ATP-binding cassette transporter A1 (ABCA1) affects total body sterol metabolism.

BACKGROUND AND AIMS: Members of the family of ABC transporters are involved in different processes of sterol metabolism, and ABCA1 was recently identified as a key regulator of high-density lipoprotein (HDL) metabolism. Our aim was to further analyze the role of ABCA1 in cholesterol metabolism. METHODS: ABCA1-deficient mice (ABCA1-/-) and wild-type mice were compared for different aspects of sterol metabolism. Intestinal cholesterol absorption was determined by a dual stable isotope technique, and analysis of fecal, plasma, and tissue sterols was performed by gas chromatography/mass spectrometry. Key regulators of sterol metabolism were investigated by Northern and Western blot analyses or enzyme activity assays. RESULTS: ABCA1-disrupted sv129/C57BL/6 hybrid mice showed a significant reduction in intestinal cholesterol absorption. The decrease in cholesterol absorption was followed by an enhanced fecal loss of neutral sterols, whereas fecal bile acid excretion was not affected. Total body cholesterol synthesis was significantly increased, with enhanced 3-hydroxy-3-methyglutaryl-coenzyme A (HMG-CoA) reductase observed in adrenals and spleen. In addition, ABCA1-/- mice showed markedly increased concentrations of cholesterol precursors in the plasma, lung, intestine, and feces. Reduced HMG-CoA reductase messenger RNA and enzyme activity in the liver suggest that enhanced cholesterol synthesis in ABCA1-/- mice occurs in peripheral tissues rather than the liver. CONCLUSIONS: The metabolism of cholesterol and cholesterol precursors is markedly affected by a lack of ABCA1 function.

Tbt-1, a new T-box transcription factor induced in activated Th1 and CD8+ T cells.

Differentiation of CD4+ T cells into T helper (Th) 1 or Th2 cells requires the cytokines interleukin (IL)-12 and IL-4, respectively. However, transcription factors that regulate expression of Th1 or Th2 cell-specific genes remain largely unclear. In the present study, a new Th1-specific transcription factor, named Tbt-1 (T-box protein expressed in T lymphocytes), was identified. Tbt-1 is a novel member of the T-box family, which is characterized by a conserved T-box DNA-binding domain. Unlike other known T-box proteins that regulate embryo development and organogenesis, Tbt-1 expression is restricted to adult lymphoid organs. Tbt-1 mRNA is only detected in peripheral lymphoid tissues such as spleen, lymph nodes, and blood leukocytes, but not in thymus or bone marrow. Tbt-1 mRNA is not detected in resting T cells but is strongly induced in differentiating Thl cells and CD8+ cytotoxic effector cells. In contrast, Tbt-1 expression was not observed in the entire process of Th2 cell differentiation. In addition, phylogenetic analyses indicate that Tbt-1 co-evolved with adaptive immune responses. Thus, Tbt-1 is the first T-box transcription factor shown to be specific for Th1 cell differentiation.

Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency.

Tangier disease (TD) and familial HDL deficiency (FHA) have recently been linked to mutations in the human ATP-binding cassette transporter 1 (hABCA1), a member of the ABC superfamily. Both diseases are characterized by the lowering or lack of high-density lipoprotein cholesterol (HDL-C) and low serum cholesterol. The murine ABCA1-/- phenotype corroborates the human TD linkage to ABCA1. Similar to TD in humans, HDL-C is virtually absent in ABCA1-/- mice accompanied by a reduction in serum cholesterol and lipid deposition in various tissues. In addition, the placenta of ABCA1-/- mice is malformed, resulting in severe embryo growth retardation, fetal loss, and neonatal death. The basis for these defects appears to be altered steroidogenesis, a direct result of the lack of HDL-C. By 6 months of age, ABCA1-/- animals develop membranoproliferative glomerulonephritis due to deposition of immunocomplexes followed by cardiomegaly with ventricular dilation and hypertrophy, ultimately succumbing to congestive heart failure. This murine model of TD will be very useful in the study of lipid metabolism, renal inflammation, and cardiovascular disease and may reveal previously unsuspected relationships between them.

Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses.

