Synthesis and evaluation of stimulatory properties of Sphingomonadaceae glycolipids

Glycosphingolipids (GSLs) from the Sphingomonadaceae family of bacteria have been reported to be potent stimulators of natural killer T cells. These glycolipids include mono-, tri- and tetraglycosylceramides. Here we have prepared the GSL-1 to GSL-4 series of glycolipids and tested their abilities to stimulate natural killer T cells. Among these glycolipids, only GSL-1 (1) is a potent stimulator. Using a series of synthetic diglycosylceramides, we show that oligoglycosylceramides from Sphingomonadaceae are not effectively truncated to GSL-1 in lysosomes in antigen-presenting cells, possibly because the higher-order GSLs are poor substrates for lysosomal acyltransfer enzymes.     

The complexation of metal cations by D-galacturonic acid: a spectroscopic study

Solid complexes of D-galacturonic acid (GalA) with cobalt(II), copper(II), nickel(II) and oxovanadium(IV) (1-4) were prepared and characterised. The metal-to-ligand molar ratio was 1:2 for complexes 1-3 and 1:1 for complex 4. The alpha- and beta-anomers of GalA were detected in all the complexes in solid state and in solutions. An addition of small amounts of the paramagnetic complexes to the D2O solution of pure ligand led to NMR line broadening of some 1H and 13C nuclei. This broadening was sensitive to the anomeric state of GalA in the case of complexes 1 and 4. NMR and vibrational spectroscopic data indicate the formation of carboxylate complexes of all the cations, while noncarboxylic oxygens are also involved into the metal bonding in some cases. VCD spectra of complexes 1-4 in D2O and Me2SO-d6 solutions confirm that GalA carboxylic group may participate in the formation of optically active species around the metal cation. Possible ways of GalA coordination by metal cations of this study were proposed and discussed.     

iNKTs foil fungi

Fungal cell wall polysaccharides are potent inducers of immune responses. Cohen et?al. (2011) demonstrate that innate recognition of fungal β-(1,3) glucan triggers effector functions of invariant natural killer T (iNKT) cells through indirect, cytokine-driven activation, a process that mediates optimal protection against the opportunistic mold Aspergillus fumigatus.     

Invariant natural killer T cells trigger adaptive lymphocytes to churn up bile

How innate immune response causes autoimmunity has remained an enigma. In this issue of Cell Host & Microbe, Mattner et al. demonstrate that invariant natural killer T cells activated by the mucosal commensal Novosphingobium aromaticivorans precipitate chronic T cell-mediated autoimmunity against small bile ducts that mirrors human primary biliary cirrhosis. These findings provide a mechanistic understanding of the role of innate immunity toward a microbe in the development of autoimmunity.     

Imbalance of peripheral B lymphocytes and NK cells in rheumatoid arthritis

The study was focused on several cellular immune disorders correlated with the imbalance between peripheral blood B lymphocytes and NK cells in severe rheumatoid arthritis. By flow cytometry we calculated the proportions of T, T helper, T cytotoxic/suppressor, B lymphocytes and natural killer cells in peripheral blood. The mitogen-induced proliferation of peripheral lymphocytes was measured by tritium-labeld uridine incorporation. Experimental data highlight a connection between annomal values of the B to natural killer cells ratio and disorders of the peripheral mononuclear cells concentration. We also showed that the polyclonal proliferation capacity of peripheral lymphocytes in rheumatoid arthritis is solely related to the B to natural killer cells ratio or to the natural killer cells proportion. The study reveals a potential role of the imbalance between proportions of peripheral B lymphocytes and natural killer cells in the immune pathogenesis of rheumatoid arthritis, thus pointing out an interrelation between the adaptive and innate immune systems.     

