Malnutrition in Healthy Individuals Results in Increased Mixed Cytokine Profiles, Altered Neutrophil Subsets and Function.

Malnutrition is commonly associated with increased infectious disease susceptibility and severity. Whereas malnutrition might enhance the incidence of disease as well as its severity, active infection can in turn exacerbate malnutrition. Therefore, in a malnourished individual suffering from a severe infection, it is not possible to determine the contribution of the pre-existing malnutrition and/or the infection itself to increased disease severity. In the current study we focussed on two groups of malnourished, but otherwise healthy individuals: moderately malnourished (BMI: 18.4-16.5) and severely malnourished (BMI <16.5) and compared several immune parameters with those of individuals with a normal BMI (≥18.5). Our results show a similar haematological profile in all three groups, as well as a similar ratio of CD4+ and CD8+ T cells. We found significant correlations between low BMI and increased levels of T helper (Th) 1 (Interferon (IFN)-γ, (interleukin (IL)-2, IL-12), Th2 (IL-4, IL-5, IL-13), as well as IL-10, IL-33 and tumor necrosis factor-α, but not IL-8 or C reactive protein. The activities of arginase, an enzyme associated with immunosuppression, were similar in plasma, peripheral blood mononuclear cells (PBMC) and neutrophils from all groups and no differences in the expression levels of CD3ζ, a marker of T cell activation, were observed in CD4+ and CD8+T cells. Furthermore, whereas the capacity of neutrophils from the malnourished groups to phagocytose particles was not impaired, their capacity to produce reactive oxygen species was impaired. Finally we evaluated the frequency of a subpopulation of low-density neutrophils and show that they are significantly increased in the malnourished individuals. These differences were more pronounced in the severely malnourished group. In summary, our results show that even in the absence of apparent infections, healthy malnourished individuals display dysfunctional immune responses that might contribute to increased susceptibility and severity to infectious diseases.

Differential impact of L-arginine deprivation on the activation and effector functions of T cells and macrophages.

The metabolism of the amino acid L-arginine is emerging as a crucial mechanism for the regulation of immune responses. Here, we characterized the impact of L-arginine deprivation on T cell and macrophage (MPhi) effector functions: We show that whereas L-arginine is required unconditionally for T cell activation, MPhi can up-regulate activation markers and produce cytokines and chemokines in the absence of L-arginine. Furthermore, we show that L-arginine deprivation does not affect the capacity of activated MPhi to up-regulate L-arginine-metabolizing enzymes such as inducible NO synthase and arginase 1. Thus, our results show that to exert their effector functions, T cells and MPhi have different requirements for L-arginine.

Gene expression in IFN-gamma-activated murine macrophages

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Gene expression in IFN-gamma-activated murine macrophages.

Macrophages are critical for natural immunity and play a central role in specific acquired immunity. The IFN-gamma activation of macrophages derived from A/J or BALB/c mice yielded two different patterns of antiviral state in murine hepatitis virus 3 infection, which were related to a down-regulation of the main virus receptor. Using cDNA hybridization to evaluate mRNA accumulation in the cells, we were able to identify several genes that are differently up- or down-regulated by IFN-gamma in A/J (267 and 266 genes, respectively, up- and down-regulated) or BALB/c (297 and 58 genes, respectively, up- and down-regulated) mouse macrophages. Macrophages from mice with different genetic backgrounds behave differently at the molecular level and comparison of the patterns of non-activated and IFN-gamma-activated A/J or BALB/c mouse macrophages revealed, for instance, an up-regulation and a down-regulation of genes coding for biological functions such as enzymatic reactions, nucleic acid synthesis and transport, protein synthesis, transport and metabolism, cytoskeleton arrangement and extracellular matrix, phagocytosis, resistance and susceptibility to infection and tumors, inflammation, and cell differentiation or activation. The present data are reported in order to facilitate future correlation of proteomic/transcriptomic findings as well as of results obtained from a classical approach for the understanding of biological phenomena. The possible implication of the role of some of the gene products relevant to macrophage biology can now be further scrutinized. In this respect, a down-regulation of the main murine hepatitis virus 3 receptor gene was detected only in IFN-gamma-activated macrophages of resistant mice.

Infection of C57BL/10ScCr and C57BL/10ScNCr mice with Leishmania major reveals a role for Toll-like receptor 4 in the control of parasite replication.

