Macrophages and Mitochondria: A Critical Interplay Between Metabolism, Signaling, and the Functional Activity.

Macrophages are phagocytic cells that participate in a broad range of cellular functions and they are key regulators of innate immune responses and inflammation. Mitochondria are highly dynamic endosymbiotic organelles that play key roles in cellular metabolism and apoptosis. Mounting evidence suggests that mitochondria are involved in the interplay between metabolism and innate immune responses. The ability of these organelles to alter the metabolic profile of a cell, thereby allowing an appropriate response to each situation, is crucial for the correct establishment of immune responses. Furthermore, mitochondria act as scaffolds for many proteins involved in immune signaling pathways and as such they are able to modulate the function of these proteins. Finally, mitochondria release molecules, such as reactive oxygen species, which directly regulate the immune response. In summary, mitochondria can be considered as core components in the regulation of innate immune signaling. Here we discuss the intricate relationship between mitochondria, metabolism, intracellular signaling, and innate immune responses in macrophages.

Regulation of murine Tap1 and Lmp2 genes in macrophages by interferon gamma is mediated by STAT1 and IRF-1.

The genes of the transporter associated with antigen processing (Tap)-1, and the low molecular weight peptide (Lmp)-2, are crucial for class I major histocompatibility complex function and share a common bidirectional promoter. In murine bone marrow-derived macrophages, interferon gamma (IFN-gamma) induced Tap-1 and upregulated Lmp-2, which is constitutively expressed at low levels. The IFN-gamma-induction was independent of early gene synthesis. The mRNA induced by IFN-gamma was very stable. In macrophages from STAT1 knockout mice, IFN-gamma did not induce the expression of Tap-1 or Lmp-2. Several areas in the promoter can be controlled by IFN-gamma, such as proximal and distal GAS boxes in the direction of the Tap-1 gene, NFgammaB and IRF-1 boxes. By making deletions of the promoter, we found that only the proximal GAS and IRF-1 boxes are required for IFN-gamma induction of Tap-1 and Lmp-2. Experiments using nuclear extracts from macrophages treated for 30 min with IFN-gamma and gel shift analysis indicated that STAT1 binds to the GAS box. The nuclear extracts from macrophages treated for at least 2 h with IFN-gamma bound to the IRF-1 box. These results indicate that both STAT1 and IRF-1 are required for the IFN-gamma induction of Tap-1 and Lmp-2 genes.

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From transcription to cell surface expression, the induction of MHC class II I-A alpha by interferon-gamma in macrophages is regulated at different levels.

Using mouse bone marrow-derived macrophages we determined the role of interferon (IFN)-gamma at the different steps in expression of the I-A alpha chain of MHC class II molecules, from transcription to the cell surface. Levels of transcription, RNA, and protein were low in cells not stimulated with IFN-gamma. Treatment with IFN-gamma for 24 or 48 h induced an increase in mRNA levels (7- and 12-fold) that did not correlate with the increase in transcription (2.5- and 2.7-fold). The half-life of mRNA was not modified by IFN-gamma. These data suggest a block at the level of translation. In fact, IFN-gamma increased ribosome loading, which confirms regulation at the translational level. Treatment with IFN-gamma increased protein synthesis (6-fold after 48 h) and level of expression at the cell surface (3- and 9-fold after 24 and 48 h, respectively). Interestingly, treatment with IFN-gamma also increased the I-A alpha protein half-life from 2 to 6-7 h. This is the first attempt to determine qualitatively and quantitatively the regulation of an inducible gene at all the putative levels of control. The data indicate that IFN-gamma plays a critical role in MHC class II protein expression in macrophages through the regulation of different steps, from transcription to surface expression.

Role of the human concentrative nucleoside transporter (hCNT1) in the cytotoxic action of 5[Prime]-deoxy-5-fluorouridine, an active intermediate metabolite of capecitabine, a novel oral anticancer drug.

We attempt to identify the plasma membrane transporter involved in the uptake of 5'-deoxy-5-fluorouridine (5'-DFUR), an intermediate metabolite of capecitabine. This novel oral fluoropyrimidine is used in cancer treatments and is a direct precursor of the cytostatic agent 5'-fluorouracil. We also examine the role of the transporter in 5'-DFUR cytotoxicity. The human concentrative nucleoside transporter (hCNT1) was cloned from human fetal liver and expressed in Xenopus laevis oocytes. The two-electrode voltage-clamp technique was used to demonstrate that 5'-DFUR, but not capecitabine or 5'-FU, is an hCNT1 substrate. Then, hCNT1 was heterologously expressed in the mammalian cell line Chinese hamster ovary-K1. Functional expression was demonstrated by monitoring transport of radiolabeled substrates and by using a monospecific polyclonal antibody generated against the transporter. hCNT1-expressing cells were more sensitive to 5'-DFUR than vector-transfected or wild-type cells. The sensitivity of the three cell types to other agents such as cisplatin or 5'-FU was identical. In conclusion, this study shows that 1) the pharmacological profile of a nucleoside transporter can be determined by an electrophysiological approach; 2) the hCNT1 transporter is involved in 5'-DFUR uptake; and 3) hCNT1 expression may increase cell sensitivity to 5'-DFUR treatment. This study also reports for the first time the generation of an antibody against hCNT1, which may be useful in the elucidation of the relationship between hCNT1 expression and tumor response to capecitabine treatment.

