Although both CB1 and CB2 are expressed on cells of the immune system, the precise nature of these receptors and the role of endogenous and exogenous cannabinoids in the regulation of the immune functions are not clear. Also, although many studies have demonstrated that cannabinoids exhibit anti‐inflammatory properties, the precise mechanisms are unclear. In this study, we demonstrate for the first time that activation of CB1 and CB2 through administration of cannabinoids such as THC, into mice, triggers massive induction of arginase 1 expressing CD11b + Gr‐1 + MDSC, with immunosuppressive properties.

Endogenous and exogenous cannabinoids signal through two major G‐protein coupled cannabinoid receptors, CB1 and CB2 17 . Delta‐9‐tetrahydrocannabinol (THC) is a natural cannabinoid compound from the plant Cannabis sativa 18 . THC and other cannabinoids have been extensively studied with respect to their immunomodulatory and anti‐inflammatory properties 17 . Cannabinoids exert their immunomodulatory effects by various mechanisms. We and others have previously shown that the immunosuppressive property of THC can be attributed in part to its ability to induce apoptosis in T lymphocytes, DC and macrophages 19 - 22 . THC has also been shown to trigger Treg 23 as well as induce anti‐inflammatory cytokine production 18 , 23 , 24 .

Recently, a suppressor cell population of myeloid lineage capable of reducing anti‐tumor as well as inflammatory immune responses has been described 1 - 5 . These cells express CD11b and Gr‐1 and have been named myeloid suppressor cells or myeloid‐derived suppressor cells (MDSC) 2 , 3 , 6 . CD11b + Gr‐1 + MDSC are a heterogeneous cell population including immature macrophages, granulocytes, DC and other myeloid cells, and possess potent immunosuppressive properties 2 , 6 . They have been shown to suppress T‐cell proliferation 7 , inhibit the cytotoxicity of CD8 T cells or NK cells both in vitro and in vivo 3 , 6 , 8 , 9 , downregulate L‐selectin expression on CD4/CD8 T cells 10 and induce antigen‐specific CD8 T‐cell tolerance in tumor‐bearing hosts 11 . L‐Arginine metabolism is the major mechanism by which MDSC cause T‐cell suppression 1 , 2 , 12 . CD11b + Gr‐1 + suppressive cells have also been identified during several inflammatory conditions 13 - 16 .

To further establish the role of cannabinoid receptors, we used CB1 and CB2 receptor KO mice. We injected WT, CB1KO (CB1 −/− ) or CB2KO (CB2 −/− or Cnr2 −/− ) mice with vehicle or THC. In CB1 and CB2 KO mice, the percentages (Fig. 8C ) as well as the absolute numbers (Fig. 8D ) of MDSC in the peritoneum were significantly reduced after THC challenge when compared with WT mice, confirming the involvement of both the receptors. Furthermore, when we injected CB1 and CB2 KO mice with CB2 or CB1 antagonist (SR2 or SR1), respectively, and then administered THC, we observed that blocking CB2 receptors in CB1 KO mice and CB1 receptors in CB2KO mice had a synergistic effect and almost completely inhibited the induction of absolute numbers of MDSC in response to THC (Fig. 8C and D ). Cumulatively, these data suggested that THC induces MDSC by signaling primarily through both CB1 and CB2 receptors.

Role of cannabinoid receptors in induction of MDSC by THC. WT mice were injected with CB1 (SR1) or CB2 antagonist (SR2) or both, 2 h prior to injecting vehicle or THC (20 mg/kg). After 12 h, peritoneal exudate cells were harvested and stained for MDSC (CD11b and Gr‐1) and analyzed by flow cytometry. (A) Representative dot plots showing percentage of MDSC. (B) Corresponding absolute MDSC numbers are shown. (C) WT, CB1KO or CB2KO mice were injected with vehicle or THC. Some CB1KO or CB2KO mice also received CB2 antagonist (SR2) or CB1 antagonist (SR1), respectively, 2 h before THC injection. Representative dot plots showing percentage of MDSC. (D) Absolute MDSC cell numbers in the peritoneum at 12 h. *** p <0.001 versus WT vehicle; † p <0.05, †† p <0.01, ††† p <0.001 versus WT‐THC. Data are mean±SEM ( n =3). Similar results were obtained in two experiments with three mice per group.

