Direct Activation of Guinea Pig Macrophages by a B-Cell Mitogen

Since polyclonal B-cell activators (PBA) require macrophages to induce modifications in the lymphocyte in vitro primary response to thymus-dependent antigens, we have investigated whether PBA act directly on macrophages. [14C]glucosamine uptake by guinea pig peritoneal adherent cells after stimulation with purified protein derivative (PPD), lipopolysaccharide (LPS), and dextran sulfate (DxS) was tested. PPD produced an increased [14C]glucosamine uptake, whereas LPS and DxS did not. According to our experiments, (a) PPD does not require the presence of lymphocytes to stimulate macrophages; furthermore, when lymphocytes were present in a concentration higher than 5%, a suppressor effect in the glucosamine uptake was found, and (b) there was no significant difference between the findings when peritoneal adherent cells were cultured in normal medium and in supernatant of lymphocyte cultures stimulated with PPD.     

Strain-Dependent Activation of Monocytes and Inflammatory Macrophages by Lipopolysaccharide of Porphyromonas gingivalis

Porphyromonas gingivalis is one of the pathogens associated with periodontal diseases, and its lipopolysaccharide (LPS) has been suggested as a possible virulence factor, acting by stimulation of host cells to secrete proinflammatory mediators. However, recent studies have shown that P. gingivalis LPS inhibited some components of the inflammatory response. The present study was designed to test the hypothesis that there are strain-dependent variations in the ability of P. gingivalis LPS to elicit the host inflammatory response. By using LPS preparations from two strains of P. gingivalis, W50 and A7346, the responses of mouse macrophages and human monocytes were evaluated by measuring the secretion of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α). Both direct and indirect (priming) effects were investigated. LPS from Salmonella typhosa was used as a reference LPS. P. gingivalis A7436 LPS induced lower secreted levels of NO from the tested cells than S. typhosa LPS but induced similar levels of TNF-α. In contrast, LPS from P. gingivalis W50 did not induce NO or TNF-α secretion. Preincubation of macrophages with LPS from S. typhosa or P. gingivalis A7436 prior to stimulation with S. typhosa LPS upregulated NO secretion and downregulated TNF-α secretion, while preincubation with P. gingivalis W50 LPS enhanced both TNF-α and NO secretory responses. These results demonstrate that LPSs derived from different strains of P. gingivalis vary in their biological activities in vitro. The findings may have an impact on our understanding of the range of P. gingivalis virulence in vivo.     

Molecular Basis of B-Cell Activation

The present experiments were performed in an attempt to investigate the nature of the surface receptor on B lymphocytes responsible for triggering of these cells. B-cell mitogenicity of unsubstituted dextrans, quantitated by activation of DNA and antibody synthesis was detected only if the ligand had a MVC higher than 7 × 10⁴. Above this threshold mitogenicity increased linearly with the log of the MW Substitution of the polymeric structure with lipid residues did not result in increased mitogenicity of the conjugate. However, sulphate substitution of the sugar units greatly enhanced the ability of the conjugates to activate DNA synthesis and. to a much smaller extent, antibody synthesis. Mitogenicity of sulphate derivatives was independent of their MW. Another polyanionic derivative (carboxymethyl) did not show increased mitogenicity. whereas a low-MW compound very similar to dextran sulphate (pentosan sulphate) was highly active. The activation induced by dextrans was immunologically nonspecific and caused induction of polyclonal antibody synthesis. The activated cells presumably belong to a subset of B cells at a rather premature stage of differentiation. These findings suggest that the mitogenic signal is delivered to the cells by single sites at the membrane. These sites appear to have the capacity to interact with polysaccharide structures or releated conformations The polymeric structure of the active ligands is not a necessary requirement for mitogenicity and seems to have the accessory function of providing multipoint binding to low-affinity receptors.     

Mechanism of B-Cell Activation and Paralysis by Thymus-Independent Antigens

Normal spleen cells showed a bell-shaped dose response profile when stimulated in vitro with die thymus independent antigen (4 -hydroxy-3,5 dinitiophenyl)acetyl (NNP)-lipopolysaccharide (LPS) with regard to the development of high-avidity plaque-forming cells to NNP The addition of suboptimal concentrations of LPS to cultures stimulated by suboptimal concentrations of NNP LPS resulted in optimal induction of B cells in that affinity fraction Addition of LPS to cultures optimally stimulated by NNP-LPS resulted in paralysis of the specific cells These results are interpreted in terms of the additive effects between the mitogenicity LPS and the mitogenicity of NNP LPS the latter being selectively focused on the specific cells, thus providing further evidence for the 'one nonspecific signal' hypothesis for immune activation of B cells     

Secretory Leukocyte Protease Inhibitor Interferes with Uptake of Lipopolysaccharide by Macrophages

