Journal of Leukocyte Biology

Cannabinoids exhibit broad immune modulating activity by targeting many cell types within the immune system, including T cells, which exhibit sensitivity, as evidenced by altered activation, proliferation, and cytokine expression. As a result of the critical role calcium plays in T cell function coupled with previous findings demonstrating disruption of the calcium-regulated transcription factor, nuclear factor of activated T cells, by cannabinoid treatment the objective of the present investigation was to perform an initial characterization of the role of the cannabinoid receptors in the regulation of the intracellular calcium concentration ([Ca2+]i) by Δ9-tetrahydrocannabinol (Δ9-THC) in T lymphocytes. Here, we demonstrate that Δ9-THC robustly elevates [Ca2+]i in purified murine splenic T cells and in the human peripheral blood acute lymphoid leukemia (HPB-ALL) human T cell line but only minimally elevates [Ca2+]i in Jurkat E6-1 (dysfunctional cannabinoid receptor 2-expressing) human T cells. Removal of extracellular calcium severely attenuated the Δ9-THC-mediated rise in [Ca2+]i in murine splenic T cells and HPB-ALL cells. Pretreatment with cannabinoid receptor antagonists, SR144528 and/or SR141716A, led to an attenuation of Δ9-THC-mediated elevation in [Ca2+]i in splenic T cells and HPB-ALL cells but not in Jurkat E6-1 cells. Furthermore, pretreatment of HPB-ALL cells with SR144528 antagonized the small rise in [Ca2+]i elicited by Δ9-THC in the absence of extracellular calcium. These findings suggest that Δ9-THC induces an influx of extracellular calcium in resting T cells in a cannabinoid receptor-dependent manner.

Immune cells have been shown to express cannabinoid receptors and to produce endogenous ligands. Moreover, activation of cannabinoid receptors on immune cells has been shown to trigger potent immunosuppression. Despite such studies, the role of cannabinoids in transplantation, specifically to prevent allograft rejection, has not, to our knowledge, been investigated previously. In the current study, we tested the effect of THC on the suppression of HvGD as well as rejection of skin allografts. To this end, we studied HvGD by injecting H-2k splenocytes into H-2b mice and analyzing the immune response in the draining ingLNs. THC treatment significantly reduced T cell proliferation and activation in draining LNs of the recipient mice and decreased early stage rejection-indicator cytokines, including IL-2 and IFN-γ. THC treatment also increased the allogeneic skin graft survival. THC treatment in HvGD mice led to induction of MDSCs. Using MDSC depletion studies as well as adoptive transfer experiments, we found that THC-induced MDSCs were necessary for attenuation of HvGD. Additionally, using pharmacological inhibitors of CB1 and CB2 receptors and CB1 and CB2 knockout mice, we found that THC was working preferentially through CB1. Together, our research shows, for the first time to our knowledge, that targeting cannabinoid receptors may provide a novel treatment modality to attenuate HvGD and prevent allograft rejection.

Langerin is a C-type lectin expressed at high level by LCs of the epidermis. Langerin is also expressed by CD8+/CD103+ XCR1+ cross-presenting DCs of mice but is not found on the homologous human CD141high XCR1+ myeloid DC. Here, we show that langerin is expressed at a low level on DCs isolated from dermis, lung, liver, and lymphoid tissue and that langerin+ DCs are closely related to CD1c+ myeloid DCs. They are distinguishable from LCs by the level of expression of CD1a, EpCAM, CD11b, CD11c, CD13, and CD33 and are found in tissues and tissue-draining LNs devoid of LCs. They are unrelated to CD141high XCR1+ myeloid DCs, lacking the characteristic expression profile of cross-presenting DCs, conserved between mammalian species. Stem cell transplantation and DC deficiency models confirm that dermal langerin+ DCs have an independent homeostasis to LCs. Langerin is not expressed by freshly isolated CD1c+ blood DCs but is rapidly induced on CD1c+ DCs by serum or TGF-β via an ALK-3-dependent pathway. These results show that langerin is expressed outside of the LC compartment of humans and highlight a species difference: langerin is expressed by the XCR1+ “DC1” population of mice but is restricted to the CD1c+ “DC2” population of humans (homologous to CD11b+ DCs in the mouse).

