Passive transfer of Leishmania lipopolysaccharide confers parasite survival in macrophages

Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. We have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study we have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here we show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, we show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.     

Effect of cytokines and lipopolysaccharide on CD14 antigen expression in human monocytes and macrophages.

The 52 kD myeloid membrane glycoprotein CD14 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rIFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rIL-1, rIL-2, rIL-3, rIL-5, rIL-6, rTNFa, rGM-CSF, rM-CSF, rTGFb1, rIFNa, lipopolysaccharide (LPS), and, as a control, rIFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. IFNg concentrations were determined in PBL culture supernatants by ELISA. rIFNa and rIL-2 reduced CD14 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rIFNa and rIL-2 were ineffective in purified monocytes or macrophages. rIL-4 strongly reduced CD14 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CD14, but when added in combination with rIFNg the effect on CD14 downregulation was more pronounced. The effect of rIFNg on CD14 in PBL cultures was dose-dependently inhibited by rIL-4 and this inhibition is probably due to an IL-4 mediated blockade of IFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced IFNg secretion. LPS strongly enhanced CD14 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rIFNg and rIL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.     

Cholera toxin B pretreatment of macrophages and monocytes diminishes their proinflammatory responsiveness to lipopolysaccharide

The cholera toxin B chain (CTB) has been reported to suppress T cell-dependent autoimmune diseases and to potentiate tolerance of the adaptive immune system. We have analyzed the effects of CTB on macrophages in vitro and have found that preincubation with CTB (10 microg/ml) suppresses the proinflammatory reaction to LPS challenge, as demonstrated by suppressed production of TNF-alpha, IL-6, IL-12(p70), and NO (p < 0.01) in cells of macrophage lines. Pre-exposure to CTB also suppresses LPS-induced TNF-alpha and IL-12(p70) formation in human PBMC. Both native and recombinant CTB exhibited suppressive activity, which was shared by intact cholera toxin. In cells of the human monocyte line Mono Mac 6, exposure to CTB failed to suppress the production of IL-10 in response to LPS. Control experiments excluded a role of possible contamination of CTB by endotoxin or intact cholera toxin. The suppression of TNF-alpha production occurred at the level of mRNA formation. Tolerance induction by CTB was dose and time dependent. The suppression of TNF-alpha and IL-6 production could be counteracted by the addition of Abs to IL-10 and TGF-beta. IFN-gamma also antagonized the actions of CTB on macrophages. In contrast to desensitization by low doses of LPS, tolerance induction by CTB occurred silently, i.e., in the absence of a measurable proinflammatory response. These findings identify immune-deviating properties of CTB at the level of innate immune cells and may be relevant to the use of CTB in modulating immune-mediated diseases.     

Diverging pathways for lipopolysaccharide and CD14 in human monocytes

BACKGROUND: CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 were studied. METHODS: The uptake and intracellular distribution of fluorescein isothiocyanate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue quenching, and confocal fluorescence microscopy. Incubation of surface-biotinylated cells with LPS at 37 degrees C or 4 degrees C and subsequent subfractionation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent assay (ELISA). RESULTS: The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 x 10(6) molecules per unstimulated monocyte) and could show that internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monocytes showed no change in biotinylated CD14 in the membrane fraction independently of the incubation temperature (37 degrees C or at 4 degrees C) used, indicating that these CD14 molecules were not taken up by an active process. CONCLUSIONS: These data indicate the presence of a large intracellular CD14 pool in monocytes with a yet unknown function, and suggest that LPS and CD14 molecules can be internalized independently after association on the cell surface.     

Parameters affecting the in vitro maturation of human monocytes to macrophages

In vitro maturation of human monocytes to macrophages was characterized by morphological criteria, cell size and lysosomal enzymes activity. Purified populations of monocytes were maintained in culture at either adherent or nonadherent conditions and their maturation to macrophages was observed in both cases. The addition of external factors such as hydrocortisone and vitamin D3 inhibited monocyte maturation. In the absence of external factors, nonadherent monocytes were inhibited in their maturation for up to 10 days when plated at crowded cell concentrations. In addition, the presence of human serum in the culture media had a higher inhibitory activity than similar concentrations of fetal calf serum. Supernates from crowded macrophages were also inhibitory for monocyte maturation. We suggest the possibility that cell crowding, as well as soluble factors found in the serum and probably secreted by macrophages, participate in the regulation of monocyte development by inhibiting their maturation. Once released from this inhibitory signal or environment, the monocytes mature to macrophages.     