To investigate whether induction of MDSC resulted from activation of cannabinoid receptors, we first used CB1 and CB2 antagonists, SR 141716A (SR1) and SR144528 (SR2), respectively. WT mice were injected with SR1 or SR2 or both, 2 h prior to administration of vehicle or THC. Mice pretreated with either SR1 or SR2 showed significantly less frequency (Fig. 8A ) and absolute number (Fig. 8B ) of MDSC when compared with mice injected with THC alone. This indicated that THC acts through both CB1 and CB2 receptors to induce the mobilization of MDSC. We did not observe any significant synergistic blocking with both the antagonists injected together.

Analysis of MDSC subsets induced by THC. To determine the granulocytic (CD11b + Ly6G high Ly6C low ) and monocytic (CD11b + Ly6G − Ly6C high ) subsets of MDSC, WT mice were injected with various doses of THC, peritoneal exudates cells were harvested after 12 h, triple stained for CD11b, Ly6G and Ly6C markers and analyzed by flow cytometry. (A) Ly6G and Ly6C expression on cells gated for CD11b + showing CD11b + Ly6G + Ly6C +(int) granulocytic (PMN) and CD11b + Ly6G neg Ly6C +(high) monocytic subsets. Representative dot plots show the percentages for granulocytic (PMN) and monocytic (MO) populations for various doses of THC. (B and C) Absolute cell numbers of each subset of MDSC for different doses of THC. ** p <0.01, *** p <0.01 versus control (THC, 0 mg/kg). Data are mean±SEM ( n =5) and represent one out of the two independent experiments.

Although MDSC are defined by their coexpression of CD11b and Gr‐1, these cells have been known to contain heterogeneous mixture of myeloid cells with suppressive function. However, recently two major subsets of MDSC have been identified based on the expression of CD11b, Ly6G and Ly6C antigens. Granulocytic subsets (PMN‐like MDSC subset) express both Ly6G and Ly6C along with CD11b (CD11b + Ly6‐G high Ly6‐C low/int ), whereas monocyte like MDSC subsets express only Ly6‐C and CD11b (CD11b + Ly6G neg Ly6C high ) 25 , 30 , 31 . We sought to identify these subsets among MDSC induced by THC. For this, we used Ab specific to Ly6G (clone: 1A8) and Ly6C (clone: HK1.4) along with anti‐CD11b. THC administration resulted in significant dose‐dependent increase in the frequency (Fig. 7A ) as well as absolute numbers of CD11b + Ly6G + Ly6C +(int) granulocytic (PMN) and CD11b + Ly6G neg Ly6C +(high) monocytic MDSC in the peritoneum (Fig. 7B and C ), suggesting that THC significantly induces both major MDSC subsets.

In naïve mice, CD11b + Gr‐1 + cells have been shown to be present in small numbers in peripheral tissues such as spleen and up to 18–50% in bone marrow depending on the mouse strain 6 , 29 . As THC induces mobilization of MDSC massively and rapidly, we speculated that the source of these cells could be bone marrow. To investigate this, we injected WT mice with THC and determined the percentage of CD11b + Gr‐1 + cells at 0 and 16 h in bone marrow and peritoneum (Fig. 6B ). We observed a significant decrease in the percentage of CD11b + Gr‐1 + cells in the bone marrow 16 h after THC administration with a corresponding increase in periphery (peritoneum). This indicated that CD11b + Gr‐1 + cells were migrating from bone marrow in response to THC. Further, we wanted to know if the MDSC were also dividing in the periphery. Mice were given vehicle or THC, 2 h after injecting with BrdU. Peritoneal cells harvested after 24 h were triple stained for CD11b, Gr‐1 and intranuclear BrdU incorporation (Fig. 6C ). Close to 37% of the MDSC in the peritoneum of the THC‐injected mice were BrdU + . As cells incorporating BrdU in the bone marrow and then migrating to peritoneum in response THC would have contributed to this percentage, we determined active proliferation in the peritoneum by cell‐cycle analysis using PI staining. We found that a significant proportion (22.5%) of THC‐induced MDSC and 23.3% of all cells induced by THC in the peritoneum were in S‐phase, indicative of active cycling when compared with 11.9% of peritoneal cells found in vehicle‐treated mice. These data suggested that MDSC accumulating in the periphery in response to THC were undergoing active proliferation.

