Distinct anti-inflammatory properties of alpha1-antitrypsin and corticosteroids reveal unique underlying mechanisms of action.

Alpha1-antitrypsin (AAT) is a serum protease inhibitor that rises during inflammation and healthy pregnancies. Plasma-derived AAT, indicated for genetic AAT deficiency, is presently being explored for additional medical indications. Unlike corticosteroids, some anti-inflammatory activities of AAT involve NF-κB-dependent outcomes, e.g., induction of IL-1R antagonist. AAT activities were compared to dexamethasone (DEX), using various in-vitro cells assays, animal studies, and NF-κB-p65 localization and activity studies. Results demonstrate a cytokine shift towards resolution in AAT-treated cells, as opposed to pan-suppression in DEX-treated cells. AAT enhanced, while DEX suppressed LPS-induced IL-1Ra production and re-epithelialization. When drugs were combined, AAT allowed the immunosuppressive DEX activities, while DEX at medium to high levels antagonized beneficial AAT effects. Interestingly, lower levels of DEX maintained the immunosuppressive effect, while allowing upregulation of IL-1Ra. Therefore, AAT may represent a distinct endogenous anti-inflammatory, resolution-promoting agent that may improve tissue well-being while preventing undesired corticostroids side effects.

Targeted therapy to the IL-2R using diphtheria toxin and caspase-3 fusion proteins modulates Treg and ameliorates inflammatory colitis.

Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL-2 fusion proteins, a diphtheria toxin (IL2-DT) and a caspase-3 (IL2-cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4(+)CD25(+)Foxp3(+) T cells in mesenteric LN, IL2-DT caused severe lymphopenia. In contrast, IL2-cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2-DT caused lymphopenia and IL2-cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25(+) T cells in vitro, with lower toxicity of IL2-cas to Foxp3(+) T cells. These data infer that targeted depletion of cells expressing the IL-2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25(+) Treg. The IL2-cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.

A fusion protein composed of IL-2 and caspase-3 ameliorates the outcome of experimental inflammatory colitis.

Targeted depletion of immune cells expressing the interleukin-2 (IL-2) receptor can exacerbate inflammatory bowel disease (IBD) through elimination of regulatory T (Treg) cells, or ameliorate its course by depletion of cytotoxic cells. To answer this question we used a fusion protein composed of IL-2 and caspase-3 (IL2-cas) in an experimental model of DSS-induced toxic colitis. In a preventive setting, co-administration of DSS with a daily therapeutic dose of IL2-cas for seven days improved all disease parameters. Although CD4(+)CD25(+) T cells were depleted in the mesenteric lymph nodes, a fractional increase in CD4(+)FoxP3(+) T cells was observed in the spleen. Likewise, IL2-cas therapy improved the outcome of established disease in a chronic model of colitis. These data demonstrate that therapies that use IL-2 as a targeting moiety exert a protective effect over the colon under conditions of inflammation. The efficacy of IL-2-targeted therapy is attributed to reduced activity of reactive T cells, which ameliorates the secondary inflammatory infiltration. IL2-cas evolves as a potential therapeutic tool in IBD.

Lysosomal recycling terminates CD1d-mediated presentation of short and polyunsaturated variants of the NKT cell lipid antigen alphaGalCer.

Short or polyunsaturated lipid variants of the NKT cell antigen alpha-galactosylceramide (alphaGC) exhibit decreased potency and a Th2 bias in vivo despite conserved TCR contact residues and stable binding to CD1d at neutral and acidic pH. Using reagents to directly visualize lipids in their free or CD1d-bound form, we determined that, contrary to predictions, these lipids reached the lysosome better than alphaGC. However, in contrast with alphaGC, they loaded CD1d at the cell surface and underwent immediate pH-dependent dissociation upon recycling to the lysosome. In cell-free assays, ultrafast dissociation of preformed complexes could be induced at acidic pH only when free competitor lipids were added, suggesting active lipid displacement. These findings provide a common cell biological explanation for the decreased stimulatory properties of short and polyunsaturated alphaGC variants. They also suggest that direct lipid displacement is a potent mechanism underlying highly dynamic lipid exchange reactions in the lysosomal compartment that shape the repertoire of lipids associated with CD1d.

Apoptosis as a mechanism of T-regulatory cell homeostasis and suppression.