Macrophages are among the most sensitive targets of bacterial endotoxin (LPS), responding to minute amounts of LPS by releasing a battery of inflammatory mediators. Transfection of macrophages with secretory leukocyte protease inhibitor (SLPI) renders these cells refractory to LPS stimulation. Here we show that uptake of LPS from soluble CD14 (sCD14)-LPS complexes by SLPI-overexpressing cells was only 50% of that seen in control cells. SLPI transfectants and mock transfectants did not differ in the surface expression of CD14 or CD18. We show, in addition, that recombinant human SLPI can bind to purified endotoxin in vitro. SLPI caused a decrease in the binding of LPS to sCD14 as assessed both by fluorescence quenching of labeled LPS and by nondenaturing polyacrylamide gel electrophoresis. These results suggest that the inhibitory effect of SLPI on macrophage responses to LPS may, in part, be due to its blockade of LPS transfer to soluble CD14 and its interference with uptake of LPS from LPS-sCD14 complexes by macrophages.     

Ovalbumin-Specific Human B-Cell Activation and Maturation

By means of a panel of monoclonal antibodies it is demonstrated that, in cultures of human peripheral blood mononuclear cells (PBMC) with the T-cell-dependent (TD) antigen ovalbumin (OA), responding B cells are activated from the resting state. The differentiation of the activated B cells to high rate-secreting plasma blasts, however, is arrested in an early activation phase, in which they can be detected as low rate-secreting plaque-forming cells. The arrest does not occur when stimulation with OA occurs in the presence of antigen-nonspecific activation and maturation factors, which are provided in the culture by the anamnestic response to the TD antigen tetanus toxoid.     

Activation of Macrophages by in Vitro Allostimulated T Cells

Murine peritoneal macrophages, parabiotically co-cultured with combinations of in vitro H-2 sensitized thymus-derived lymphocytes obtained from drug-pretreated mice, possessed an increased cytotoxicity against alloantibody-coated target cells. This heightened activity appeared to be accentuated by and dependent on T-cell synergy. After 5 days of in vitro allosensitization at 37 degrees C, cortisone-resistant thymocytes allosensitized in combination with cyclophosphamide-pretreated splenic T cells released molecules that produced strong antibody-dependent macrophage-mediated cytotoxicity (ADCC). This enhanced ADCC correlated with increased macrophage rosetting with IgG-sensitized erythrocytes. These heightened activities resulted from soluble mediators released by the activated T cells which diffused across a 0.22-microns Millipore filter and were not dependent on lymphocyte-macrophage contact. Evidence that these molecules originated from the highly enriched T-cell populations and were not synthesized de novo by macrophages was supported by results of pretreatment with protein and RNA synthesis inhibitors. Evidence that soluble Fc receptors released from the alloactivated T cells were responsible for the increased macrophage EA binding and ADCC was obtained in affinity chromatography experiments in which activity could be depleted by passage over a Sepharose-Fc-coupled column and recovered in the column eluate.     

Differential Recovery of Macrophages from Endotoxin-Tolerant States Elicited by Lipopolysaccharide and Enzymatic Treatments

Exposure of macrophages to endotoxin (lipopolysaccharide, LPS) leads to a suppression of their capacity to bind LPS and to produce cytokines after reexposure to LPS. This phenomenon is termed endotoxin tolerance, or LPS-induced desensitization. LPS also stimulates the secretion of serine proteases in macrophages, and activates membrane phospholipases. We have investigated the role of trypsin (a serine protease) and of a phosphatidylinositol-specific phospholipase C (PI-PLC, which cleaves GPI-anchored molecules such as CD14), on LPS-induced desensitization. The results obtained by treatment with PI-PLC or in the presence of protease inhibitors, suggested that activation of phospholipases and proteases are not involved in LPS-induced desensitization. However, trypsin treatment of macrophages abolished both LPS binding and cytokine responses. The recovery of macrophages from this trypsin-induced tolerance (restoration of TNF-α synthesis without reexpression of LPS-binding sites) was very different from that following LPS-induced tolerance (reexpression of LPS-binding sites without restoration of TNF-α synthesis). The results are consistent with the hypothesis that signaling LPS-receptors might be synthesized de novo after trypsin degradation, whereas non-signaling LPS-receptors might be internalized and recycled after preexposure to LPS.     

The Role of Gamma Interferon in B-Cell Activation

This study evaluates the role of gamma interferon in in vitro B-cell activation. Two clones of an alloreactive helper T-cell line were equally effective in activating B cells polyclonally to proliferate and to mature to immunoglobulin secretion. One of these clones produced high levels of gamma interferon, while the other clone did not. From these data we conclude that gamma interferon plays no limiting role in T-dependent B-cell activation.     