(A) THC‐induced MDSC express functional arginase. Arginase activity in the lysates of peritoneal exudate cells from vehicle or THC‐injected WT mice determined on the basis of conversion of L‐arginine to L‐ornithine as described in the Materials and methods section. *** p <0.001 versus vehicle control. Data are mean±SEM ( n =3). Two experiments with three mice per group were performed. Each measurement was performed in triplicates. (B) CD11b + Gr‐1 + cells migrate from bone marrow in response to THC. Representative dot plots of peritoneal and bone marrow cells harvested 0 or 16 h after THC treatment, showing percentage of CD11b + Gr‐1 + cells (gated), indicate possible migration of MDSC from bone marrow. Data are representative of two independent experiments with three mice per group. (C) THC‐induced MDSC proliferate in periphery. In vivo BrdU labeling showing proliferation of THC‐induced MDSC in peritoneum at 24 h. Representative histograms on right show percentage of BrdU + cells among gated CD11b + Gr‐1 + MDSC shown on left. Two experiments with three mice per group showed comparable results.

L‐Arginine metabolism is an important pathway used by MDSC to blunt anti‐tumor immunity 1 or reduce inflammatory responses 13 - 16 . Expression of arginase, the enzyme which metabolizes and depletes essential amino acid L‐arginine, is the hallmark characteristic of MDSC. In order to test if THC‐induced MDSC express functional arginase, we injected mice with vehicle or THC and determined arginase enzyme activity by spectrophotometric assay based on the principle of conversion of substrate L‐arginine to L‐ornithine. THC‐induced cells from the peritoneum accounted for several fold higher levels of arginase activity when compared with vehicle controls thereby suggesting that THC‐induced MDSC expressed functionally active arginase (Fig. 6A ).

To further confirm the role of G‐CSF in the induction of MDSC by THC in vivo , we pretreated mice with anti‐mouse G‐CSF Ab before injecting THC. We observed that as low as 10 μg/mouse of anti‐G‐CSF was able to significantly block the accumulation of MDSC in the peritoneum in response to THC (Fig. 5C ), indicating that G‐CSF plays a crucial role in this process. These data suggest that THC may trigger migration and expansion of MDSC primarily by inducing G‐CSF.

GM‐CSF has been shown to play an important role in induction MDSC associated with tumors 28 . However, we could not detect increase in GM‐CSF in sera in the current model. To further investigate the role of GM‐CSF and G‐CSF in the induction of MDSC by THC, we determined the levels of these cytokines directly in the peritoneal exudates of vehicle and THC‐injected mice. Although there was a robust increase in the levels of G‐CSF in the peritoneum of THC‐injected mice as compared with vehicle controls, the GM‐CSF levels were low and there was no significant difference between the two groups (Fig. 5B ).

Role of G‐CSF in the induction of MDSC by THC in vivo . (A) WT mice were injected with vehicle or THC (20 mg/kg), blood was collected at 6, 12 and 24 h, and serum chemokines G‐CSF and KC were determined by Bioplex assay. * p <0.05, ** p <0.01 and *** p <0.001 versus vehicle control. Data are mean±SEM ( n =5) and represent one out of the two independent experiments with similar results. (B) Detection of G‐CSF and GM‐CSF in the peritoneal exudates by sandwich ELISA. *** p <0.001 and n.s. (not significant) versus vehicle control. Data are mean±SEM ( n =3) and representative of two separate experiments. (C) Ab to G‐CSF blocks the accumulation MDSC in response to THC. Absolute numbers of CD11b + Gr‐1 + MDSC in the peritoneum 16 h after injecting THC in mice pretreated with control IgG or anti‐G‐CSF Ab. ** p <0.01 versus IgG control. Data are mean±SEM ( n =3). Two experiments with three mice per group showed similar results.