Galectin-3 is a member of a growing family of beta-galactoside-binding animal lectins. Previous studies have demonstrated a variety of biological activities for this protein in vitro, including activation of cells, modulation of cell adhesion, induction of pre-mRNA splicing, and regulation of apoptosis. To assist in fully elucidating the physiological and pathological functions of this protein, we have generated galectin-3-deficient (gal3(-/-)) mice by targeted interruption of the galectin-3 gene. Gal3(-/-) mice consistently developed fewer inflammatory cell infiltrations in the peritoneal cavities than the wild-type (gal3(+/+)) mice in response to thioglycollate broth treatment, mainly due to lower numbers of macrophages. Also, when compared to cells from gal3(+/+) mice, thioglycollate-elicited inflammatory cells from gal3(-/-) mice exhibited significantly lower levels of NF-kappaB response. In addition, dramatically different cell-spreading phenotypes were observed in cultured macrophages from the two genotypes. Whereas macrophages from gal3(+/+) mice exhibited well spread out morphology, those from gal3(-/-) mice were often spindle-shaped. Finally, we found that peritoneal macrophages from gal3(-/-) mice were more prone to undergo apoptosis than those from gal3(+/+) mice when treated with apoptotic stimuli, suggesting that expression of galectin-3 in inflammatory cells may lead to longer cell survival, thus prolonging inflammation. These results strongly support galectin-3 as a positive regulator of inflammatory responses in the peritoneal cavity.

Impaired immunoproteasome assembly and immune responses in PA28-/- mice.

In vitro PA28 binds and activates proteasomes. It is shown here that mice with a disrupted PA28b gene lack PA28a and PA28b polypeptides, demonstrating that PA28 functions as a hetero-oligomer in vivo. Processing of antigenic epitopes derived from exogenous or endogenous antigens is altered in PA28-/- mice. Cytotoxic T lymphocyte responses are impaired, and assembly of immunoproteasomes is greatly inhibited in mice lacking PA28. These results show that PA28 is necessary for immunoproteasome assembly and is required for efficient antigen processing, thus demonstrating the importance of PA28-mediated proteasome function in immune responses.

Defective CD8+ T cell activation and cytolytic function in the absence of LFA-1 cannot be restored by increased TCR signaling.

Signaling through the TCR as well as engagement of costimulatory molecules are required for efficient T cell activation and progression into differentiated effector cells. The beta2 integrin LFA-1 (CD11a/CD18) has been implicated in TCR costimulation as well as in cell-cell adhesion function, but its exact role is still ambiguous. The present study focuses on the requirement for LFA-1 in CD8+ T cell activation and effector function using LFA-1-deficient cells expressing the 2C transgenic TCR as a model system. The lack of LFA-1 expression in 2C T cells resulted in severely diminished proliferative response toward allogeneic BALB/c splenocytes. Increase in TCR signaling alone by pulsing stimulators with high affinity peptides, p2Ca or QL9, had minimal effects in restoring proliferation. Addition of exogenous IL-2, however, enhanced the effect of peptide pulsing on proliferation of LFA-1-deficient 2C T cells. LFA-1-deficient 2C CTLs generated from alloantigen stimulation exhibited a defective cytotoxic activity when tested on a variety of target cells. Cytolysis could be improved, but not fully rectified by peptide pulsing of target cells. Thus, in the 2C TCR model, LFA-1 has a requisite role for optimal CD8+ T cell activation and effector function, which cannot be overcome by increasing peptide/MHC density on either the APCs or target cells, respectively.

Defective interleukin (IL)-18-mediated natural killer and T helper cell type 1 responses in IL-1 receptor-associated kinase (IRAK)-deficient mice.

Interleukin (IL)-18 is functionally similar to IL-12 in mediating T helper cell type 1 (Th1) response and natural killer (NK) cell activity but is related to IL-1 in protein structure and signaling, including recruitment of IL-1 receptor-associated kinase (IRAK) to the receptor and activation of c-Jun NH2-terminal kinase (JNK) and nuclear factor (NF)-kappaB. The role of IRAK in IL-18-induced responses was studied in IRAK-deficient mice. Significant defects in JNK induction and partial impairment in NF-kappaB activation were found in IRAK-deficient Th1 cells, resulting in a dramatic decrease in interferon (IFN)-gamma mRNA expression. In vivo Th1 response to Propionibacterium acnes and lipopolysaccharide in IFN-gamma production and induction of NK cytotoxicity by IL-18 were severely impaired in IRAK-deficient mice. IFN-gamma production by activated NK cells in an acute murine cytomegalovirus infection was significantly reduced despite normal induction of NK cytotoxicity. These results demonstrate that IRAK plays an important role in IL-18-induced signaling and function.

Cardiovascular responses mediated by protease-activated receptor-2 (PAR-2) and thrombin receptor (PAR-1) are distinguished in mice deficient in PAR-2 or PAR-1.

