Polyanions Inhibit Murine Macrophage Fc Receptor Mediated ADCC and Binding

We examined the effect of various polyanions on mouse complement hemolytic activity, Fc receptor (FcR) subclass mediated antibody-dependent cellular cytotoxicity (ADCC) as well as binding by mouse peritoneal macrophages (MΦ) of sheep erythrocyte targets. All the polyanions tested (dextran sulfate, carrageenan, polyvinyl sulfate, pentosan polysulfate and polyanethol sulfonic acid) inhibited the hemolytic activity of mouse serum complement to varying degrees. Polyanions inhibited ADCC mediated by either IgG2a or IgG2b in a reversible manner. FcR subclass mediated binding studies at 4°C indicated that the various polyanions compete for FcR binding of sheep erythrocytes opsonized with murine IgG2a, IgG2b and polyclonal IgG. Polyanethol sulfonic acid was uniformly the most potent inhibitor of mouse CH50 and FcR dependent ADCC and binding functions, but: did not affect C3b receptor mediated binding.     

IgA Binding Factors and Fc Receptors for IgA: Comparative Studies Between IgA and IgE Fc Receptor Systems

The expression of Fc receptors (FcR) for IgA (Fc_αR) as well as for IgE (Fc_?R) on T lymphocytes (T cells) is enhanced or up regulated by the corresponding class of immunoglobulins (Ig). The production of class-specific regulatory factors binding to IgA and IgE (IgA binding factor [IgA-BF]; IgE binding factor [IgE-BF]) is also induced by these respective ligands.

Murine IgA-BFs produced by a T hybridoma T2D4 and concanavalin A-activated spleen cells suppressed the in vitro IgA antibody responses of pokeweed mitogen-stimulated mouse spleen cells class-specifically. Human IgA antibody response was also suppressed by the murine IgA-BF. Similar suppressive IgA-BF is also produced by a human natural killer (NK)-like cell line (YT), which has no rearrangement of the T cell receptor beta-chain gene, indicating that non-T non-B/LGL cells may also be involved in the regulation of the class-specific antibody responses. It appears that, in human as well as murine systems, T-and NK-cells have the capacity to co-express multiple class-specific FcRs and to produce the corresponding immunoglobulin binding factors.

While the Fc_?R expression is abnormally enhanced in the diseases with hyperimmuno-globulinemia E, disregulation of Fc_αR is associated with certain human diseases involving the altered IgA regulation. In IgA nephropathy, which is characterized by increased serum IgA level and IgA deposition in the mesangium, there is an enhancement of the expression of Fc_aR. In contrast, IgA failed to induce Fc_αR significantly on the lymphocytes from the patients with selective IgA deficiency, indicating that Fc_αR plays an important role in the IgA regulation in vivo.     

Comparison of the Binding of Radiolabelled Human IgG and Fc Fragments to Murine Spleen Cells

The binding properties of an IgG1 human myeloma protein, normal IgG, and the Fc and Fab fragments of each were compared in cultures of murine spleen cells. Both 125I-labelled IgG and Fc fragments bound to splenic lymphocytes, whereas Fab fragments did not bind significantly at the highest concentrations tested. On a molar basis, more Fc bound than intact IgG. According to Scatchard plot analysis, the affinity constand of IgG1 was 1.5 x 10(6) +/- 1 x 10(5) L/M and that of the Fc fragments was 7.8 x 10(5) +/- 2.6 x 10(5) L/M. Approximately 25,000 binding sites/cell were calculated for IgG1 and 102,000 for Fc. Deaggregation of the Fc preparation did not change these values, suggesting that the difference in binding of IgG and Fc did not result from Fc aggregation. Unlabelled IgG inhibited about 25% of the labelled Fc binding, whereas unlabelled Fc inhibited approximately 80% of the labelled Fc binding. IgG antigen-antibody complexes, however, inhibited 75% of the Fc binding. In the reciprocal experiment both intact IgG and Fc inhibited the binding of labelled IgG by 100%. The major cell population that bound IgG and Fc fragments in the spleen cell preparation were the B lymphocytes. Removal of macrophages did not significantly affect the binding of labelled Fc fragments. In addition, T-cell-enriched populations bound an insignificant quantity of Fc fragments.     

PWM依赖的小鼠腹腔巨噬细胞介导的细胞毒作用的研究

本文采用间接MTT法,对凝集素美洲商陆(PWM)依赖的小鼠腹腔巨噬细胞(PEMφ)介导的细胞毒作用(PDMC)进行检测分析,发现凝集素(PWM、ConA)预处理靶细胞(Raji、U_(14)),对细胞毒作用的影响视凝集素、靶细胞的不同而异;在PWM分别预处理效、靶细胞的实验中,PWM对Raji靶细胞的作用显更重要;在PWM直接作用下,其PDMC活性可进一步提高,后者可为GlcNAc有效地抑制.观察PDMC中Raji靶细胞的形态学变化,发现坏死与凋亡现象并存.     