THC is known to suppress inflammatory cytokines induced by infectious agents or during inflammatory conditions in vivo 23 , 24 . However, we sought to determine if THC can induce any cytokine/chemokines in vivo resulting in the recruitment of MDSC. To this end, we injected WT mice with a single dose of THC or vehicle and collected sera at 6, 12 and 24 h. We analyzed sera for IL‐1α, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐5, IL‐6, IL‐9, IL‐10, IL‐12 (p40), IL‐12 (p70), IL‐13, IL‐17, IFN‐γ, TNF‐α, MIP‐1α, MIP‐1β, RANTES, MCP‐1, GM‐CSF, granulocyte CSF (G‐CSF), keratinocyte chemoattractant (KC) and eotaxin by Bioplex assay. THC had little or no effect on majority of these cytokines and chemokines at 6, 12 and 24 h as compared with basal levels induced by vehicle (data not shown), except for KC (CXCL1) and G‐CSF. THC induced significant levels of chemokine, KC at 12 h and very high and persistent levels of G‐CSF (∼1500 pg/mL) at 12 and 24 h (Fig. 5A ). It is noteworthy that THC induced G‐CSF levels significantly at an early time point, 6 h, and to a very high level at 12–24 h. It is possible that the initial upregulation of G‐CSF by THC leads to migration and activation of MDSC which in turn secrete more G‐CSF causing increased mobilization and expansion of these cells.

THC induces MDSC in TLR4‐deficient mice. To rule out the involvement of TLR4 ligands, we injected TLR4‐deficient C3H and C57BL10/ScN mice with vehicle or THC (20 mg/kg, i.p. ) and after 12 h, peritoneal exudate cells were harvested, and stained and analyzed for the coexpression of CD11b and Gr‐1 by flow cytometry. (A) Representative dot plots showing percentage of gated MDSC. (B) Mean absolute number of MDSC±SEM ( n =4) represent data from one out of the two independent experiments. *** p <0.001 versus vehicle control.

Previous studies have shown that TLR ligands can trigger induction MDSC 25 - 27 . The pure THC preparation used in this study had undetectable levels of endotoxins. However, to further rule out the effect of endotoxins, as well as to investigate the possibility if THC acted via TLR4 in this case, we tested the same batch of THC in TLR4‐deficient C57BL10/ScN mice and TLR4‐mutant C3H mice. Both C57BL10/ScN and C3H mice showed significant increase in frequency as well as absolute number of CD11b + Gr‐1 + MDSC (Fig. 4A and B ) thereby suggesting that THC‐induced recruitment of MDSC was independent of TLR4.

We studied the morphology of THC‐induced MDSC by Wright Giemsa staining. Cytospin preparations of peritoneal exudate cells from mice injected with vehicle or THC were stained and analyzed by light microscopy (Fig. 3C ). Vehicle‐injected mice showed the characteristics mainly of lymphocytes and macrophages. On the contrary, cells from THC‐injected mice consisted of PMN‐like cells with characteristic circular (or donut shaped) nucleus as well as monocyte‐like cells. We also looked at cellular infiltrates in parietal peritoneum sections. H&E stained cross‐sections of peritoneal tissue showed significant infiltration of cells in the THC group, whereas vehicle‐injected mice showed no infiltration of cells (Fig. 3D ).