Lipopolysaccharide Accelerates Neuropilin-1 Protein Degradation by Activating the Large GTPase Dynamin-1 in Macrophages

Inflammation - Neuropilin-1 (Nrp1) is highly expressed in macrophages and plays a critical role in acute and chronic inflammation-associated diseases, such as sepsis, type II diabetes, and...     

Induction of Adrenomedullin mRNA and Protein by Lipopolysaccharide and Paclitaxel (Taxol) in Murine Macrophages

Lipopolysaccharide (LPS), a potent inflammatory stimulus derived from the outer membrane of gram-negative bacteria, has been implicated in septic shock. Plasma levels of adrenomedullin (AM), a potent vasorelaxant, are increased in septic shock and possibly contribute to the characteristic hypotension. As macrophages play a central role in the host response to LPS, we studied AM production by LPS-stimulated macrophages. When peritoneal exudate macrophages from C3H/OuJ mice were treated with protein-free LPS (100 ng/ml) or the LPS mimetic paclitaxel (Taxol; 35 μM), an ~10-fold increase in steady-state AM mRNA levels was observed, which peaked between 2 and 4 h. A three- to fourfold maximum increase in the levels of immunoreactive AM protein was detected after 6 to 8 h of stimulation. While LPS-hyporesponsive C3H/HeJ macrophages failed to respond to protein-free LPS with an increase in steady-state AM mRNA levels, increased levels were observed after stimulation of these cells with a protein-rich (butanol-extracted) LPS preparation. In addition, increased AM mRNA was observed following treatment of either C3H/OuJ or C3H/HeJ macrophages with soluble Toxoplasma gondii tachyzoite antigen or the synthetic flavone analog 5,6-dimethylxanthenone-4-acetic acid. Gamma interferon also stimulated C3H/OuJ macrophages to express increased AM mRNA levels yet was inhibitory in the presence of LPS or paclitaxel. In vivo, mice challenged intraperitoneally with 25 μg of LPS exhibited increased AM mRNA levels in the lungs, liver, and spleen; the greatest increase (>50-fold) was observed in the liver and lungs. Thus, AM is produced, by murine macrophages, and furthermore, LPS induces AM mRNA in vivo in a number of tissues. These data support a possible role for AM in the pathophysiology of sepsis and septic shock.     

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages

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CD14 Expression and Binding of Lipopolysaccharide to Alveolar Macrophages and Monocytes

We have studied the expression of the lipopolysaccharide (LPS) receptor CD14 on monocytes (Mo) and alveolar macrophages (AM), including density- and size-defined subpopulations. Bronchoalveolar lavage (BAL) was performed on eleven healthy non-smokers and blood sampled from 5 of them, and the levels of cell CD14 expression was investigated using flow cytometry. The influence of LPS stimulation on the CD14 expression of AM was studied at various intervals during prolonged incubation. Further, the relationship between CD14 expression and LPS binding to Mo and subpopulations of AM was studied by measuring fluorescein isothiocyanate (FITC)-LPS binding (flow cytometry) and binding of radioiodinated LPS (^I25I-LPS). The CD14 expression was 13-fold higher (P < 0.02) on Mo than on unfractionated and high density AM. The CD14 level on the latter was higher than on low density AM, and also higher (P < 0.05) on small AM compared to large (flow cytometrically defined) AM. LPS stimulation had a downregulating effect on AM CD14 level, but after several hours of continuing decreased expression, an increased (P < 0.05) CD14 expression was demonstrated, indicating de novo synthesis. The binding of LPS to subpopulations of AM and isolated Mo was not significantly different, but the binding of FITC-LPS to Mo in whole blood was higher than to AM (P < 0.02). The presented results indicate that AM of different size and maturity have different and variable (activation dependent) CD14 levels. The LPS binding capacity was, however, not proportional to the CD14 expression, indicating that LPS binding mechanisms unrelated to CD14 levels were also operable.     

Cell Surface Structures Required for B-Cell Activation with Haptenated Syngeneic Cells

The requirement for H-2-coded antigens on the cell surface of stimulator cells used for induction of B-cell responsiveness against haptenated (fluorescein isothiocyanate (FITC)) syngeneic cells was studied using H-2-less F9 teratocarcinoma cell lines. I found that FITC-labelled F9 cells, in contrast to normal spleen cells, could not induce hapten-specific antibody synthesis. The effect of treatment of stimulator cells with glutaraldehyde or trypsin before or after hapten labelling was also analysed. It was found that regardless of the order of treatment, hapten-specific antibody synthesis could not be induced by cells treated with glutaraldehyde or trypsin. In addition, hapten-specific B-cell unresponsiveness could not be induced by FITC-labelled glutaraldehyde-treated syngeneic lymphocytes. However, cold targets treated with trypsin or glutaraldehyde efficiently blocked T-cell-mediated cytotoxicity. The requirement for H-2-coded antigens for B-cell activation against haptenated syngeneic lymphocytes is discussed.