CLRs on DCs play important roles in immunity and are expressed selectively on certain DC subsets. Murine DCAL2 (myeloid inhibitory C-type lectin/Clec12a) is a type-II CLR with an ITIM. Using a mouse DCAL2-specific mAb, we found that DCAL2 is expressed at relatively high levels on APCs and that DCAL2 expression can be used to divide CD8α– DCs into DCAL2+DCIR2– and DCAL2–DCIR2+ subpopulations. CD8α–DCAL2+ DC, CD8α–DCIR2+ DC, and CD8α+DCAL2+ DC subsets each express different levels of TLRs and respond to unique classes of TLR ligands by producing distinct sets of cytokines. Whereas CD8α–DCAL2+ DCs robustly produce cytokines, including IL-12, in response to CpG, CD8α–DCIR2+ DCs produce only TNF-α and IL-10 in modest amounts when stimulated with zymosan. However, CD8α–DCIR2+ DCs, unlike the other DC subsets, strongly up-regulate OX40L when stimulated with bacterial flagellin. As predicted from their cytokine expression, CD8α–DCAL2+ DCs efficiently induced Th1 responses in the presence of CpG in vitro and in vivo, whereas CD8α–DCIR2+ DCs induced Th2 cells in response to flagellin. Thus, CD8α–DCAL2+ DCs comprise a distinct CD8α– DC subset capable of supporting Th1 responses. DCAL2 is a useful marker to identify a Th1-inducing CD8α– DC population.

We examined the role of MCP-1, a potent chemotactic and activating factor for macrophages, in perfusion, inflammation, and skeletal muscle regeneration post-ischemic injury. MCP-1−/− or C57Bl/6J control mice [wild-type (WT)] underwent femoral artery excision (FAE). Muscles were collected for histology, assessment of tissue chemokines, and activity measurements of lactate dehydrogenase (LDH) and myeloperoxidase. In MCP-1−/− mice, restoration of perfusion was delayed, and LDH and fiber size, indicators of muscle regeneration, were decreased. Altered inflammation was observed with increased neutrophil accumulation in MCP-1−/− versus WT mice at Days 1 and 3 (P≤0.003), whereas fewer macrophages were present in MCP-1−/− mice at Day 3. As necrotic tissue was removed in WT mice, macrophages decreased (Day 7). In contrast, macrophage accumulation in MCP-1−/− was increased in association with residual necrotic tissue and impaired muscle regeneration. Consistent with altered inflammation, neutrophil chemotactic factors (keratinocyte-derived chemokine and macrophage inflammatory protein-2) were increased at Day 1 post-FAE. The macrophage chemotactic factor MCP-5 was increased significantly in WT mice at Day 3 compared with MCP-1−/− mice. However, at post-FAE Day 7, MCP-5 was significantly elevated in MCP-1−/− mice versus WT mice. Addition of exogenous MCP-1 did not induce proliferation in murine myoblasts (C2C12 cells) in vitro. MCP-1 is essential for reperfusion and the successful completion of normal skeletal muscle regeneration after ischemic tissue injury. Impaired muscle regeneration in MCP-1−/− mice suggests an important role for macrophages and MCP-1 in tissue reparative processes.

To clarify interleukin (IL)-6 roles in wound healing, we prepared skin excisions in wild-type (WT) and IL-6-deficient BALB/c [knockout (KO)] mice. In WT mice, the wound area was reduced to 50% of original size at 6 days after injury. Microscopically, leukocyte infiltration was evident at wound sites. Furthermore, the re-epithelialization rate was ∼80% at 6 days after injury with increases in angiogenesis and hydroxyproline contents. The gene expression of IL-1, chemokines, adhesion molecules, transforming growth factor-β1, and vascular endothelial growth factor was enhanced at the wound sites. In contrast, the enhanced expression of these genes was significantly reduced in KO mice. Moreover, in KO mice, the reduction of wound area was delayed with attenuated leukocyte infiltration, re-epithelialization, angiogenesis, and collagen accumulation. Finally, the administration of a neutralizing anti-IL-6 monoclonal antibody significantly delayed wound closure in WT mice. These observations suggest that IL-6 has crucial roles in wound healing, probably by regulating leukocyte infiltration, angiogenesis, and collagen accumulation.

B cell neoplasms present heterogeneous patterns of lymphoid organ involvement, which may be a result of the differential expression of chemokine receptors. We found that chemokine receptor (CCR)7, CXC chemokine receptor (CXCR)4, or CXCR5, the main chemokine receptors that mediate B cell entry into secondary lymphoid tissues and their homing to T cell and B cell zones therein, were highly expressed in B malignancies with widespread involvement of lymph nodes. Conversely, those pathologies with little or no nodular dissemination showed no expression to very low levels of CCR7 and CXCR5 and low to moderate levels of CXCR4. These findings provide evidence for the role of CCR7, CXCR4, and CXCR5 in determining the pattern of lymphoid organ involvement of B tumors. Functional studies were performed on B malignancies expressing different levels of CCR7, CXCR5, and CXCR4. Multiple myeloma (MM) cells did not express CCR7 nor CXCR5 and did not migrate in response to their ligands; a moderate expression of CXCR4 on MM cells was accompanied by a migratory response to its ligand, CXCL12. By contrast, cells from B cell chronic lymphocytic leukemia (B-CLL) expressed the highest levels of these chemokine receptors and efficiently migrated in response to all ligands of CCR7, CXCR4, and CXCR5. In addition, the migration index of B-CLL cells in response to both of the CCR7 ligands correlated with the presence of clinical lymphadenopathy, thus indicating that the high expression of functional chemokine receptors justifies the widespread character of B-CLL, representing a clinical target for the control of tumor cell dissemination.