IFN-gamma-dependent transcription of MHC class II IA is impaired in macrophages from aged mice.

To determine the effect of aging on IFN-gamma-induced MHC class II antigen expression, we produced bone marrow-derived macrophages in vitro. In these conditions, we analyzed the effect of aging on the genomic expression of macrophages without the influence of other cell types that may be affected by aging. Although macrophages from young and aged mice showed an identical degree of differentiation, after incubation with IFN-gamma, the expression at the cell surface of the IA complex and the levels of IAbeta protein and mRNA were lower in aged macrophages. Moreover, the transcription of the IAbeta gene was impaired in aged macrophages. The amount of transcription factors that bound to the W and X, but not to the Y, boxes of the IAbeta promoter gene was lower in aged macrophages. Similar levels of CIITA mRNA were found after IFN-gamma treatment of both young and aged macrophages. This shows that neither the initial cascade that starts after the interaction of IFN-gamma with the receptor nor the second signals involved in the expression of CIITA are impaired in aged macrophages. These data indicate that aging is associated with low levels of MHC class II gene induction by IFN-gamma because of impaired transcription.

Molecular mechanisms involved in macrophage survival, proliferation, activation or apoptosis.

Macrophages play a critical role during the immune response. Like other cells of the immune system, macrophages are produced in large amounts and most of them die through apoptosis. Macrophages survive in the presence of soluble factors, such as IFN-gamma, or extracellular matrix proteins like decorin. The mechanism toward survival requires the blocking of proliferation at the G1/S boundary of the cell cycle that is mediated by the cyclin-dependent kinase (cdk) inhibitor, p27kip and the induction of a cdk inhibitor, p21waf1. At the inflammatory loci, macrophages need to proliferate or become activated in order to perform their specialized activities. Although the stimuli inducing proliferation and activation follow different intracellular pathways, both require the activation of extracellular signal-regulated kinases (ERKs) 1 and 2. However, the kinetics of ERK-1/2 activation is different and is determined by the induction of the MAP-kinase phosphatase-1 (MKP-1) that dephosphorilates ERK-1/2. This phosphatase plays a critical role in the process of proliferation versus activation of the macrophages.

The expression of MHC class II genes in macrophages is cell cycle dependent.

Using different drugs, we stopped the cell cycle of bone marrow-derived macrophages at different points. After IFN-gamma stimulation, macrophages arrested at the G(1) phase of the cell cycle did not increase cell surface expression of the MHC class II IA. This inhibition is specific, because, under the same conditions, IFN-gamma induces the expression of Fcgamma receptors and the inducible NO synthase mRNA. Treatments that inhibit macrophage proliferation by blocking the cell cycle at the G(1) phase, such as adenosine, forskolin, or LPS, blocked the IFN-gamma induction of IA. Under IFN-gamma treatment, the steady-state levels of IAalpha and IAss mRNA did not increase in cells arrested at the G(1) phase and the half-life of the MHC mRNA was not modified. These data suggest that the cell cycle modulation of IFN-gamma-induced MHC II gene expression occurs at the transcriptional level. The expression of the class II transactivator mRNA induced by IFN-gamma was also blocked when macrophages were arrested at the G(1) phase of the cell cycle, suggesting that the lack of IFN-gamma response occurs at the early steps of MHC class II expression. Finally, macrophages arrested at the G(1) phase showed increased basal levels of cell surface IA due to an increase of the translational efficiency. These data show that the expression of MHC class II genes is regulated by the cell cycle.

An ORF from Bacillus licheniformis encodes a putative DNA repressor.

The complete sequence of a reading frame adjacent to the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis is reported. It encodes a putative 171 amino acid residues protein with either, low significant sequence similarity in data banks or the corresponding orthologue in the recently sequenced Bacillus subtilis genome. Computer analyses predict a canonical Helix-Turn-Helix motif characteristic of bacterial repressors/DNA binding proteins. A maxicells assay shows that the encoded polypeptide is expressed. A DNA-protein binding, assay performed by gel electrophoresis shows that the expressed protein specifically binds to Bacillus licheniformis DNA.

LPS induces apoptosis in macrophages mostly through the autocrine production of TNF-alpha.