We developed mice deficient in protease-activated receptor-2 (PAR-2) or PAR-1 to explore the pathophysiological functions of these receptors. In this report, we evaluated mean arterial pressure and heart rate (HR) changes in response to PAR-1 or PAR-2 activation in anesthetized wild-type (WT), PAR-1-deficient (PAR-1(-/-)), and PAR-2-deficient (PAR-2(-/-)) mice. In WT mice, TFLLRNPNDK, a PAR-1 selective activating peptide, caused hypotension and HR decreases at 1 micromol/kg. TFLLRNPNDK also caused secondary hypertension following L-NAME pretreatment. These responses were absent in PAR-1(-/-) mice. In WT mice, SLIGRL, a PAR-2 selective activating peptide, caused hypotension without changing HR at 0.3 micromol/kg. SLIGRL did not induce hypertension following Nomega-nitrol-arginine-methyl ester-HCl (L-NAME). The response to SLIGRL was absent in PAR-2(-/-) mice. SFLLRN, a nonselective receptor activating peptide caused hypotension and HR decreases in WT mice at 0.3 micromol/kg, as well as secondary hypertension following L-NAME. SFLLRN still induced hypotension in PAR-1(-/-) mice, but HR decrease and secondary hypertension following L-NAME were absent. The hypotensive and bradycardic responses to SFLLRN and TFLLRNPNDK in PAR-2(-/-) mice were accentuated compared with WT mice. By using mouse strains deficient in either PAR-1 or PAR-2, we confirmed the in vivo specificity of TFLLRNPNDK and SLIGRL as respective activating peptides for PAR-1 and PAR-2, and the distinct hemodynamic responses mediated by activation of PAR-1 or PAR-2. Moreover, the accentuated response to PAR-1 activation in PAR-2-deficient mice suggests a compensatory response and potential receptor cross-talk.

Characterization of the mouse proteasome regulator PA28b gene.

The proteasome regulator PA28, which can be upregulated by IFN, is important in the modulation of proteasome activity. Since the proteasome has been implicated in the processing of the major histocompatibility complex (MHC) class I antigens, it was of interest to determine the regulatory elements of PA28 at the genomic level. Although PA28 has been found in different species, the gene layout on the chromosome was not determined. In this study, the genetic organization of mouse PA28b was characterized. Two copies of the PA28b gene, namely b1 and b2, were found by restriction fragment mapping and Southern hybridization. By fluorescence in situ hybridization, the location of the two PA28b genes was determined on chromosomes 11 and 14. PA28b1 has 11 exons, whereas PA28b2 has no introns and appears to be a nonfunctional pseudogene. The 5' promoter region of PA28b1 contains several transcriptional factor binding sites including two IFN responsive elements. The expression levels of PA28 and other gene products involved in MHC class I antigen presentation appear to be correlated in various tissues. Notably, PA28 is expressed at high levels in immunological tissues such as spleen and peripheral blood leukocytes. Taken together, PA28 seems to be co-regulated with other molecules involved in MHC class I antigen presentation.

The role of H2-O and HLA-DO in major histocompatibility complex class II-restricted antigen processing and presentation.

The function of major histocompatibility complex (MHC) class II molecules is to sample exogenous antigens for presentation to CD4+ T helper cells. After synthesis in the endoplasmic reticulum, class II molecules are directed into the endosomal system by association with the invariant chain (Ii), which is sequentially cleaved, generating class II dimers loaded with Ii-derived peptides (CLIP). These class II-peptide complexes are physiological substrates for H2-M/HLA-DM, a resident of the endosomal/lysosomal system which facilitates the removal of CLIP from newly synthesised class II alpha beta dimers. Exchange of CLIP for antigenic class II-binding peptides is also promoted by the action of H2-M/HLA-DM, resulting in stable peptide-class II complexes that are transported to the cell surface for presentation to CD4+ T cells. Recent evidence suggests that this H2-M/HLA-DM-mediated 'peptide editing' is influenced by another MHC class II-encoded molecule, H2-O/HLA-DO. This non-polymorphic alpha beta heterodimer is associated with H2-M/HLA-DM during intracellular transport and within the endosomal system of B cells. H2-O/HLA-DO alters the peptide exchange function of H2-M/HLA-DM in a pH-dependent manner, so that H2-M/HLA-DM activity is limited to more acidic conditions, corresponding to lysosomal compartments. Indeed, H2-O/HLA-DO may serve to limit the presentation of antigens after fluid phase uptake by B cells, while augmenting presentation of antigens internalised via membrane Ig receptors. Such a mechanism may maintain the fidelity of the B-cell-CD4+ T-cell interaction, counteracting self reactivity arising from less stringent lymphocyte activation. Here, data evaluating the role of H2-O/HLA-DO shall be reviewed and its putative function discussed.