To date, chronic lymphocytic leukemia (CLL) remains incurable with current treatments, which include the monoclonal antibodies (mAbs) rituximab and alemtuzumab. The efficacy of rituximab is modest when used as single agent, and alemtuzumab induces severe immunosuppression. To develop more potent and specific therapies, we propose the CC chemokine receptor 7 (CCR7) as an attractive target molecule to treat CLL, as it not only fulfills the requirements of a high-surface expression and a good level of tissue specificity, but it also plays a crucial role in mediating the migration of the tumor cells to lymph nodes (LNs) and thus, in the development of clinical lymphadenopathy. In the current work, murine anti-human CCR7 mAb mediated a potent, complement-dependent cytotoxicity (CDC) against CLL cells while sparing normal T lymphocytes from the same patients. The sensitivity to CDC was related to the antigenic density of CCR7. Moreover, these mAb blocked the in vitro migration of CLL cells in response to CC chemokine ligand 19 (CC219), one of the physiological ligands of CCR7. Conversely, CLL cells were poorly lysed through antibody-dependent, cell-mediated cytotoxicity (ADCC), probably as a result of the murine origin and the isotype of the anti-CCR7 mAb used. Molecular engineering techniques will allow us to obtain chimeric or humanized anti-CCR7 mAb to reach the best clinical response for this common and yet incurable leukemia.

Experimental autoimmune uveoretinitis (EAU) is a T helper type 1 cell-mediated autoimmune disease, which serves as a model of human chronic uveitis. In this model, cells of a monocyte/macrophage lineage and retinal antigen (Ag)-specific T cells infiltrate into the retina and cause inflammatory lesion, where proinflammatory cytokines and various stimuli activate a transcriptional factor, nuclear factor-κB (NF-κB), which modulates inflammation and enhances immune responses. In the present study, the therapeutic effect of administration of a NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC), was examined in a murine EAU model. It was shown that PDTC ameliorated the clinical symptoms of EAU mice and significantly reduced the histopathological score compared with those in untreated mice. mRNA expressions of tumor necrosis factor α and interleukin-1β were suppressed in eyes of PDTC-treated EAU mice. However, when T cells from PDTC-treated EAU mice, Ag-presenting cells (APC), and the retinal Ag peptides were cocultured, these T cells showed the same level of proliferation as those from control mice. Furthermore, addition of PDTC in the culture of T cells from EAU mice, Ag, and APC completely abrogated the T cell-proliferative response and cytokine production. Pretreatment of Ag-primed T cells or APC with PDTC in vitro also reduced these responses. These results indicate that the inhibitory effect of PDTC is attributed mainly to the suppression of effector-phase responses including inflammation but not to the inhibition of T cell priming. Regulation of NF-κB pathway in the lesion could be a novel target for the successful control of uveoretinitis.

Osteoclasts are large, multinucleated cells, which originate from the fusion of macrophages. They play a central role in bone development and remodeling via the resorption of bone and are thus important mediators of bone loss, which leads to osteoporosis. IL-1R-associated kinase (IRAK)-M is a pseudokinase, which acts as a negative modulator of innate immune responses mediated by TLRs and IL-1R. Recently, it has been reported that IRAK-M also participates in the control of macrophage differentiation into osteoclasts. In addition, it was shown that IRAK-M knockout mice develop a strong osteoporosis phenotype, suggesting that down-regulation of this molecule activates osteoclast-mediated bone resorption. We studied the effect of the osteoporosis-inducing glucocorticoid, 6-methylprednisolone (6-MP), on IRAK-M expression in osteoclasts. Our results showed that osteoclasts, derived from THP-1 and RAW cells as well as human blood monocytes, differentiated into osteoclasts, express high levels of IRAK-M at mRNA and protein levels. In addition, 6-MP down-regulates IRAK-M expression, which correlates with an increased activation of bone resorption. These findings suggest a mechanism of corticosteroid-induced osteoporosis and open new avenues for treating this endemic disease of Western societies.