Mycobacterium leprae-Induced Alterations in Macrophage Fc Receptor Expression and Monocyte-Lymphocyte Interaction in Familial Contacts of Leprosy Patients

Macrophage Fc receptor expression and monocyte-lymphocyte interaction in the presence of Mycobacterium leprae were examined in familial contacts of leprosy patients. Defective Mφ functions similar to those of borderline and lepromatous patients could he observed in approximately 71% of consanguineous contacts and 43% of of spouses of index patients. Although the values in the latter group were markedly lower than those of the consanguineous contacts, they tended to be higher than those of normal individuals (20%). These in vitro Mφ functions were independent of age, sex, and age at onset of exposure and were only weakly associated with duration of exposure. The outcome of the monocyte-lymphocyte interaction lest paralleled to a large extent the in vivo Mitsuda lepromin response. Four contacts with defective Mφ functions also showed signs of leprosy. The value of these in vitro tests as markers of 'susceptibility' could therefore prove significant.     

A Tumor Necrosis Factor Mimetic Peptide Activates a Murine Macrophage Cell Line To Inhibit Mycobacterial Growth in a Nitric Oxide-Dependent Fashion

The control of mycobacterial infections depends on the cytokine-mediated activation of mononuclear phagocytes to inhibit the growth of intracellular mycobacteria. Optimal activation requires the presence of T-cell-derived gamma interferon (IFN-γ) and other signals, including tumor necrosis factor (TNF). Recently, an 11-mer peptide based on amino acids 70 to 80 of the human TNF sequence, TNF_(70-80), was found to have TNF mimetic properties, which include the activation of human and mouse neutrophils to kill Plasmodia spp. Therefore, we investigated the capacity of TNF_(70-80) to activate the murine macrophage cell line RAW264.7 infected with the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin (BCG). When RAW264.7 cells were pretreated with human TNF or TNF_(70-80) in the presence of IFN-γ, there was a dose-dependent reduction in the replication of BCG as measured by the uptake of ³H-labeled uracil and a concomitant release of nitric oxide as measured by the nitrite in the culture supernatants. TNF- or TNF_(70-80)-induced macrophage activation was dependent on IFN-γ and was inhibited by neutralizing monoclonal antibody to human TNF and by anti-IFN-γ antisera. Both nitrite release and BCG growth inhibition were abrogated by competitive inhibitors of l-arginine, which blocked the activation of inducible nitric oxide synthase. A soluble form of the Type 1 TNF receptor blocked the activation of BCG-infected macrophages by human TNF and TNF_(70-80), demonstrating that the effect of TNF_(70-80) is dependent on signaling through TNF receptor I. The mimetic effects of TNF_(70-80) on macrophage activation in vitro suggest that treatment with TNF_(70-80) may modulate mycobacterial infections in vivo.Mycobacteria are intracellular parasites which replicate within the shielded environment of monocyte-derived tissue macrophages. Activation of antibacterial killing mechanisms within these cells by cytokines is essential for the control of mycobacterial infections (24). Gamma interferon (IFN-γ) plays a central role in this since it is produced by a variety of lymphocytes responding to mycobacterial infections, including CD4⁺ and CD8⁺ αβ T cells and γδ T cells. Administration of recombinant IFN-γ protects mice against lethal Mycobacterium tuberculosis infection in some but not all experimental models (13, 20), whereas neutralization with anti-IFN-γ antibodies exacerbates the infection (13). The failure of mice deficient in IFN-γ or IFN-γ receptors to control M. tuberculosis infection confirms that this cytokine is essential for killing M. tuberculosis (12, 20). Studies with human and murine macrophages, however, have demonstrated that additional signals are required to fully activate mycobacterial killing (24). Potential activators include other cytokines, such as tumor necrosis factor (TNF), interleukin-4 (IL-4), IL-6, and granulocyte-macrophage colony-stimulating factor (15, 18, 19), and in humans 1,25-dihydroxy-vitamin D₃, the biologically active form of vitamin D₃ (14). TNF alone cannot activate macrophages sufficiently to kill mycobacteria, but it does synergize with IFN-γ to increase the antimycobacterial activity of infected macrophages in vitro (18). Administration of anti-TNF antibodies decreases the resistance of mice to infection with M. bovis bacillus Calmette-Guérin (BCG) (25) and M. tuberculosis (21). TNF is a necessary requirement for effective antimycobacterial immunity, since mice deficient in the 55-kDa TNF receptor I (TNFRI) develop progressive M. tuberculosis infection (21). The protective effects of TNF and the lethal consequences of anti-TNF antibodies have been observed in other models of intracellular bacterial infection, including infections by Listeria monocytogenes, Salmonella typhimurium, and Legionella spp. (37). Although in mycobacterial infections, such as leprosy, high levels of TNF have been associated with tissue damage and systemic toxicity, local TNF synthesis is essential for the control of mycobacterial infections (35).Studies with neutralizing anti-human TNF monoclonal antibodies (MAb) demonstrated that the sequence from amino acids 65 to 85 of the TNF molecule was involved in binding to the TNF receptor (32). By use of truncated peptides, amino acids 70 to 80 were identified as essential for TNF activity (33). When this peptide sequence was modified by substitution of leucine-76 for isoleucine, the subsequent peptide TNF_(70-80) had increased stability in vitro in the presence of serum (32a) and possessed TNF mimetic properties both in vitro and in vivo (27). TNF_(70-80) stimulated a reactive oxygen burst in human and murine neutrophils (27) and activated human neutrophils to kill Plasmodium falciparum (27). In a murine model of Plasmodium chabaudi infection, systemic therapy with TNF_(70-80) increased the rate of recovery and clearance of parasites (27). More recently, TNF_(70-80) was found to reduce the weight loss and systemic effects in mice chronically infected with Pseudomonas aeruginosa (32a). The demonstrated properties of TNF_(70-80) and the known requirement of TNF for activating macrophages led us to examine whether this mimetic peptide would have antimycobacterial activity on a murine macrophage cell line. We now report that TNF_(70-80) synergizes with IFN-γ to activate murine macrophages to inhibit the growth of M. bovis BCG and that this property is dependent on its activation of inducible nitric oxide synthase (iNOS).     