Effect of interleukin 2 on the inhibition of human natural killer activity by monolayer cells

We have previously shown that human endogenous natural killer activity against K562 is inhibited by primary cultures of natural killer-resistant monolayer target cells. In this study we have analyzed the sensitivity of activated killer cells to this inhibitory effect. Interleukin-2 (IL-2), when present during an 18-hr contact of peripheral blood lymphocytes with monolayers, did not affect the inhibition of natural killer cell activity. Pretreatment of effector cells with IL-2 for 24-62 hr before the contact with monolayer cells eradicated the inhibition caused by malignant cells, benign cells remaining inhibitory. The IL-2-pretreated effector cells killed preferentially malignant target cells, although significant cytotoxicity was also detectable against benign cell cultures. The results indicate that activation of killer cells in vitro by IL-2 involves the desensitization of effector cells to the inhibitory signals of target cells, and that the selectivity of IL-2-activated killer cells toward malignant target cells involves weaker inhibition of activated killer cells by malignant cells.     

Asymmetric transport of D-glucose anomers across the human erythrocyte membrane

The anomeric preference in the influx and efflux of D-glucose across the human erythrocyte membrane was studied. beta-D-Glucose was transported 1.5 times faster than alpha-D-glucose into the cells, when washed cells were incubated at 20 degrees C in medium containing either alpha- or beta-D-glucose (100 mM). On the other hand, no difference between half-times of efflux of the two anomers was distinguishable. The result demonstrates the presence of influx-efflux asymmetry in anomeric preference in D-glucose transport across the human erythrocyte membrane, and is consistent with the view (Barnett et al., Biochem. J. 145, 417-429, 1975) that the C-1 hydroxyl group of D-glucose interacts with the D-glucose transport protein only in the influx, but not in the efflux.     

Reorganization of actin cytoskeleton in 3T3-SV40 cells and their sensitivity to lysis by natural killer cells

The present work was aimed to examine whether the actin reorganization of 3T3-SV40 cells influences their sensitivity to natural killer (NK) cells activity. The effects of N-acetylcystein (NAC) and latrunculin B, actin depolimerizator, on both cellular parameters were studied. Experiments with NAC demonstrated that 3T3-SV40 sensitivity to NK cells activity remained unchanged under the disordered microfilaments but decreased upon the appearance of structured stress-fibres. The data on latrunculin B action resulted in the opposite conclusion: the more microfilaments disorganization in the presence of latrunculin B the lesser 3T3-SV40 sensitivity to lysis by NK cells. These facts suggest that relations between microfilament integrity in 3T3-SV40 cells and their sensitivity to NK cells are rather independent. The latter confirms our previous conclusion (Gamaley et al., 2006). Decrease in 3T3-SV40 sensitivity to NK cells activity accompanied by actin reorganization resulted from both latrunculin B and NAC action suggests changes in cellular surface, which ultimately lead to inactivation (or loss) of the molecules being activating signals to NK cells.     

Characterization of the natural killer-like lymphocytes in rheumatoid synovial fluid

IgG Fc- cytotoxic cells found in the synovial fluid of patients with rheumatoid arthritis have natural killer (NK)-like characteristics but can kill NK-resistant cell lines as well. The phenotype of these cells was defined by complement-mediated lysis with monoclonal antibodies. The synovial fluid killer cell activity was significantly reduced by treatment with complement and OKT11 and 4F2, but the cytotoxic T cells did not express the NK-related antigens OKM1 and Leu-7, nor the cytotoxic T lymphocyte-specific antigen, OKT8. These results demonstrate that the synovial fluid killer cells resemble the activated T cells generated in an autologous mixed leukocyte reaction or in the treatment of peripheral blood mononuclear cells with interleukin 2, and they are distinct from the conventional NK cells found in blood.     