Time course of THC‐induced MDSC accumulation in peritoneum and spleen. Peritoneal exudate cells and splenocytes harvested at indicated time points after the administration of THC in WT mice ( i.p. , 20 mg/kg) were analyzed for CD11b + Gr‐1 + MDSC by flow cytometry. (A) Representative dot plots showing percentages of MDSC (gated). (B) Absolute MDSC cell numbers were calculated based on the percentages and total cell numbers. *** p <0.001, ** p <0.01 and * p <0.05 versus control (0 h). Data are mean±SEM ( n =5) and representative of two separate experiments. (C) Morphological analysis. Representative photomicrographs (40× objective) of Wright–Giemsa‐stained cytospin preparations of peritoneal exudate cells harvested after 16 h from mice injected with vehicle or THC. MDSC induced by THC consist of PMN‐like cells with characteristic circular nuclei (red arrows) and monocyte‐type cells (blue arrows). (D) Representative H & E‐stained cross‐sections of parietal peritoneum from mice injected with vehicle or THC showing significant cellular infiltrates in THC‐injected mice. Similar results were obtained in two experiments with three mice per group (C and D).

The kinetics of MDSC induction by THC in peritoneum and spleen was determined by injecting mice with a single dose of THC, and analyzing recruitment of MDSC at different time points (0–24 h). A significant increase in frequency (Fig. 3A ) and absolute number of MDSC (Fig. 3B ) in peritoneum and spleen was detected as early as 8 h after THC injection, which peaked around 16 h and started to decline by 24 h.

Next, we used a well‐established model of T‐cell‐mediated liver inflammation induced by ConA to test the immunosuppressive properties of THC‐induced CD11b + Gr‐1 + cells in vivo . We adoptively transferred CD11b + Gr‐1 + cells purified from the peritoneum of THC‐injected mice into naïve mice before inducing hepatitis using ConA. The transferred CD11b + Gr‐1 + cells significantly suppressed liver inflammation as indicated by decreased liver enzyme, alanine transaminase (ALT) levels, a commonly used marker for liver inflammation (Fig. 2E ). These data together suggested that the CD11b + Gr‐1 + cells induced by THC exhibited the functional characteristics of MDSC.

THC‐induced CD11b + Gr‐1 + cells are immunosuppressive in vitro and in vivo . In vitro T‐cell suppression determined by (A) coculturing purified THC‐induced MDSC with WT T cells stimulated using ConA or (B) OT‐II T cells stimulated using ova peptide in the presence of irradiated APC. T‐cell proliferation was measured by [3H]‐thymidine incorporation (CPM, counts per minute) at 72 h . T cells stimulated in the absence of any MDSC served as positive control. (C) Sorted CD11b + Gr‐1 + cells from the peritoneum of THC or TGB‐injected mice were used in T‐cell proliferation assay stimulated using ConA. (D) Percentage T‐cell suppression for TGB‐neutrophils and THC‐MDSC. * p <0.05, ** p <0.01 and *** p <0.001 versus control for all above. Data are mean±SEM of triplicate determinations and representative of two experiments. (E) Serum ALT levels indicative of T‐cell‐induced hepatitis measured 16 h after ConA challenge in mice with or without adoptively transferred purified THC‐induced peritoneal MDSC (5×10 6 /mouse). *** p <0.001 versus vehicle; †† p <0.01 versus ConA. Data are mean±SEM ( n =4). Two independent experiments with three to four mice per group showed similar results.

Next, we investigated if THC‐induced CD11b + Gr‐1 + cells were MDSC by studying their ability to mediate inhibition of T‐cell proliferation, which is one of the most important characteristics of MDSC. To this end, we performed a series of in vitro and in vivo studies. For in vitro assays, we determined the suppressive activity of purified peritoneal CD11b + Gr‐1 + cells induced by THC on the proliferation of T cells stimulated with polyclonal as well as antigen‐specific stimuli. T‐cell proliferation was assessed by [3H]‐thymidine uptake. THC‐induced CD11b + Gr‐1 + cells caused a dose‐dependent decrease in proliferation of T cells stimulated with ConA with almost complete inhibition at a 1:2 ratio of CD11b + Gr‐1 + cells to T cells (Fig. 2A ). THC‐induced CD11b + Gr‐1 + cells also significantly decreased proliferation of OT‐II ova‐transgenic T cells stimulated using agonist ova peptide (Fig. 2B ). As a control, we purified CD11b + Gr‐1 + cells from peritoneal exudates induced by thioglycollate broth (TGB), which at early time points (4 h) triggers primarily neutrophils, and compared their ability to suppress T‐cell proliferation. Side‐by‐side comparison of suppressive activity showed that THC‐induced CD11b + Gr‐1 + cells were highly immunosuppressive compared with the CD11b + Gr‐1 + neutrophils (Fig. 2C and D ).