鼠腹腔巨噬细胞诱生TNF的条件探讨

本文研究了用鼠腹腔巨噬细胞诱生TNF的适合条件。结果显示LFS刺激8～30h内TNF的产量最高,5～10μg/ml的LPS为合适的刺激剂量;较好的巨噬细胞浓度在2×10~6/ml左右和静止的腹腔定居巨噬细胞不能产生明显的TNF。特别引人注意的是有效的TNF诱生有赖于巨噬细胞培养中LPS的持续存在以及培养液中的小牛血清对TNF的产生有明显的抑制作用。本实验为进一步研究TNF及巨噬细胞的免疫学提供了有用的数据。     

Soluble Epoxide Hydrolase Inhibitor Suppresses the Expression of Triggering Receptor Expressed on Myeloid Cells-1 by Inhibiting NF-kB Activation in Murine Macrophage

Triggering receptors expressed on myeloid cell-1 (TREM-1) is a superimmunoglobulin receptor expressed on myeloid cells. TREM-1 amplifies the inflammatory response. Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 enzyme, have anti-inflammatory properties. However, the effects of EETs on TREM-1 expression under inflammatory stimulation remain unclear. Therefore, inhibition of soluble epoxide hydrolase (sEH) with a highly selective inhibitor [1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea, TPPU] was used to stabilize EETs. LPS was intratracheally injected into mice to induce pulmonary inflammation, after TPPU treatment for 3 h. Histological examination showed TPPU treatment-alleviated LPS-induced pulmonary inflammation. TPPU decreased TREM-1 expression, but not DAP12 or MyD88 expression. Murine peritoneal macrophages were challenged with LPS in vitro. We found that TPPU reduced LPS-induced TREM-1 expression in a dose-dependent manner, but not DAP12 or MyD88 expression. TPPU also decreased downstream signal from TREM-1, reducing pro-inflammatory cytokine TNF-α and IL-1β mRNA expression. Furthermore, TPPU treatment inhibited IkB degradation in vivo and in vitro. Our results indicate that the inhibition of sEH suppresses LPS-induced TREM-1 expression and inflammation via inhibiting NF-kB activation in murine macrophage.     

Influence of Intestinal Microflora on Murine Bone Marrow and Spleen Macrophage Precursors

To investigate the adjuvant effect of intestinal flora on macrophage–colony-stimulating factor-responsive macrophage progenitors from spleen and bone marrow, we compared progenitor numbers and phenotypic characteristics of in vitro matured macrophages in germ-free and flora-associated mice (conventional, Escherichia coli -monoassociated and conventionalized mice). The data obtained show that the flora affected differentially bone marrow and spleen progenitors. It increased the numbers of progenitors in the spleen but not in the bone marrow. It did not modify the expression of F4/80, Mac-1 and major histocompatibility complex (MHC) class II on bone-marrow-derived macrophages (BMDM), while it clearly up-regulated MHC class II expression on spleen-derived macrophages (SDM). This effect was more pronounced in flora-associated ex germ-free mice than in conventional mice and it was greatly enhanced in the absence of M-CSF. In vitro stimulation by lipopolysaccharide had no effect on marker expression of BMDM, while it decreased F4/80 and enhanced MHC class II molecules on SDM from germ-free and flora-associated mice. However, the expression of MHC class II remained lower in germ-free mice. Enhancement of MHC class II molecule expression on SDM may contribute to the protective role of flora, because successful immune responses are dependent on the expression of these molecules.     