Activation-induced cell death is a general mechanism of immune homeostasis through negative regulation of clonal expansion of activated immune cells. This mechanism is involved in the maintenance of self- and transplant tolerance through polarization of the immune responses. The Fas/Fas-ligand interaction is a major common executioner of apoptosis in lymphocytes, with a dual role in regulatory T cell (Treg) function: Treg cell homeostasis and Treg cell-mediated suppression. Sensitivity to apoptosis and the patterns of Treg-cell death are of outmost importance in immune homeostasis that affects the equilibrium between cytolytic and suppressor forces in activation and termination of immune activity. Naive innate (naturally occurring) Treg cells present variable sensitivities to apoptosis, related to their turnover rates in tissue under steady state conditions. Following activation, Treg cells are less sensitive to apoptosis than cytotoxic effector subsets. Their susceptibility to apoptosis is influenced by cytokines within the inflammatory environment (primarily interleukin-2), the mode of antigenic stimulation and the proliferation rates. Here, we attempt to resolve some controversies surrounding the sensitivity of Treg cells to apoptosis under various experimental conditions, to delineate the function of cell death in regulation of immunity.

Involvement of IL-2 in homeostasis of regulatory T cells: the IL-2 cycle.

A large body of evidence on the activity of regulatory T (Treg) cells was gathered during the last decade, and a similar number of reviews and opinion papers attempted to integrate the experimental findings. The abundant literature clearly delineates an exciting area of research but also underlines some major controversies. A linear cause-result interpretation of experimental maneuvers often ignores the fact that the activity of Treg cells is orchestrated with the effector T (Teff) cells within an intricate network of physiological immune homeostasis. Every modulation of the activity of the effector (cytotoxic) immune system revolves to affect the activity of regulatory (suppressive) cells through elaborate feedback loops of negative and positive regulation. The lack of IL-2 production by innate Treg cells makes this cytokine a prime coupler of the effector and suppressive mechanisms. Here we attempt to integrate evidence that delineates the involvement of IL-2 in primary and secondary feedback loops that regulate the activity of suppressive cells within the elaborate network of physiological immune homeostasis.

A distal effect of microsomal triglyceride transfer protein deficiency on the lysosomal recycling of CD1d.

Microsomal triglyceride transfer protein (MTP) is an endoplasmic reticulum (ER)-resident lipid transfer protein involved in the biosynthesis and lipid loading of apolipoprotein B. MTP was recently suggested to directly regulate the biosynthesis of the MHC I-like, lipid antigen presenting molecule CD1d, based on coprecipitation experiments and lipid loading assays. However, we found that the major impact of MTP deficiency occurred distal to the ER and Golgi compartments. Thus, although the rates of CD1d biosynthesis, glycosylation maturation, and internalization from the cell surface were preserved, the late but essential stage of recycling from lysosome to plasma membrane was profoundly impaired. Likewise, functional experiments indicated defects of CD1d-mediated lipid presentation in the lysosome but not in the secretory pathway. These intriguing findings suggest a novel, unexpected role of MTP at a late stage of CD1d trafficking in the lysosomal compartment.

The Niemann-Pick type C2 protein loads isoglobotrihexosylceramide onto CD1d molecules and contributes to the thymic selection of NKT cells.

The Niemann-Pick type C2 (NPC2) protein is a small, soluble, lysosomal protein important for cholesterol and sphingolipid transport in the lysosome. The immunological phenotype of NPC2-deficient mice was limited to an impaired thymic selection of Valpha14 natural killer T cells (NKT cells) and a subsequent reduction of NKT cells in the periphery. The remaining NKT cells failed to produce measurable quantities of interferon-gamma in vivo and in vitro after activation with alpha-galactosylceramide. In addition, thymocytes and splenocytes from NPC2-deficient mice were poor presenters of endogenous and exogenous lipids to CD1d-restricted Valpha14 hybridoma cells. Importantly, we determined that similar to saposins, recombinant NPC2 was able to unload lipids from and load lipids into CD1d. This transfer activity was associated with a dimeric form of NPC2, suggesting a unique mechanism of glycosphingolipid transfer by NPC2. Similar to saposin B, NPC2 dimers were able to load isoglobotrihexosylceramide (iGb3), the natural selecting ligand of NKT cells in the thymus, into CD1d. These observations strongly suggested that the phenotype observed in NPC2-deficient animals was directly linked to the efficiency of the loading of iGb3 into CD1d molecules expressed by thymocytes. This conclusion was supported by the rescue of endogenous and exogenous iGb3 presentation by recombinant NPC2. Thus, the loading of endogenous and exogenous lipids and glycolipids onto CD1d is dependent on various small, soluble lipid transfer proteins present in the lysosome.

Characterization of the natural killer T-cell response in an adoptive transfer model of atherosclerosis.