THC induces CD11b + Gr‐1 + cells in vivo . (A) B6 WT mice were injected i.p. with vehicle or THC (20 mg/kg). Total viable peritoneal exudate cells harvested after 16 h were quantified by trypan blue exclusion. *** p <0.001 versus vehicle control. Data are mean±SEM ( n =6). Similar results were obtained in four independent experiments with four to six mice per group. (B) Cells were analyzed by flow cytometry. Representative dot plots showing forward scatter (FSC) and side scatter (SSC) analysis for granularity and size by flow cytometry. Oval gate represents larger and more granular cells. (C) Phenotypic characterization. Cells were stained for various cell surface markers using fluorescently labeled mAb and analyzed by flow cytometry. Representative histograms from three mice per group show percentage of positive cells for each antigen. Results represent similar observations from two independent experiments with three to five mice per group. (D) WT mice were injected with various doses of THC and after 12 h peritoneal exudate cells were stained using fluorescently labeled mAb to CD11b and Gr‐1 antigens for MDSC and analyzed by flow cytometry. Representative dot plots show percentage of MDSC (gated double‐positive cells). (E) Absolute numbers of MDSC calculated based on the percentages and total cell numbers. ** p <0.01 versus vehicle control. Data are mean±SEM ( n =5). (D and E) Similar results were obtained in three independent experiments with three to five mice per group.

C57BL/6 (B6) WT mice were injected with vehicle or THC i.p . The exudate cells in the peritoneal cavity were harvested after 16 h and analyzed. To our surprise, the administration of THC induced massive local accumulation of cells in the peritoneal cavity when compared with vehicle control (Fig. 1A ). Flow cytometric analysis using forward and side scatter revealed that majority of cells induced by THC were granular and larger in size (Fig. 1B ). We further phenotyped these cells using mAb to cell‐surface markers, CD3, Gr‐1, CD11b and F4/80 (Fig. 1C ). Cells from vehicle‐injected mice showed phenotypic characteristics expected from a normal peritoneum. CD11b high cells in the peritoneum typically represent mature macrophages, which also express high F4/80. Interestingly, majority of cells (>90%) from THC‐injected mice were positive for CD11b and Gr‐1 markers. Close to one third proportion of the population also showed low or intermediate expression of F4/80. We performed a dose–response study by injecting ( i.p. ) WT mice with different doses of THC and analyzed the peritoneal exudate cells for the coexpression of CD11b and Gr‐1 by flow cytometry. THC triggered a dose‐dependent increase in the percentage (Fig. 1D ) as well as absolute numbers (Fig. 1E ) of CD11b + Gr‐1 + cells in the peritoneum. In control mice, the CD11b high (Gr‐1 − ) cell population represents mature macrophages. This is typical of naïve peritoneum where mature macrophages form a significant proportion, whereas, CD11b + Gr‐1 + cells induced by THC expressed intermediate levels of CD11b. The CD11b high (Gr‐1 − ) macrophages disappear in the dot plots with increasing doses of THC (Fig. 1D ) likely because of the dilution effect resulting from several fold induction of CD11b + Gr‐1 + cells or due to THC‐induced killing of mature macrophages as reported previously 20 . The robust induction of CD11b + Gr‐1 + cells seen here in response to THC also shows that unlike lymphocytes 19 - 21 or macrophages 20 , these cells are resistant to THC‐induced killing.