A phenyl-beta-galactoside-specific monoclonal antibody reactive with murine and rat NK cells

The phenyl-beta-galactoside (phi-beta-gal)-specific monoclonal antibody (mAb) 49H.8 cross-reacts with the terminal disaccharide structure of the asialo GM1 (AGM1) molecule. It was found to react with phi-beta-gal determinants on murine and rat splenic natural killer (NK) cells, as measured by complement depletion studies. Flow cytometric analysis identified the antigen on two IL 2-dependent cloned murine NK cell lines and the rat large granular lymphocyte leukemia RNK. We have compared the 49H.8 reactivity to that of anti-AGM1 antisera (alpha-AGM1) on NK cells and a panel of NK related killer cells, including bone marrow-derived killer cells, lymphokine-activated killer cells (LAK), and anomalous killer cells (AK). We found that the 49H.8 specificity closely paralleled that of alpha-AGM1. When tested against Con A-reactive T cells, the 49H.8 mAb was less reactive than the alpha-AGM1, indicating that it may be a more specific marker for splenic NK populations than the alpha-AGM1.     

Complement Activation by Bacterial Surface Glycolipids: A Study with Planar Bilayer Membranes

Planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the outer membrane of Gram-negative bacteria to study complement (C) activation by various bacterial surface glycolipids with the aim of defining the C activation pathway. As glycolipids the lipopolysaccharides of Salmonella enterica serovar Minnesota R mutant strains R595 (Re LPS) and R4 (Rd₂ LPS), pentaacyl lipid A from the LPS of the Escherichia coli Re mutant F515, and glycosphingolipid GSL-1 of Sphingomonas paucimobilis IAM 12576 were used. Methylester and carboxyl-reduced derivatives of GSL-1 were used to elucidate the role of the carboxyl group as common functional group of LPS and GSL-1 for C activation. The formation of lytic pores was monitored via the measurement of changes in membrane current. For all glycolipids we observed a considerable increase in membrane current soon after addition of whole human serum due to the formation of lytic pores in the membranes. Pore formation was dependent on the presence of C9, indicating that the observed current changes were due to C activation. We found that in our reconstitution system of the outer membrane lipid A, Re LPS, and Rd₂ LPS activated the classical pathway, the activation being independent of specific anti-LPS antibodies. In contrast, GSL-1 and the methylester derivative of GSL-1 activated the alternative pathway even at the low serum concentrations used in this study (about 0.2% v/v). Interestingly, the carboxyl reduced GSL-1 activated the classical pathway. Received: 16 July 1998/Revised: 28 October 1998     

Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice

Interleukin-18 (IL-18) produced by activated antigen-presenting cells stimulates natural killer (NK) cells, natural killer T (NKT) cells, and T cells to secrete gamma interferon (IFN-gamma). In this study, injection of a single 10- micro g dose of recombinant murine IL-18 rapidly, reversibly, and noncytopathically inhibited hepatitis B virus (HBV) replication in the livers of HBV transgenic mice. Furthermore, HBV replication was inhibited by as little as 1 micro g of IL-18 injected repetitively, and also by a single 0.1- micro g dose of IL-18 injected together with 1 ng of IL-12, neither of which inhibited HBV replication individually, demonstrating synergy between these cytokines in this system. The antiviral effect of IL-18 was mediated by its ability to activate resident intrahepatic NK cells and NKT cells to produce IFN-gamma and by its ability to induce IFN-alpha/beta production in the liver. These results suggest that IL-18 has the potential to contribute to the control of HBV replication during self-limited infection and that it may have therapeutic value for the treatment of patients with chronic hepatitis.     

Glucocorticoid receptor mediated suppression of natural killer cell activity: identification of associated deacetylase and corepressor molecules