The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized. In the present study, we investigated the contribution of TLR4 to the course of cutaneous leishmaniasis in vivo. We used C57BL/10ScNCr (TLR4(0/0)) and C57BL/10ScCr [TLR4/interleukin-12 (IL-12)Rbeta2(0/0)] mice and compared the course of Leishmania major infection, parasite load, cell recruitment, and cytokine profile with those of wild-type C57BL/10ScSn mice. Our results confirm the importance of IL-12 receptor-mediated signaling in resistance to L. major infections. Importantly, we show that the lack of TLR4 results in an increased permissiveness for parasite growth during the innate and adaptive phase of the immune response and in delayed healing of the cutaneous lesions. The use of the tlr4 transgenic mouse strain TCr5 demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.

Factors influencing Leishmania major infection in IL-4-deficient BALB/c mice.

The outcome of Leishmania major infection in IL-4-deficient BALB/c mice has been a controversial subject. We have shown that IL-4-deficient BALB/c mice infected with Leishmania major developed progressive lesions and could not contain the replication of the parasites, whereas other studies have reported that IL-4-deficient mice were able to resist infection. Therefore, we examined different factors that can influence the course of Leishmania major infection. We tested different lines of IL-4-deficient BALB/c mice and show that the reported differences in the outcome of infection were not due to the different genetic origin of the embryonic stem cells used to disrupt the IL-4 gene. In addition, we infected IL-4-deficient mice with different isolates of L. major parasites and show that none of the parasite strains tested were cleared, although some of them caused milder pathology. Interestingly, this milder pathology was paralleled by a reduced arginase activity of the parasites. We also tested the influence of age on the course of Leishmania major infection in IL-4-deficient BALB/c mice and show that older mice express a transient resistance. Thus, we conclude that differences in the age of the mice and in the arginase activity of the different isolates of parasites are factors that can influence the non-healing phenotype of IL-4-/- BALB/c mice.

Differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of L-arginine metabolism.

Type 2 cytokines regulate fibrotic liver pathology in mice infected with Schistosoma mansoni. Switching the immune response to a type 1-dominant reaction has proven highly effective at reducing the pathologic response. Activation of NOS-2 is critical, because type 1-deviated/NO synthase 2 (NOS-2)-deficient mice completely fail to control their response. Here, we demonstrate the differential regulation of NOS-2 and arginase type 1 (Arg-1) by type 1/type 2 cytokines in vivo and for the first time show a critical role for arginase in the pathogenesis of schistosomiasis. Using cytokine-deficient mice and two granuloma models, we show that induction of Arg-1 is type 2 cytokine dependent. Schistosome eggs induce Arg-1, while Mycobacterium avium-infected mice develop a dominant NOS-2 response. IFN-gamma suppresses Arg-1 activity, because type 1 polarized IL-4/IL-10-deficient, IL-4/IL-13-deficient, and egg/IL-12-sensitized animals fail to up-regulate Arg-1 following egg exposure. Notably, granuloma size decreases in these type-1-deviated/Arg-1-unresponsive mice, suggesting an important regulatory role for Arg-1 in schistosome egg-induced pathology. To test this hypothesis, we administered difluoromethylornithine to block ornithine-aminodecarboxylase, which uses the product of arginine metabolism, L-ornithine, to generate polyamines. Strikingly, granuloma size and hepatic fibrosis increased in the ornithine-aminodecarboxylase-inhibited mice. Furthermore, we show that type 2 cytokine-stimulated macrophages produce proline under strict arginase control. Together, these data reveal an important regulatory role for the arginase biosynthetic pathway in the regulation of inflammation and demonstrate that differential activation of Arg-1/NOS-2 is a critical determinant in the pathogenesis of granuloma formation.

Lipopolysaccharide-induced apoptosis of macrophages determines the up-regulation of concentrative nucleoside transporters Cnt1 and Cnt2 through tumor necrosis factor-alpha-dependent and -independent mechanisms.