The deleterious effects of lipopolysaccharide (LPS) during endotoxic shock are associated with the secretion of tumor necrosis factor (TNF) and the production of nitric oxide (NO), both predominantly released by tissue macrophages. We analyzed the mechanism by which LPS induces apoptosis in bone marrow-derived macrophages (BMDM). LPS-induced apoptosis reached a plateau at about 6 hours of stimulation, whereas the production of NO by the inducible NO-synthase (iNOS) required between 12 and 24 hours. Furthermore, LPS-induced early apoptosis was only moderately reduced in the presence of an inhibitor of iNOS or when using macrophages from iNOS -/-mice. In contrast, early apoptosis was paralleled by the rapid secretion of TNF and was almost absent in macrophages from mice deficient for one (p55) or both (p55 and p75) TNF-receptors. During the late phase of apoptosis (12-24 hours) NO significantly contributed to the death of macrophages even in the absence of TNF-receptor signaling. NO-mediated cell death, but not apoptosis induced by TNF, correlated with the induction of p53 and Bax genes. Thus, LPS-induced apoptosis results from 2 independent mechanisms: first and predominantly, through the autocrine secretion of TNF-alpha (early apoptotic events), and second, through the production of NO (late phase of apoptosis). (Blood. 2000;95:3823-3831)

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Interferon gamma induces the expression of p21waf-1 and arrests macrophage cell cycle, preventing induction of apoptosis.

Incubation of bone marrow macrophages with lipopolysaccharide (LPS) or interferon gamma (IFN gamma) blocks macrophage proliferation. LPS treatment or M-CSF withdrawal arrests the cell cycle at early G1 and induces apoptosis. Treatment of macrophages with IFN gamma stops the cell cycle later, at the G1/S boundary, induces p21Waf1, and does not induce apoptosis. Moreover, pretreatment of macrophages with IFN gamma protects from apoptosis induced by several stimuli. Inhibition of p21Waf1 with antisense oligonucleotides or using KO mice shows that the induction of p21Waf1 by IFN gamma mediates this protection. Thus, IFN gamma makes macrophages unresponsive to apoptotic stimuli by inducing p21Waf1 and arresting the cell cycle at the G1/S boundary. Therefore, the cells of the innate immune system could only survive while they were functionally active.

Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids.

We have identified a new human cDNA, L-amino acid transporter-2 (LAT-2), that induces a system L transport activity with 4F2hc (the heavy chain of the surface antigen 4F2, also named CD98) in oocytes. Human LAT-2 is the fourth member of the family of amino acid transporters that are subunits of 4F2hc. The amino acid transport activity induced by the co-expression of 4F2hc and LAT-2 was sodium-independent and showed broad specificity for small and large zwitterionic amino acids, as well as bulky analogs (e.g. BCH (2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid)). This transport activity was highly trans-stimulated, suggesting an exchanger mechanism of transport. Expression of tagged N-myc-LAT-2 alone in oocytes did not induce amino acid transport, and the protein had an intracellular location. Co-expression of N-myc-LAT-2 and 4F2hc gave amino acid transport induction and expression of N-myc-LAT-2 at the plasma membrane of the oocytes. These data suggest that LAT-2 is an additional member of the family of 4F2 light chain subunits, which associates with 4F2hc to express a system L transport activity with broad specificity for zwitterionic amino acids. Human LAT-2 mRNA is expressed in kidney >>> placenta >> brain, liver > spleen, skeletal muscle, heart, small intestine, and lung. Human LAT-2 gene localizes at chromosome 14q11.2-13 (13 cR or approximately 286 kb from marker D14S1349). The high expression of LAT-2 mRNA in epithelial cells of proximal tubules, the basolateral location of 4F2hc in these cells, and the amino acid transport activity of LAT-2 suggest that this transporter contributes to the renal reabsorption of neutral amino acids in the basolateral domain of epithelial proximal tubule cells.

IFN-gamma up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation.

Adenosine is a potent endogenous anti-inflammatory agent released by cells in metabolically unfavorable conditions, such as hypoxia or ischemia. Adenosine modulates different functional activities in macrophages. Some of these activities are believed to be induced through the uptake of adenosine into the macrophages, while others are due to the interaction with specific cell surface receptors. In murine bone marrow-derived macrophages, the use of different radioligands for adenosine receptors suggests the presence of A2B and A3 adenosine receptor subtypes. The presence of A2B receptors was confirmed by flow cytometry using specific Abs. The A2B receptor is functional in murine macrophages, as indicated by the fact that agonists of A2B receptors, but not agonists for A1, A2A, or A3, lead to an increase in cAMP levels. IFN-gamma up-regulates the surface protein and gene expression of the A2B adenosine receptor by induction of de novo synthesis. The up-regulation of A2B receptors correlates with an increase in cAMP production in macrophages treated with adenosine receptor agonist. The stimulation of A2B receptors by adenosine or its analogues inhibits the IFN-gamma-induced expression of MHC class II genes and also the IFN-gamma-induced expression of nitric oxide synthase and of proinflammatory cytokines. Therefore, the up-regulation of the A2B adenosine receptor expression induced by IFN-gamma could be a feedback mechanism for macrophage deactivation.