Natural killer T (NKT) cells have recently been implicated in atherogenesis, primarily for their ability to recognize and respond to lipid antigens. Because the atherosclerotic lesion is characterized by the retention and modification of lipids in the vascular wall, NKT cells may be involved in promoting the local vascular inflammatory response. Here, we investigate the proatherogenic role of NKT cells in an adoptive transfer model of atherosclerosis, using as recipients immune-deficient, atherosclerosis-susceptible RAG1(-/-)LDLR(-/-) mice. The adoptive transfer of an NKT cell-enriched splenocyte population from Valpha14Jalpha18 T-cell receptor transgenic mice resulted in a 73% increase in aortic root lesion area compared with recipients of NKT cell-deficient splenocytes derived from CD1d(-/-) mice after 12 weeks of Western-type diet feeding. The total serum from hypercholesterolemic mice leads to a small but significant activation of Valpha14Jalpha18 T-cell receptor-expressing hybridoma line by dendritic cells that is CD1d-dependent. Therefore, these studies demonstrate that NKT cells are proatherogenic in the absence of exogenous stimulation, and this activity is likely associated with endogenous lipid antigens carried by lipoproteins in the circulation and perhaps also in the atherosclerotic plaque.

Cutting edge: impaired glycosphingolipid trafficking and NKT cell development in mice lacking Niemann-Pick type C1 protein.

Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal transmembrane protein involved in the cellular transport of glycosphingolipids and cholesterol that is mutated in a majority of patients with Niemann-Pick C neurodegenerative disease. We found that NPC1-deficient mice lacked Valpha14-Jalpha18 NKT cells, a major population of CD1d-restricted T cells that is conserved in humans. NPC1-deficient mice also exhibited marked defects in the presentation of Sphingomonas cell wall Ags to NKT cells and in bacterial clearance in vivo. A synthetic fluorescent alpha-glycosylceramide analog of the Sphingomonas Ag trafficked to the lysosome of wild-type cells but accumulated in the late endosome of NPC1-deficient cells. These findings reveal a blockade of lipid trafficking between endosome and lysosome as a consequence of NPC1 deficiency and suggest a common mechanism for the defects in lipid presentation and development of Valpha14-Jalpha18 NKT cells.

Lysosomal glycosphingolipid recognition by NKT cells.

NKT cells represent a distinct lineage of T cells that coexpress a conserved alphabeta T cell receptor (TCR) and natural killer (NK) receptors. Although the TCR of NKT cells is characteristically autoreactive to CD1d, a lipid-presenting molecule, endogenous ligands for these cells have not been identified. We show that a lysosomal glycosphingolipid of previously unknown function, isoglobotrihexosylceramide (iGb3), is recognized both by mouse and human NKT cells. Impaired generation of lysosomal iGb3 in mice lacking beta-hexosaminidase b results in severe NKT cell deficiency, suggesting that this lipid also mediates development of NKT cells in the mouse. We suggest that expression of iGb3 in peripheral tissues may be involved in controlling NKT cell responses to infections and malignancy and in autoimmunity.

Editing of CD1d-bound lipid antigens by endosomal lipid transfer proteins.

It is now established that CD1 molecules present lipid antigens to T cells, although it is not clear how the exchange of lipids between membrane compartments and the CD1 binding groove is assisted. We report that mice deficient in prosaposin, the precursor to a family of endosomal lipid transfer proteins (LTP), exhibit specific defects in CD1d-mediated antigen presentation and lack Valpha14 NKT cells. In vitro, saposins extracted monomeric lipids from membranes and from CD1, thereby promoting the loading as well as the editing of lipids on CD1. Transient complexes between CD1, lipid, and LTP suggested a "tug-of-war" model in which lipid exchange between CD1 and LTP is on the basis of their respective affinities for lipids. LTPs constitute a previously unknown link between lipid metabolism and immunity and are likely to exert a profound influence on the repertoire of self, tumor, and microbial lipid antigens.

The COOH terminus of GATE-16, an intra-Golgi transport modulator, is cleaved by the human cysteine protease HsApg4A.

Docking of a vesicle at the appropriate target membrane involves an interaction between integral membrane proteins located on the vesicle (v-SNAREs) and those located on the target membrane (t-SNAREs). GATE-16 (Golgi-associated ATPase enhancer of 16 kDa) was shown to modulate the activity of SNAREs in the Golgi apparatus and is therefore an essential component of intra-Golgi transport and post-mitotic Golgi re-assembly. GATE-16 contains a ubiquitin fold subdomain, which is terminated at the carboxyl end by an additional amino acid after a conserved glycine residue. In the present study we tested whether the COOH terminus of GATE-16 undergoes post-translational cleavage by a protease which exposes the glycine 116 residue. We describe the isolation and characterization of HsApg4A as a human protease of GATE-16. We show that GATE-16 undergoes COOH-terminal cleavage both in vivo and in vitro, only when the conserved glycine 116 is present. We then utilize an in vitro assay to show that pure HsApg4A is sufficient to cleave GATE-16. The characterization of this protease may give new insights into the mechanism of action of GATE-16 and its other family members.

Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion.

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function.