In murine bone marrow macrophages, lipopolysaccharide (LPS) induces apoptosis through the autocrine production of tumor necrosis factor-alpha (TNF-alpha), as demonstrated by the fact that macrophages from TNF-alpha receptor I knock-out mice did not undergo early apoptosis. In these conditions LPS up-regulated the two concentrative high affinity nucleoside transporters here shown to be expressed in murine bone marrow macrophages, concentrative nucleoside transporter (CNT) 1 and 2, in a rapid manner that is nevertheless consistent with the de novo synthesis of carrier proteins. This effect was not dependent on the presence of macrophage colony-stimulating factor, although LPS blocked the macrophage colony-stimulating factor-mediated up-regulation of the equilibrative nucleoside transport system es. TNF-alpha mimicked the regulatory response of nucleoside transporters triggered by LPS, but macrophages isolated from TNF-alpha receptor I knock-out mice similarly up-regulated nucleoside transport after LPS treatment. Although NO is produced by macrophages after LPS treatment, NO is not involved in these regulatory responses because LPS up-regulated CNT1 and CNT2 transport activity and expression in macrophages from inducible nitric oxide synthase and cationic amino acid transporter (CAT) 2 knock-out mice, both of which lack inducible nitric oxide synthesis. These data indicate that the early proapoptotic responses of macrophages, involving the up-regulation of CNT transporters, follow redundant regulatory pathways in which TNF-alpha-dependent- and -independent mechanisms are involved. These observations also support a role for CNT transporters in determining extracellular nucleoside availability and modulating macrophage apoptosis.

Down-regulation of Bgp1(a) viral receptor by interferon-gamma is related to the antiviral state and resistance to mouse hepatitis virus 3 infection.

Together with the evidence that the reduced virus growth and the antiviral state induced by interferon (IFN)-gamma, occurring only in macrophages from resistant animals, correlated with the decrease of MHV3 binding to macrophage membrane proteins, we show here the expression of cellular and viral genes in resistant (A/J) and susceptible (BALB/c) mouse macrophages after IFN-gamma activation/infection. The expression of interferon response gene 47 and interferon regulatory factor 1 genes takes place after IFN-gamma activation in both macrophages, indicating their activation. The expression of the biliary glycoprotein 1(a) (Bgp1(a), the main virus receptor) decreased only in IFN-gamma-activated A/J mouse macrophages, in contrast to the expression of the Bgp2 (alternative receptor), which was not influenced by IFN-gamma activation. The synthesis of both viral mRNA and virus particles was delayed only in IFN-gamma-activated A/J mouse macrophages compared with susceptible BALB/c macrophages. Besides the evidence that IFN-gamma may modulate the expression of the Bgp1(a) isoform of carcinoembryonic antigen family, these data show that IFN-gamma, which induces resistance against MHV3 infection, may be involved in the down-regulation of the main viral receptor expression, a key step forward in our understanding of the molecular basis of resistance against virus infection.

Involvement of mitochondria and caspase-3 in ET-18-OCH(3)-induced apoptosis of human leukemic cells.

The induction of cell death in leukemic HL-60 cells by the ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) followed the typical apoptotic changes in ultrastructural morphology, including blebbing, chromatin condensation, nuclear membrane breakdown and extensive vacuolation. Using a cytofluorimetric approach, we found that ET-18-OCH(3) induced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) followed by production of reactive oxygen species (ROS) and DNA fragmentation in leukemic cells. ET-18-OCH(3) also induced caspase-3 activation in human leukemic cells, as assessed by cleavage of caspase-3 into the p17 active form and cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP). ET-18-OCH(3) analogues unable to induce apoptosis failed to disrupt DeltaPsi(m) and to activate caspase-3. ET-18-OCH(3)-resistant Jurkat cells generated from sensitive Jurkat cells showed no caspase-3 activation and did not undergo DeltaPsi(m) disruption upon ET-18-OCH(3) incubation. Cyclosporin A partially inhibited DeltaPsi(m) dissipation, caspase activation and apoptosis in ET-18-OCH(3)-treated leukemic cells. Overexpression of bcl-2 by gene transfer prevented DeltaPsi(m) collapse, ROS generation, caspase activation and apoptosis in ET-18-OCH(3)-treated leukemic T cells. Pretreatment with the caspase inhibitor Z-Asp-2, 6-dichlorobenzoyloxymethylketone prevented ET-18-OCH(3)-induced PARP proteolysis and DNA fragmentation, but not DeltaPsi(m) dissipation. ET-18-OCH(3) did not affect the expression of caspases and bcl-2-related genes. ET-18-OCH(3)-induced apoptosis did not require protein synthesis. Our data indicate that DeltaPsi(m) dissipation and caspase-3 activation are critical events of the apoptotic cascade triggered by the antitumor ether lipid ET-18-OCH(3), and that the sequence of events in the apoptotic action of ET-18-OCH(3) on human leukemic cells is: DeltaPsi(m) disruption, caspase-3 activation and internucleosomal DNA degradation.

Intracellular triggering of Fas, independently of FasL, as a new mechanism of antitumor ether lipid-induced apoptosis.

Antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET-18-OCH(3) required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET-18-OCH(3) does not promote Fas or FasL expression and ET-18-OCH(3)-induced apoptosis is not inhibited by pre-incubation with an anti-Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET-18-OCH(3)-resistant normal human Fas-positive fibroblasts do not incorporate ET-18-OCH(3), but undergo apoptosis upon ET-18-OCH(3) microinjection. Murine fibroblasts L929 and L929-Fas, stably transfected with human Fas cDNA, do not incorporate ET-18-OCH(3) and are resistant to its action when added exogenously. Microinjection of ET-18-OCH(3) induces apoptosis in L929-Fas cells, but not in wild-type L929 cells. Confocal laser scanning microscopy shows that ET-18-OCH(3) induces Fas clustering and capping during triggering of ET-18-OCH(3)-induced apoptosis. Microinjection-induced apoptosis and Fas clustering are specific for the molecular structure of ET-18-OCH(3). Our data indicate that ET-18-OCH(3) induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET-18-OCH(3) on tumors since only cancer cells incorporate sufficient amounts of the drug.

Th1/Th2-regulated expression of arginase isoforms in murine macrophages and dendritic cells.

Activated murine macrophages metabolize arginine by two alternative pathways involving the enzymes inducible NO synthase (iNOS) or arginase. The balance between the two enzymes is competitively regulated by Th1 and Th2 T helper cells via their secreted cytokines: Th1 cells induce iNOS, whereas Th2 cells induce arginase. Whereas the role of macrophages expressing iNOS as inflammatory cells is well established, the functional competence of macrophages expressing arginase remains a matter of speculation. Two isoforms of mammalian arginases exist, hepatic arginase I and extrahepatic arginase II. We investigated the regulation of arginase isoforms in murine bone marrow-derived macrophages (BMMPhi) in the context of Th1 and Th2 stimulation. Surprisingly, in the presence of either Th2 cytokines or Th2 cells, we observe a specific induction of the hepatic isoform arginase I in BMMPhi. Induction of arginase I was shown on the mRNA and protein levels and obeyed the recently demonstrated synergism among the Th2 cytokines IL-4 and IL-10. Arginase II was detectable in unstimulated BMMPhi and was not significantly modulated by Th1 or Th2 stimulation. Similar to murine BMMPhi, murine bone marrow-derived dendritic cells, as well as a dendritic cell line, up-regulated arginase I expression and arginase activity upon Th2 stimulation, whereas arginase II was never detected. In addition to revealing the unexpected expression of arginase I in the macrophage/monocyte lineage, these results uncover a further intriguing parallelism between iNOS and arginase: both have a constitutive and an inducible isoform, the latter regulated by the Th1/Th2 balance.

Induction of apoptosis in human mitogen-activated peripheral blood T-lymphocytes by the ether phospholipid ET-18-OCH3: involvement of the Fas receptor/ligand system.

1. Activated T-cells constitute a target for treatment of autoimmune diseases. We have found that the antitumour ether phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) induced dose- and time-dependent apoptosis in human mitogen-activated peripheral blood T-lymphocytes, but not in resting T-cells. T-lymphocytes were stimulated with phytohemagglutinin and interleukin-2 or with concanavalin A. Apoptosis was assessed by DNA fragmentation through cell cycle and TUNEL analyses, as well as through visualization of internucleosomal DNA fragmentation in agarose gels. 2. The ET-18-OCH3-mediated apoptotic response in activated T-lymphocytes was less intense than in human leukaemic T cell lines, such as Jurkat cells and Peer cells; namely about 25% apoptosis in activated T-cells versus about 46-61% apoptosis in T leukaemic cells after 24 h treatment with 10 microM ET-18-OCH3. 3. The ET-18-OCH3 thioether analogue BM 41.440 (ilmofosine) showed a similar apoptotic capacity to that found with ET-18-OCH3 in activated T-cells, whereas the phospholipid analogue hexadecylphosphocholine (miltefosine) failed to promote this response. 4. The uptake of [3H]-ET-18-OCH3 was much larger in activated T-cells than in resting lymphocytes. 5. Using a cytofluorimetric approach we have found that ET-18-OCH3 induced disruption of the mitochondrial transmembrane potential and production of reactive oxygen species in activated T-cells, but not in resting lymphocytes. 6. ET-18-OCH3 induced an increase in Fas (APO-1/CD95) ligand mRNA expression in activated T-cells, and incubation with a blocking anti-Fas (APO-1/CD95) antibody partially inhibited the ET-18-OCH3-induced apoptosis of activated T-lymphocytes. 7. These results demonstrate that mitogen-activated T-cells, unlike resting lymphocytes, are able to take up significant amounts of ET-18-OCH3, and are susceptible to undergo apoptosis by the ether lipid via, in part, the Fas (APO-1/CD95) receptor/ligand system. This ET-18-OCH3 apoptotic action can be of importance in the therapeutic action of this ether lipid in certain autoimmune diseases.