Interleukin (IL)-1 receptor-associated kinase (IRAK) requirement for optimal induction of multiple IL-1 signaling pathways and IL-6 production.

Interleukin (IL)-1 is a proinflammatory cytokine with pleiotropic effects in inflammation. IL-1 binding to its receptor triggers a cascade of signaling events, including activation of the stress-activated mitogen-activated protein (MAP) kinases, c-Jun NH2-terminal kinase (JNK) and p38 MAP kinase, as well as transcription factor nuclear factor kappaB (NF-kappaB). IL-1 signaling results in cellular responses through induction of inflammatory gene products such as IL-6. One of the earliest events in IL-1 signaling is the rapid interaction of IL-1 receptor-associated kinases, IRAK and IRAK-2, with the receptor complex. The relative roles of IRAK and IRAK-2 in IL-1 signaling pathways and subsequent cellular responses have not been previously determined. To evaluate the importance of IRAK in IL-1 signaling, IRAK-deficient mouse fibroblast cells were prepared and studied. Here we report that IL-1-mediated activation of JNK, p38, and NF-kappaB were all reduced in embryonic fibroblasts deficient in IRAK expression. In addition, IL-6 production in response to IL-1 was also dramatically reduced in IRAK-deficient embryonic fibroblasts and in skin fibroblasts prepared from IRAK-deficient mice. Our results demonstrate that IRAK plays an essential proximal role in coordinating multiple IL-1 signaling pathways for optimal induction of cellular responses.

Altered antigen presentation in mice lacking H2-O.

HLA-DM catalyzes the release of MHC class II-associated invariant chain-derived peptides (CLIP) from class II molecules. Recent evidence has suggested that HLA-DO is a negative regulator of HLA-DM in B cells, but the physiological function of HLA-DO remains unclear. Analysis of antigen presentation by B cells from mice lacking H2-O (the mouse equivalent of HLA-DO), together with biochemical analysis using purified HLA-DO and HLA-DM molecules, suggests that HLA-DO/H2-O influences the peptide loading of class II molecules by limiting the pH range in which HLA-DM is active. This effect may serve to decrease the presentation of antigens internalized by fluid-phase endocytosis, thus concentrating the B cell-mediated antigen presentation to antigens internalized by membrane immunoglobulin.

Thymic selection by a single MHC/peptide ligand produces a semidiverse repertoire of CD4+ T cells.

The influence of individual peptides in thymic selection was examined in H2-M- mice, in which positive selection is directed to a single peptide, class II-associated invariant chain peptide (CLIP) bound to H2-A(b). Two sensitive in vivo approaches showed that 70%-80% of CD4+ T cells undergoing positive selection to CLIP+H2-A(b) have self-reactivity to the various peptides expressed on wild-type H2-M+ antigen-presenting cells. When these self-reactive T cells were depleted, the residual CD4+ cells displayed a polyclonal repertoire in terms of alloreactivity, responses to foreign protein antigens, and Vbeta usage. Nevertheless, studies with two T cell receptor transgenic lines suggested that the repertoire of CD4+ cells induced by CLIP was less diverse than the repertoire of CD4+ cells in normal mice. Generation of a fully diverse T cell repertoire thus requires positive selection against multiple peptides.

Peptide-induced positive selection of TCR transgenic thymocytes in a coreceptor-independent manner.

T cell receptor (TCR) transgenic thymocytes specific for the LCMV gp peptide are normally positively selected to the CD8 lineage. Transgenic thymocyte development was substantially reduced in the absence of these CD8 coreceptors. However, efficient positive selection was restored when TCR transgenic CD8-/- fetal thymic lobes were cultured with a peptide variant of the wild-type ligand. These mature thymocytes were functional, as shown by their ability to respond against strong peptide agonists. Additional experiments demonstrated that transgenic positive selection was peptide-specific. These results prove that CD8 does not possess essential signaling properties that are necessary for T cell development. In addition, the unilateral commitment of transgenic thymocytes to mature CD4-TCR(hi) T cells expressing intracellular perforin suggests that there must be some instructive component to CD4 down-regulation and lineage commitment during thymocyte selection.

Impaired immune responses toward alloantigens and tumor cells but normal thymic selection in mice deficient in the beta2 integrin leukocyte function-associated antigen-1.