Eotaxin-2 generation is differentially regulated by lipopolysaccharide and IL-4 in monocytes and macrophages

The eotaxins are a family of CC chemokines that coordinate the recruitment of inflammatory cells, in particular eosinophils, to sites of allergic inflammation. The cDNA for eotaxin-2 (CC chemokine ligand 24) was originally isolated from an activated monocyte library. In this study, we show for the first time that peripheral blood monocytes generate bioactive eotaxin-2 protein constitutively. Eotaxin-2 production was significantly up-regulated when monocytes were stimulated with the proinflammatory cytokine IL-1beta and the microbial stimuli, LPS and zymosan. In contrast, the Th2 cytokines, IL-4 and IL-13, and the proinflammatory cytokine, TNF-alpha, acting alone or in combination, did not enhance the generation of eotaxin-2 by monocytes. Indeed, IL-4 suppressed the generation of eotaxin-2 by LPS-stimulated monocytes. Although other chemokines, including macrophage-inflammatory protein-1alpha, monocyte chemoattractant protein-1, macrophage-derived chemokine, and IL-8 were generated by monocytes, eotaxin-1 (CC chemokine ligand 11) could not be detected in the supernatants of monocytes cultured in the presence or absence of any of the stimuli used in the above experiments. Furthermore, human dermal fibroblasts that produce eotaxin-1 did not generate eotaxin-2 under basal conditions or when stimulated with specific factors, including IL-4, IL-13, TNF-alpha, and LPS. When monocytes were differentiated into macrophages, their constitutive generation of eotaxin-2 was suppressed. Moreover, IL-4, but not LPS, up-regulated the production of eotaxin-2 by macrophages. Taken as a whole, these results support a role for macrophage-derived eotaxin-2 in adaptive immunity, with a Th2 bias. In contrast, a role for monocyte-derived eotaxin-2 is implicated in innate immunity.     

Differential tolerance induction by lipoarabinomannan and lipopolysaccharide in human macrophages

Various bacterial cell wall components have been shown to induce hyporesponsiveness in macrophages (MAC). Here, mycobacterial glycolipids were employed to determine whether they induce a state of 'tolerance/hyporesponsiveness' in MAC in vitro in order to assess whether mycobacterial components negatively affect the immune response to Mycobacterium tuberculosis. Arabinosylated lipoarabinomannan (ARA-LAM) stimulated hyporesponsiveness by reducing TNF-alpha, GM-CSF, G-CSF, IL-10, and IL-6 release similarly to LPS, but caused no changes in IL-8 secretion. Mannose-capped LAM (MAN-LAM) acted in a different way in that TNF-alpha, GM-CSF, and IL-10 were upregulated after restimulation of MAC. Blocking experiments by mannan suggest mannose-receptor involvement in MAN-LAM activation only. Cross-stimulation experiments demonstrated a hierarchy of signaling, with LPS being the most potent stimulator and mediating abrogation of ARA-LAM-stimulated tolerance but not vice versa. MAN-LAM was the least potent stimulator of either MAC activation and induction of hyporesponsiveness. Similarly to LPS, ARA-LAM upregulated CD14 surface expression after restimulation. Recurrent MAN-LAM treatment either downmodulated or did not induce any change in CD14 expression. The role of MAN-LAM regulated cytokine secretion as well as implications regarding M. tuberculosis infection will be discussed.     

Ultrastructural immunocytochemical localization of lysozyme in human monocytes and macrophages

Summary In order to investigate the mechanism of synthesis and secretion of lysozyme (LZ) by human mononuclear phagocytes, the ultrastructural localization of LZ was studied by a pre-embedding direct immunoperoxidase method. Blood monocytes showed a reaction product for LZ in cytoplasmic granules, whereas cultured monocytes showed the reaction product in phagosomes as well as granules at 5 h of culture and in numerous large granules at 3 days of culture. In Kupffer cells, LZ was present in cytoplasmic granules, vacuoles and phagosomes. Some Kupffer cells showed a positive reaction for LZ in the rough endoplasmic reticulum, perinuclear cisterna and Golgi apparatus. Macrophages in the lymph nodes contained LZ in cytoplasmic granules. Bone marrow macrophages contained numerous phagosomes with electron-dense degradation products of erythrocytes, but the reaction product for LZ could not be clearly identified. The present study demonstrated that LZ is present in the granules of human mononuclear phagocytes and released into phagosomes. An in-vitro culture study, furthermore, demonstrated that macrophages produce LZ-containing large granules distinct from those of monocytes. However, findings that indicate the synthesis and secretion of LZ by cultured monocytes, as suggested previously by other investigators, were not observed in this study.     