A new antioxidative vitamin B6 analogue modulates pathophysiological cell proliferation and damage.

The new large scale synthesis of the yellow colored vitamin B6 analogue 5'-O-phosphono-pyridoxylidenerhodanine (2) (B6PR) leads to oligohydrates of its monosodium salt (4). The light-red hemiheptadecahydrate (8 1/2 hydrate) (4a) was crystallized and its three-dimensional structure determined by X-ray crystallography. Special nucleotide and protein interaction properties together with scavenging antioxidative function are combined in this simple water-soluble vitamin B6 analogues B6PR. High (mM) concentrations were untoxic to 'healthy' not affected cells and primary tissues. Complexation of ions (e.g. Ca2+, Fe2+, and Zn2+), modulation of nitric oxide synthases (NOS I-III), nitric oxide (NO) metabolism, and reactive oxygen species (ROS) was found. Special cytoprotecting, immunomodulating, stimulating and inhibiting activities were observed in vitro, not in comparison with some natural and synthetic pyridoxines. Low B6PR suppressed proliferation, high induced selective cell death of some cancer cell lines. Low B6PR protected HIV-1-infected CD4+ HUT 78 cells against HIV-1-mediated destruction (complete inhibition of HIV-1-induced syncytia formation and cell death) and reduced p24 level. Autoreactive S100beta-specific T cells of Lewis rat, a model of multiple sclerosis, could be influenced. Oxidative damage and age, acquired and inherited disease related pathophysiological disorders can be treated by this new cytopathology-selective versatile acting B6PR.

Murine macrophages secrete interferon gamma upon combined stimulation with interleukin (IL)-12 and IL-18: A novel pathway of autocrine macrophage activation.

Interferon (IFN)-gamma, a key immunoregulatory cytokine, has been thought to be produced solely by activated T cells and natural killer cells. In this study, we show that murine bone marrow- derived macrophages (BMMPhi) secrete large amounts of IFN-gamma upon appropriate stimulation. Although interleukin (IL)-12 and IL-18 alone induce low levels of IFN-gamma mRNA transcripts, the combined stimulation of BMMPhi with both cytokines leads to the efficient production of IFN-gamma protein. The macrophage-derived IFN-gamma is biologically active as shown by induction of inducible nitric oxide synthase as well as upregulation of CD40 in macrophages. Our findings uncover a novel pathway of autocrine macrophage activation by demonstrating that the macrophage is not only a key cell type responding to IFN-gamma but also a potent IFN-gamma-producing cell.

Alternative metabolic states in murine macrophages reflected by the nitric oxide synthase/arginase balance: competitive regulation by CD4+ T cells correlates with Th1/Th2 phenotype.

Activated murine macrophages metabolize L-arginine via two main pathways that are catalyzed by the inducible enzymes nitric oxide synthase (iNOS) and arginase. We have previously shown that CD4+ T cell-derived cytokines regulate a competitive balance in the expression of both enzymes in macrophages; Thl-type cytokines induce iNOS while they inhibit arginase, whereas the reverse is the case for Th2-type cytokines. Here we addressed the regulation of both metabolic pathways by CD4+ T cells directly. Macrophages were used as APCs for established Th1 and Th2 T cell clones as well as for in vitro polarized Th1 or Th2 T cells of transgenic mice bearing an MHC class II-restricted TCR. Both systems revealed a similar dichotomy in the macrophages; Th1 T cells led to an exclusive induction of iNOS, whereas Th2 T cells up-regulated arginase without inducing iNOS. Arginase levels induced by Th2 T cells far exceeded those inducible by individual Th2 cytokines. Similarly, high arginase levels could be induced by supernatants of Th2 cells stimulated in various ways. Ab blocking experiments revealed the critical importance of IL-4 and IL-10 for arginase up-regulation. Finally, strong synergistic effects between IL-4/IL-13 and IL-10 were observed, sufficient to account for the extraordinarily high arginase activity induced by Th2 cells. Our results suggest that the iNOS/arginase balance in macrophages is competitively regulated in the context of Th1- vs Th2-driven immune reactions, most likely by cytokines without the requirement for direct cell interaction.