We have generated mice deficient in the beta2 integrin LFA-1 by targeted disruption of the CD11a gene in embryonic stem cells. In vitro LFA-1 -/- cells exhibit a delayed proliferative response toward alloantigens in the MLR. In vivo the host-vs-graft reaction toward injected allogeneic cells is also reduced. Alloantigen-specific CTLs generated from LFA-1 -/- mice are impaired in their cytotoxic activity toward allogeneic spleen cells as well as cell line targets. The proliferative response of LFA-1 -/- splenocytes following stimulation by LPS, PMA plus ionomycin, or immobilized anti-CD3epsilon mAb is normal, but Con A-stimulated proliferation is greatly diminished. We observe typical edema formation in a delayed type hypersensitivity reaction to SRBC with normal extravasation of leukocytes and demonstrate recruitment of neutrophils to an LPS-induced inflammatory site in these mice, suggesting that LFA-1 does not play an essential role in lymphocyte homing and leukocyte extravasation. We further show that LFA-1 -/- mice are susceptible to metastasis of B16 melanoma tumors, although their in vitro NK cell activity appears normal. A study of LFA-1 -/- mice expressing transgenic TCRs indicates that thymic maturation and selection of T cells are unaffected by the loss of LFA-1. Our results indicate that LFA-1 is important for alloantigen-triggered T cell proliferation and cytotoxicity, for Con A stimulation of T cells, and in tumor rejection. It does not appear to play an essential role in lymphocyte homing and leukocyte extravasation or in T cell maturation and selection in the thymus.

Biological consequences of thrombin receptor deficiency in mice.

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

HLA-DO is a lysosomal resident which requires association with HLA-DM for efficient intracellular transport.

The murine MHC class II molecule H2-O is expressed in B-cells and in thymic epithelium but the human equivalent, HLA-DO (DO), has not been detected, though the corresponding genes, HLA-DNA and HLA-DOB, are well known. Here we show DO to be a lysosomal resident in B-cells. Surprisingly, DO forms stable complexes with HLA-DM (DM), another lysosomal class II-like molecule which is important for class II-restricted antigen presentation. Association with DM is necessary for efficient exit of DO from the endoplasmic reticulum (ER) and thus for accumulation in lysosomes. The association is evolutionarily conserved and in mice lacking H2-M, the mouse equivalent of DM, the amount of intracellular H2-O is decreased and only minor amounts of H2-O appear to leave the ER. The DO-DM complexes survive in the lysosomal system suggesting that DO and DM functions may be intertwined.

Adoptively transferred CD4+ lymphocytes from CD8 -/- mice are sufficient to mediate the rejection of MHC class II or class I disparate skin grafts.

Recent studies revealed that CD4+ cells initiate allograft rejection through direct recognition of allogeneic MHC class II Ags and indirect recognition of MHC peptides processed by self APCs. Both pathways were shown to help CD8+ cells that eventually lysed allogeneic MHC class I-presenting targets. There was little evidence, however, that CD4+ cells are sufficient for graft rejection. We studied skin graft rejection by CD8-deficient (CD8 -/-) mice. We showed that BALB/cJ(H-2d) CD8 -/- mice could reject allogeneic skin from C57BL/6J(H-2b) mice deficient in MHC class I or in MHC class II Ags. To understand the role of CD4+ cells in this process, we isolated them from CD8 -/- mice and transferred them to BALB/cJ nude mice that had been grafted with allogeneic skin (H-2b) from animals deficient in MHC class I or MHC class II. Nude mice injected with CD4+ cells rejected MHC class II and, albeit more slowly, MHC class I disparate skins. We showed in vitro evidence that CD4+ cells were not cytotoxic toward MHC class I or MHC class II disparate targets and that they recognized MHC class I allogeneic targets through indirect recognition. CD4+ cells produced Th1 cytokines, but not IL-4, following stimulation with allogeneic cells. Furthermore, intragraft TNF-alpha was elevated in skin grafted onto nude mice reconstituted with CD4+ cells compared with nonreconstituted mice. This suggests that MHC class II- or MHC class I-guided CD4+ cells alone are sufficient to induce rejection by the generation of cytokine-induced lesions.

Antigen presentation and T cell development in H2-M-deficient mice.

HLA-DM (DM) facilitates peptide loading of major histocompatibility complex class II molecules in human cell lines. Mice lacking functional H2-M, the mouse equivalent of DM, have normal amounts of class II molecules at the cell surface, but most of these are associated with invariant chain-derived CLIP peptides. These mice contain large numbers of CD4+ T cells, which is indicative of positive selection in the thymus. Their CD4+ cells were unresponsive to self H2-M-deficient antigen-presenting cells (APCs) but were hyperreactive to wild-type APCs. H2-M-deficient APCs failed to elicit proliferative responses from wild-type T cells.