TNF mediates the sustained activation of Nrf2 in human monocytes

Modulation of monocyte function is a critical factor in the resolution of inflammatory responses. This role is mediated mainly by the production of TNF-α. Investigations of the actions of TNF have mostly focused on acute activation of other cell types such as fibroblasts and endothelial cells. Less is known about the effects of TNF on monocytes themselves, and little is known about the regulation of cell responses to TNF beyond the activation of NF-κB. In this study, we investigated the regulation of NF-E2-related factor 2 (Nrf2) cyctoprotective responses to TNF in human monocytes. We found that in monocytes TNF induces sustained Nrf2 activation and Nrf2 cytoprotective gene induction in a TNFR1-dependent manner. Under TNF activation, monocytes increased their expression of Nrf2-dependent genes, including NAD(P)H:quinone oxidoreductase 1 and glutamyl cysteine ligase modulatory, but not heme oxygenase-1. We also showed that autocrine TNF secretion was responsible for this sustained Nrf2 response and that Nrf2 activation by TNF was mediated by the generation of reactive oxygen species. Moreover, we showed that Nrf2-mediated gene induction can modulate TNF-induced NF-κB activation. These results show for the first time, to our knowledge, that TNF modulates prolonged Nrf2-induced gene expression, which in turn regulates TNF-induced inflammatory responses.     

Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy

Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-kappaB-dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4(+) T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.     

Overexpressed human mitochondrial thioredoxin confers resistance to oxidant-induced apoptosis in human osteosarcoma cells

Oxidative damage to mitochondria is a central mechanism of apoptosis induced by many toxic chemicals. Thioredoxin family proteins share a conserved Cys-X-X-Cys motif at their active center and play important roles in control of cellular redox state and protection against oxidative damage. In addition to the well studied cytosolic and extracellular form (Trx1), rat and avian mitochondrial forms of thioredoxin (mtTrx) have been reported. In this study, we cloned the full-length human mtTrx cDNA and performed localization and functional studies in 143B human osteosarcoma cells. The coding sequence of human mtTrx consists of a region with homology to Trx1 as well as a putative mitochondrial localization signal (MLS) at its N terminus. In stably transfected cell lines, mtTrx had a mitochondrial localization as measured by subcellular fractionation studies and by confocal fluorescence microscopy. Deletion of the MLS rendered mtTrx to be solely expressed in the cytosolic fraction. On SDS-PAGE, transfected mtTrx had the same apparent molecular weight as the MLS truncated form, indicating that the leader sequence is cleaved during or after mitochondrial import. Treatment with the oxidant tert-butylhydroperoxide induced apoptosis in 143B cells. This oxidant-induced apoptosis was inhibited by overexpressing the full-length mtTrx in 143B cells. Thus, human mtTrx is a member of the thioredoxin family of proteins localized to mitochondria and may play important roles in protection against oxidant-induced apoptosis.     

Effect of different tumor necrosis factor (TNF) reactive agents on reverse signaling of membrane integrated TNF in monocytes

Reverse signaling of transmembrane TNF (mTNF) contributes to the versatility of this cytokine superfamily. Previously, we could demonstrate that mTNF acting as receptor confers resistance to bacterial lipopolysaccharide in monocytes and macrophages (MO/MPhi). Reverse signaling can be induced by incubation with the monoclonal anti-TNF antibody 195F and other TNF antagonists, such as the humanized monoclonal antibody infliximab and the humanized soluble TNF receptor construct etanercept, respectively, all in former or present clinical use. Here, we addressed the question whether there are differences in modulating the LPS response in MO/MPhi among these three antagonists. Whereas 195F and infliximab suppress both, the release of an LPS-induced endothelial cell apoptotic factor and proinflammatory cytokines, etanercept only protected against the LPS-triggered apoptosis activity, but left the LPS-induced cytokine release unchanged. These data could have clinical impact with regard to TNF neutralization strategies.