Identification and characterization of a membrane protein (y+L amino acid transporter-1) that associates with 4F2hc to encode the amino acid transport activity y+L. A candidate gene for lysinuric protein intolerance.

We have identified a new human cDNA (y+L amino acid transporter-1 (y+LAT-1)) that induces system y+L transport activity with 4F2hc (the surface antigen 4F2 heavy chain) in oocytes. Human y+LAT-1 is a new member of a family of polytopic transmembrane proteins that are homologous to the yeast high affinity methionine permease MUP1. Other members of this family, the Xenopus laevis IU12 and the human KIAA0245 cDNAs, also co-express amino acid transport activity with 4F2hc in oocytes, with characteristics that are compatible with those of systems L and y+L, respectively. y+LAT-1 protein forms a approximately 135-kDa, disulfide bond-dependent heterodimer with 4F2hc in oocytes, which upon reduction results in two protein bands of approximately 85 kDa (i.e. 4F2hc) and approximately 40 kDa (y+LAT-1). Mutation of the human 4F2hc residue cysteine 109 (Cys-109) to serine abolishes the formation of this heterodimer and drastically reduces the co-expressed transport activity. These data suggest that y+LAT-1 and other members of this family are different 4F2 light chain subunits, which associated with 4F2hc, constitute different amino acid transporters. Human y+LAT-1 mRNA is expressed in kidney >> peripheral blood leukocytes >> lung > placenta = spleen > small intestine. The human y+LAT-1 gene localizes at chromosome 14q11.2 (17cR approximately 374 kb from D14S1350), within the lysinuric protein intolerance (LPI) locus (Lauteala, T., Sistonen, P. , Savontaus, M. L., Mykkanen, J., Simell, J., Lukkarinen, M., Simmell, O., and Aula, P. (1997) Am. J. Hum. Genet. 60, 1479-1486). LPI is an inherited autosomal disease characterized by a defective dibasic amino acid transport in kidney, intestine, and other tissues. The pattern of expression of human y+LAT-1, its co-expressed transport activity with 4F2hc, and its chromosomal location within the LPI locus, suggest y+LAT-1 as a candidate gene for LPI.

Two missense point mutations in different alleles in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene produce 3-hydroxy-3-methylglutaric aciduria in a French patient.

Two novel point mutations in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene were found in a French patient with double heterozygous 3-hydroxy-3-methylglutaric aciduria. Amplification by reverse transcriptase-polymerase chain reaction of the mRNA using five different pairs of oligonucleotides produced no differences in the fragments amplified with respect to the control. Single-strand conformation polymorphism analysis showed that only one amplified fragment was different in the patient vs. control. Sequencing of the amplified fragments showed two missense point mutations, A698G and T788C, each of them mixed with the wild-type sequence. These mutations produced the changes H233R and L263P, leading to changes in the enzyme activity, which was largely abolished. The father and one brother of the proband were heterozygous for the L263P mutation and the mother and one daughter were heterozygous for the H233R mutation, which confirms the double-heterozygous character of the patient. Another sibling was free of the mutations. An enzymatic restriction analysis has been proposed to screen the occurrence of these two mutations in future patients.

Dexamethasone enhances macrophage colony stimulating factor- and granulocyte macrophage colony stimulating factor-stimulated proliferation of bone marrow-derived macrophages.

Glucocorticoids are effective repressors of the immune system. We have examined the effect of glucocorticoids on the proliferation of murine macrophages. Dexamethasone by itself did not affect proliferation of differentiated or undifferentiated bone marrow-derived macrophages (BMM) and elicited peritoneal macrophages. However, dexamethasone enhanced the proliferation induced by macrophage colony stimulating factor (M-CSF) of these cells. The effect of dexamethasone was not restricted to M-CSF-dependent proliferation. Similarly, dexamethasone enhanced granulocyte macrophage colony stimulating factor (GM-CSF)-dependent proliferation of BMM. In agreement, macrophages transfected with the glucocorticoid receptor showed an enhancement of M-CSF-dependent proliferation. The enhancement of proliferation by dexamethasone or the glucocorticoid receptor was abolished by RU 486, an antagonist of the glucocorticoid receptor. Moreover, the addition of antibodies against M-CSF inhibits the effect of dexamethasone, suggesting that dexamethasone increases the autocrine production of M-CSF. This only occurs when M-CSF or GM-CSF, which induce M-CSF, are present in the media. In tissues, dexamethasone may enhance macrophage proliferation and contribute to the resolution of the inflammatory states.