th17-associated cytokines including IL-6, IL-2, TNF-α, and the IgG response. Addition of 2-AG to activated popliteal lymph node (PopLN) cell cultures also inhibited lymphocyte proliferation. Together, these data show for the first time that activated T and B cells produce 2-AG, which plays a negative regulatory role to decrease DTH via inhibition of T-cell activation and proliferation. Moreover, these findings suggest that exogenous 2-AG treatment can be used therapeutically in Th1- or Th17-driven disease.

Keywords: Anti-inflammatory; Cannabinoid receptor; Delayed-type hypersensitivity (DTH); Endocannabinoids; Th1; Th17; T cell suppression.

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Conflict of interest statement

The authors declare no commercial or finical conflict of interest.

Figures

Figure 1
Figure 1. Lymphocyte activation causes dysregulation of endocannabinoids
(A) Raw chromatogram for naïve C57BL/6 (BL6) T cells and B cells after LC/MS/MS detection. (B) Quantified AEA and 2-AG in naïve lymphocytes (n=2 individual samples ran in triplicate-pooled from multiple mice). (C, D) Spleens were collected from naïve BL6 mice and activated with ConA (2.5μg/mL) or LPS (5μg/mL). 2-AG was quantified after (C) ConA or (D) LPS activation using LC/MS/MS detection (n=6 individual samples ran in triplicate). Delayed type hypersensitivity (DTH) induction, 24 hours after rechallenge, was determined using footpad swelling and lymphocyte activation (n=15 per treatment group). (E) Relative percent swelling of mBSA rechallenged footpad compared to naïve mouse footpad. (F) Popliteal lymph node (PopLN) absolute cell counts (per mouse) was determined by hemocytometer viability counts. (G) Circulating 2-AG in plasma (individual samples ran in triplicate). Absolute cell counts from flow cytometer proportions applied to viability counts (H) CD3+CD69+ activated T cells and (I) CD19+CD69+ activated B cells (gated on live cells). Data shown as mean + SEM of duplicate measurements (n=5 per treatment group per experiment). Student’s t-test or ANOVA/Tukey * p<0.05 ** p<0.01 ***p<0.001 ****p>0.0001
Figure 2
Figure 2. Endocannabinoid 2-AG reduces lymphocyte driven inflammation
2-AG treatment (40 mg/kg n=9) or vehicle (ethanol diluted in 1×PBS n=11) was given on days 4–6 after sensitization (n=5 individual samples pooled and ran in triplicate for ELISA and proliferation assays). (A) Schematic of model and treatment regimen. (B) Splenocytes from in vivo mBSA sensitized mice (n=3 per treatment group) were pooled and co-cultured in triplicate with 2 uCi of 3H-thymidine to assess spontaneous proliferation (C) Relative percent swelling of treated mouse footpads compared to naïve mouse footpads (BL6). (D–F) PopLN absolute cell counts using hemocytometer viability (D), applied to flow cytometer proportions of CD3+CD69+ activated T cells (E), and CD19+CD69+ B cells (F). Cytokine secretion was assessed by sandwich ELISA using culture supernatants from splenocytes co-cultured with PMA and Ca Ionomycin (G) IL-17a/f, (H) IFN-γ, (I) TNF-α, (J) IL-6, and (K) IL-2. (L) Plasma was collected 24 hours after mBSA rechallenge and assessed for IgG. Splenocytes were stimulated with (M) mBSA rechallenge (10 mg/mL in vivo), (N) ConA (2.5μg/mL), or (O) LPS (5.0μg/mL), treated with 2-AG, and co-cultured with 2 uCi of 3H-thymidine to assess proliferation. Data shown as mean + SEM (individual samples ran in triplicate-pooled from n=5 mice per treatment group). Student’s t-test or ANOVA/Tukey * p<0.05 ** p<0.01 ***p<0.001 ****p<0.0001
Figure 3
Figure 3. Loss of CB1 receptor function increases footpad swelling
cDNA (miRNeasy-Qiagen and iScript-BioRad) was isolated form PopLN of naïve BL6 and DTH mice (n=5 mice per treatment group). mRNA was assessed for CB1 (A) and CB2 (B) mRNA message using Sso Advanced SYBR green (BioRad). CB1KO mice (BL6 background) were sensitized and rechallenged following the previously outlined protocol. Disease parameters, as described previously, were assessed (n=5 per treatment group). (C) Relative percent swelling of the treated footpad compared with that of a naïve mouse. Symbols represent individual mice and data shown as mean ± S.E.M. ANOVA/Tukey * p<0.05 ** p<0.01 ***p<0.001 ****p<0.0001
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References

    1. Tanasescu R, Constantinescu CS. Cannabinoids and the immune system: an overview. Immunobiology. 2010;215:588–597. - PubMed
    1. Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M, Kaminski NE, Schatz AR, Gopher A, Almog S, Martin BR, Compton DR, et al. Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol. 1995;50:83–90. - PubMed
    1. Sugiura T, Kondo S, Sukagawa A, Nakane S, Shinoda A, Itoh K, Yamashita A, Waku K. 2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain. Biochem Biophys Res Commun. 1995;215:89–97. - PubMed
    1. Sugiura T, Kondo S, Kishimoto S, Miyashita T, Nakane S, Kodaka T, Suhara Y, Takayama H, Waku K. Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid CB2 receptor. Comparison of the agonistic activities of various cannabinoid receptor ligands in HL-60 cells. J Biol Chem. 2000;275:605–612. - PubMed
    1. Prescott SM, Majerus PW. Characterization of 1,2-diacylglycerol hydrolysis in human platelets. Demonstration of an arachidonoyl-monoacylglycerol intermediate. J Biol Chem. 1983;258:764–769. - PubMed

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