Physical and psychological stressors reduce natural killer cell function. This reduction in cellular function results from stress-induced release of glucocorticoids. Glucocorticoids act upon natural killer cells to deacetylate and transrepress immune response genes through epigenetic processes. However, other than the glucocorticoid receptor, the proteins that participate in this process are not well described in natural killer cells. The purpose of this study was to identify the proteins associated with the glucocorticoid receptor that are likely epigenetic participants in this process. Treatment of natural killer cells with the synthetic glucocorticoid, dexamethasone, produced a significant time dependent reduction in natural killer cell activity as early as 8h post treatment. This reduction in natural killer cell activity was preceded by nuclear localization of the glucocorticoid receptor with histone deacetylase 1 and the corepressor, SMRT. Other class I histone deacetylases were not associated with the glucocorticoid receptor nor was the corepressor NCoR. These results demonstrate histone deacetylase 1 and SMRT to associate with the ligand activated glucocorticoid receptor within the nuclei of natural killer cells and to be the likely participants in the histone deacetylation and transrepression that accompanies glucocorticoid mediated reductions in natural killer cell function.     

Increased expression of the NK cell receptor KLRG1 by virus-specific CD8 T cells during persistent antigen stimulation

The killer cell lectin-like receptor G1 (KLRG1) is a natural killer cell receptor expressed by T cells that exhibit impaired proliferative capacity. Here, we determined the KLRG1 expression by virus-specific T cells. We found that repetitive and persistent antigen stimulation leads to an increase in KLRG1 expression of virus-specific CD8+ T cells in mice and that virus-specific CD8+ T cells are mostly KLRG1+ in chronic human viral infections (human immunodeficiency virus, cytomegalovirus, and Epstein-Barr virus) but not in resolved infection (influenza virus). Thus, by using KLRG1 as a T-cell marker, our results suggest that the differentiation status and function of virus-specific CD8+ T cells are directly influenced by persistent antigen stimulation.     

Effect of low intensity of electromagnetic radiation in the centimeter and millimeter range on proliferative and cytotoxic activity of murine spleen lymphocytes

It was found that single total-body exposure to electromagnetic centimeter waves (8.15-18 GHz, 1 microW/cm2, 5 h) stimulated the proliferation of mouse T and B splenic lymphocytes. The same effects were observed upon in vivo treatment of rats for 5 h with millimeter waves (42.2 GHz, amplitude modulation 10 Hz, 1 microW/cm2). The whole-body irradiation with centimeter or millimeter waves did not cause any significant changes in natural activity of killer cells. The cellular responses induced by the irradiation of isolated animal cells in vitro did not coincide with those revealed after the total-body irradiation of animals. Thus, the in vitro irradiation of natural killer cells to millimeter waves for 1 h increased their cytotoxic activity whereas, after treatment to centimeter waves for the same time, the activity of killer cells did not change. On the contrary, irradiation of T and B lymphocytes with millimeter waves (42.2 GHz, amplitude modulation 10 Hz, 1 microW/cm2, 1 h) suppressed the blasttransformation of cells. The results show a higher immunostimulative potential of centimeter waves as compared to millimeter waves.     

Alteration of T cell subsets and induction of suppressor T cell activity in normal subjects after exposure to sunlight

The effects of exposure to natural sunlight on the immune system were studied in 15 normal human subjects. Exposure was for 1 hr each day for 12 days over 2 wk and tests were carried out before, on completion, and 2 wk after completion. In comparison to concurrent studies on 13 age- and sex-matched controls, sun-exposed subjects had a significant increase in their circulation of T cells recognized by OKT8 monoclonal antibodies and a decrease in OKT4 positive T cells. Suppressor T cell activity measured in pokeweed mitogen-stimulated cultures of T and B cells was significantly increased against IgG and IgM production. These changes were still evident in many of the subjects 2 weeks after completion of the sun exposure. A trend for depression of natural killer cell activity against a melanoma target cell was noted in the present study, but this did not appear as marked as that noted previously in subjects exposed to radiation in solariums. The differences between the effect of radiation from solariums and natural sunlight on the immune system may result from the higher dosage of UV-A in radiation from solariums. The results suggest that exposure to sunlight may favor the induction of suppressor pathways in response to antigenic stimuli and that this may limit immune responses against tumor cells such as melanoma. They support the idea from animal studies that systemic changes in the immune system may be an important factor in the association of UV radiation with malignancy.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.