Discussion

Immunosuppressive and anti‐inflammatory properties of cannabinoids, including THC, have been well established 17. THC is known to cause immune suppression by various mechanisms including induction of apoptosis in effector T cells, suppressing the inflammatory cytokine production under certain activating conditions, as well as arresting cell proliferation 18. However, the effect of cannabinoids on important suppressor cell population, namely, MDSC is not known. MDSC were initially identified in large numbers in tumor‐bearing mice and were found to effectively suppress anti‐tumor immune responses 4-6. There is strong evidence showing that these cells are immunosuppressive and inactivate T cells 1, 2. Several recent studies have shown their role in modulation of inflammatory diseases 14-16. Induction of MDSC in cancer has also been attributed to be a consequence of chronic inflammation 32, 33.

In this study, we injected THC i.p. in naïve mice in the absence of any activating conditions. Initially, we were perplexed to see the dramatic accumulation of cellular infiltrates in the peritoneal cavity in response to THC, which were found to be large and granular cells by preliminary assessment. Given the potent immunosuppressive properties of THC, we speculated that these may not be inflammatory infiltrates, but some type of suppressor cells. Further phenotypic and functional characterization revealed that THC‐induced cells coexpressed CD11b and Gr‐1 markers. We further observed that THC‐induced CD11b+Gr‐1+ cells expressed functional arginase and caused significant inhibition in the proliferation of T cells stimulated with polyclonal (ConA) as well as antigen‐specific (ova) stimuli in vitro, and conA‐induced T‐cell‐mediated liver inflammation in vivo, all hallmark characteristics of MDSC. It should be noted that unlike THC‐induced CD11b+Gr‐1+ cells, purified CD11b+Gr‐1+ neutrophils isolated 4 h after thioglycollate administration failed to exhibit similar immunosuppressive properties.

We and others have shown that THC suppresses inflammatory cytokines elevated during an inflammatory response 23, 34. However, the effect of THC on various cytokines in naïve mice has not been thoroughly investigated. Among an array of 23 different cytokines and chemokines tested, we made a striking observation that THC caused a dramatic induction in the levels of G‐CSF, and to some extent KC, in sera of naïve mice. Very high induction of G‐CSF was also observed directly in the peritoneum. We noted that blocking of G‐CSF by anti‐G‐CSF Ab was able to significantly decrease THC‐induced accumulation of MDSC in vivo. G‐CSF is a crucial cytokine required for the differentiation of granulocytes such as neutrophils from bone marrow precursors, and which also regulates their migration, proliferation as well as function 35. Anti‐inflammatory properties of G‐CSF have also been known 36. A very recent study using a spontaneous metastatic memory carcinoma model showed that the number of MDSC in the spleen directly correlated with G‐CSF transcript levels 37. Differential accumulation of tumor infiltrating monocytic MDSC over granulocytic MDSC in tumor‐bearing hosts mediated by chemokines, particularly G‐CSF has also been demonstrated 38.

We further showed that the effect of THC was independent of TLR4 and mediated directly through activation of both CB1 and CB2. This observation was supported by the findings that CB1 or CB2 select antagonists could block MDSC induction by THC partially but significantly and was confirmed using CB1‐ and CB2‐deficient mice. It has been shown that bone marrow is the source of large numbers of CD11b+Gr‐1+ precursors 6, 29. In this study, THC seemed to cause the migration of CD11b+Gr‐1+ MDSC from bone marrow. THC also induced the proliferation of migrated MDSC in the periphery as evidenced by significant proportion of actively proliferating MDSC in the peritoneum of THC injected mice. Various cell types including macrophages, endothelial cells and fibroblasts are known to express CB1 and CB2 39. THC may activate CB1/CB2 on these cells to induce the secretion of mediators such as G‐CSF and KC (CXCL1). Inasmuch as, G‐CSF and KC have been shown to induce the development, migration and expansion of granulocytes, particularly neutrophils, it is highly likely that these molecules may play a crucial role in MDSC induction. We have noted that unlike the i.p. administration of THC, i.v. injection does not result in robust induction of MDSC in spleen, lungs and peritoneum although a significant induction in the liver was seen (data not shown). This may result from the fact that i.p. injection of THC may activate resident cells in the peritoneal cavity such as fibroblasts or immature macrophages that are known to produce G‐CSF 40. This is also supported by our observation that high levels of G‐CSF were found in the peritoneal cavity washings after THC injection by the i.p. route. Although G‐CSF is primarily known to promote neutrophil differentiation from bone marrow precursors, it is possible that G‐CSF by itself or other factors such as KC induced by THC or a combination of G‐CSF and THC may promote mobilization of CD11b+Gr‐1+ MDSC and arrest their differentiation into mature neutrophils or macrophages. Currently, we are further testing this possibility.

