α‐Galactosylceramide suppresses murine eosinophil production through interferon‐γ‐dependent induction ofNOsynthase andCD95

Abstract

BACKGROUND AND PURPOSEα-Galactosylceramide (α-GalCer), a pleiotropic immunomodulator with therapeutic potential in neoplastic, autoimmune and allergic diseases, activates invariant natural killer T-cells throughCD1-restricted receptors for α-GalCer on antigen-presenting cells, inducing cytokine secretion. However the haemopoietic effects of α-GalCer remain little explored. EXPERIMENTAL APPROACHα-GalCer-induced modulation of eosinophil production in IL-5-stimulated bone marrow cultures was examined in wild-type (BALB/c, C57BL/6) mice and their mutants lacking CD1, inducible NOS (iNOS), CD95 and IFN-γ, along with the effects of lymphocytes; IFN-γ; caspase and iNOS inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs) and LTD4; and dexamethasone. KEY RESULTSα-GalCer (10 , i.v.) suppressed colony formation by GM-CSF-stimulated bone marrow progenitors in semi-solid cultures. α-GalCer and dexamethasone synergistically promoted eosinophil maturation. Suppression of eosinophil production by α-GalCer was prevented by aminoguanidine and was undetectable in bone marrow lacking iNOS, CD95, CD28; or CD1d. Separation on Percoll gradients and depletion of CD3+ cells made bone marrow precursors unresponsive to α-GalCer. Responsiveness was restored with splenic lymphocytes. Experiments with (i) IFN-γ-deficient bone marrow, alone or co-cultured with spleen T-cells from wild-type, but not from CD1d-deficient, donors; (ii) IFN-γ neutralization; and (iii) recombinant IFN-γ, showed that these effects of α-GalCer were mediated by IFN-γ. Effects of α-GalCer on eosinophil production were blocked by LTD4 and NSAIDs. CONCLUSIONS AND IMPLICATIONSα-GalCer activation of IFN-γ-secreting, CD1d-restricted lymphocytes induced iNOS-CD95-dependent apoptosis in developing eosinophils. This pathway is initiated by endogenous regulatory lymphocytes, antagonised by LTD4, NSAIDs and aminoguanidine, and modified by dexamethasone. AbbreviationsDEC, diethylcarbamazine; α-GalCer, α-galactosylceramide; iNKT cells, invariant natural killer T lymphocytes

Odd couple: The unexpected partnership of glucocorticoid hormones and cysteinyl-leukotrienes in the extrinsic regulation of murine bone-marrow eosinopoiesis

“…These alternatives have clearly distinct counterparts in an experimental setting: In the first case, bone-marrow effects can be elicited by intravenous transfer of plasma from the appropriate donors to naive recipients[ 22 ], and effects of this transfer will be restricted to the bone-marrow as long as there are no responsive targets elsewhere; in the second, transfer of leukocyte populations capable of homing to the bone-marrow compartment will be sufficient, and responses will both be limited to the bone-marrow compartment, and associated with the physical presence of the transferred leukocytes in this compartment[ 23 ].…”

“…Further insight on the underlying cellular mechanisms is provided by the effects of DEC. Interestingly, DEC requires not only 5-LO to be effective[ 47 ] but inducible NO synthase (iNOS) as well[ 54 ]. This enzyme, which produces large amounts of NO in the course of cellular immune responses to a number of intracellular pathogens, had already been shown to be required for the suppressive effects of PGE2 on bone-marrow eosinopoiesis[ 72 ]; more recently, it was shown to account for similar effects of α-galactosylceramide (α-GalCer; an anticancer agent and immunomodulator)[ 23 ] and IL-17 (a powerful proinflammatory cytokine)[ 26 ] on bone-marrow. These observations, therefore, establish DEC as a pharmacological link between 5-LO and iNOS in bone-marrow, as discussed below.…”

“…It is therefore important that CysLT can counteract the effects of both IL-17[ 26 ] and α-GalCer[ 23 ] on eosinopoiesis, just as CysLT counteract those of PGE2[ 70 ]. Like GC, therefore, CysLT target the iNOS-CD95-dependent proapoptotic mechanism that suppresses eosinopoiesis, as part of their eosinophilia-promoting actions.…”

“…Coupling of GC to CysLT is portrayed, based on the available evidence, as a transient relationship between events taking place within a single cell target (eosinophil precursor inside the bone-marrow), since these events all impinge upon a signaling sequence that begins at surface receptors and ends at apoptotic cell death[ 72 , 73 ] which is limited to immature eosinophils and their immediate precursos[ 36 ]. In this continuous sequence of signaling events, GC and CysLT act as suppressors of apoptosis (indicated in light blue colored boxes and arrows) by blocking distinct signaling steps upstream from iNOS; conversely, inhibitors of CysLT production or action, including DEC, promote apoptosis (indicated in orange-colored boxes and arrows, for DEC as well as a wide panel of pharmacological agents which act at CysLT-unrelated steps) by acting on targets upstream from iNOS[ 23 , 26 , 45 , 47 , 54 , 70 , 72 , 73 ]. In addition to its systemic effects on adrenal release of GC during allergen challenge, which are not shown in the picture, TNF-α is hypothesized to have separate effects on GC and CysLT-mediated responses: A constitutive effect permissive for GC action on eosinophil precursors (solid lavender line), and an adaptive (coupling) effect permissive for GC control of CysLT responses in the same cell target (discontinuous lavender line).…”

“… A graphical abstract of the main events identified in extrinsic regulation of bone-marrow eosinopoiesis, and of the hypothetical interactions of tumor necrosis factor-α with the underlying mechanisms. Colored boxes and arrows identify different classes of agents and their actions as follows: Orange, extrinsic suppressors of murine bone-marrow eosinopoiesis both in vitro [ 23 , 26 , 72 , 73 ] and in vivo [ 47 , 54 ]; light blue, extrinsic enhancers of eosinopoiesis in vitro [ 25 , 36 , 51 , 70 ] and in vivo [ 45 , 47 , 52 , 53 ]; yellow, essential components of a proapototic sequence (iNOS-CD95L-dependent pathway[ 54 , 72 , 73 ]) which is susceptible to activation by the first (orange-labeled) and blockade by the second (light blue-labeled) sets of extrinsic regulators; lavender, TNF-α, presenting both constitutive (continuous arrow) and challenge-induced (discontinous arrow) effects, inside the bone-marrow, besides its extramedullary actions[ 45 ] as activator of the HPA axis (not shown). TNF-α: Tumor necrosis factor-α.…”