We observed that a single dose of THC, as low as 5–10 mg/kg, was able to induce significant levels of MDSC in mice, which is clinically relevant. Based on the body surface area normalization method 41, this translates to human equivalent dose of 15–30 mg/m2 (0.40–0.81 mg/kg). In cancer patients, the dose of THC (dronabinol or Marinol®) recommended is usually 2.5–20 mg/m2/day (0.068–0.54 mg/kg), as an anti‐emetic treatment during chemotherapy. Chan et al. demonstrated that rats injected with 50 mg/kg body weight of THC had a serum concentration of 10 μM THC within 10 h of administration 42. Azorlosa et al. showed that levels as high as 1 μM could be attained in the plasma of humans after recreational use of marijuana 43, and in a separate report, it was shown that THC can get preferentially concentrated 15‐ to 20‐fold in some tissues 44. Such levels of THC may lead to significant suppression of the immune response, leading to increased susceptibility to opportunistic infections and cancer. In fact, it was shown that treatment of mice with doses of 8 mg/kg significantly suppressed the responses of mice to infection with Legionella pneumophila 34. In light of these, our results suggest that THC, at a pharmacological or recreational dose, may induce MDSC and cause immunosuppression.

Studies from our laboratory suggest that cannabinoids are a double‐edged sword. On one hand, cannabinoids can suppress malignancies of the immune system by inducing apoptosis of tumors that express cannabinoid receptors 45. On the other hand, because they suppress anti‐tumor immune response, they promote growth and metastasis of cannabinoid receptor‐deficient tumors such as breast cancer that are resistant to cannabinoid‐induced apoptosis 46. Epidemiological studies have shown that marijuana smokers are more susceptible to certain types of cancer 47, 48. It is possible that induction of MDSC by cannabinoids may make an individual more susceptible to certain types of cancer, particularly those which do not express cannabinoid receptors. We previously showed that THC promotes the growth and metastasis of 4T1 mammary tumors that lack cannabinoid receptors by suppressing anti‐tumor immunity 46. Recent studies have suggested that 4T1 tumor growth is associated with MDSC induction 49. Thus, it is tempting to speculate that THC further enhances induction of MDSC which may be responsible for the accelerated growth and metastasis of 4T1 tumors and studies are in progress to address this in detail.

This study unravels a new mechanism by which cannabinoids trigger immune suppression. These findings are important in several ways: (i) THC is currently used clinically to ameliorate nausea and vomiting as well as stimulate appetite during chemotherapy in cancer patients, and HIV/AIDS‐infected patients and to lower intraocular eye pressure to treat glaucoma 50. Thus, it is important to know if such a treatment would impact the immune system and pose health hazards. As MDSC are known to promote tumor growth, our observations indicate that THC treatment in cancer patients may potentiate adverse effects. (ii) As CB2 is expressed only on immune cells, CB2 select agonists may constitute better anti‐inflammatory agents with less nonspecific toxicity than the currently available anti‐inflammatory agents. Thus, the demonstration that CB2 activation leads to MDSC which in turn may help suppress inflammation may provide novel avenues to develop anti‐inflammatory agents. (iii) Our studies shed light on the potential role of endocannabinoids that activate cannabinoid receptors on MDSC induction and consequent regulation of inflammation.