Identification of an Oolemmal IgG Fc Receptor: Its Role in Promoting Binding of Antibody-Labelled Human Sperm to Zona-Free Hamster Eggs

ABSTRACT: Antisperm antibodies (ASAs) present in sera of infertile men and women have been shown either to promote or inhibit penetration of zona-free hamster eggs by antibody-labelled human spermatozoa. Increased numbers of oolemmal-bound sperm have been noted in association with increased sperm penetration frequencies, following antibody labelling, when compared with antibody-free sperm. The promotion of adherence of ASA-labelled sperm to the oolemma could be mediated through the binding of antibodies to common epitopes present on the sperm and egg surfaces or through Fc-mediated binding to an oolemmal Fc receptor. In support of the latter hypothesis, we report that zona-free hamster eggs bind aggregated human IgG and IgG Fc fragments. The presence of an oolemmal IgG Fc receptor has been confirmed using a rat monoclonal antibody (2.4G2) directed against a murine IgG Fc receptor (Fc γ R_II) as judged both by indirect immunofluorescence and by immunobead binding. In addition, the pre-incubation of zona-free hamster eggs with IgG Fc diminished both adhesion to and penetration of the oolemma by human spermatozoa.     

Immunomodulation of RAW 264.7 Murine Macrophage Functions and Antioxidant Activities of 11 Plant Extracts

A group of 11 medicinal plants, including Lavandula pubescens, Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Silene succulenta, Silene villosa, Bogonvillea glabra, Cakile maritime, Gomphrene celesoids, Mirabilis jalaba, and Silene nocturna growing in Egypt, were extracted and examined for their immunomodulatory and antioxidant activities. RAW 264.7 cells were recruited to investigate the immunomodulatory effect through multiple parameters analysis. First, the proliferation index of macrophages cells was evaluated revealing that Trigonella foenugricium, Silene succulenta and Silene villosa have a significant cytotoxic effect on RAW cells. Interestingly, we observed enhancement of macrophages phagocytic function of by all extracts except Cakile maritime, Gomphrena celosioides and Silene nocturna. Afterwards, macrophages were challenged by incubation with LPS and the effect of various extracts on inflammatory responses was investigated; the generation of NO from activated macrophage was substantially suppressed by 7 extracts namely, Trigonella foenugricium, Calligonum comosum, Silene succulenta, Bougainvillea glabra, Mirabilis jalaba, Gomphrena celosioides and Silene nocturna. TNF-α was decreased by percentage range from 3.8 to 85.8% and Trigonella foenugricium extract showed the highest inhibition of TNF-α release. All extracts except Trigonella foenugricium, Salsola schweinforthi, Silene succulenta and Mirabilis jalaba significantly inhibited COX-2 production from stimulated macrophage. Moreover, evaluating the potential antioxidant activity of these extracts showed that Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Bogonvillea glabra and Mirabilis jalaba exhibited some antioxidant activities. Taken together, our results suggest that some of these extracts may have a considerable antinflammatory and antioxidant effects and may be a potential therapeutic choice in the treatment of inflammatory diseases.     

Macrophage Fc gamma 2b receptor expression and receptor-mediated phospholipase activity: regulation by endogenous eicosanoids

The expression of Fc gamma 2b receptors and receptor-mediated arachidonic acid metabolism by murine peritoneal macrophages was examined in vitro. The expression of Fc gamma 2b receptors was found to increase progressively with time in culture and this increase was dependent on protein synthesis and glycosylation. The increase in Fc gamma 2b receptor expression was inhibited by hydrocortisone and by BW755C, an inhibitor of both the lipoxygenase and cyclo-oxygenase pathways of arachidonic acid metabolism. Inhibition by BW755C was found to be reversed in the presence of exogenous leukotriene D4. In contrast, selective inhibition of the cyclo-oxygenase pathway by indomethacin enhanced the increase in receptor expression. This enhancement was only partially reversed by exogenous prostaglandin (PG)E2. Interaction of the Fc gamma 2b receptor with ligand in the form of erythrocytes specifically sensitized with IgG2b resulted in the release and subsequent metabolism of arachidonic acid. PGE2 was found to be the principal product. Occupation of the Fc gamma 2a receptor did not result in arachidonic acid release. Down regulation of Fc gamma 2b receptor expression produced a commensurate reduction in receptor-mediated phospholipase activity measured by arachidonic acid release. Macrophages cultured for 24 h in the presence of fetal calf serum without additional stimuli produced substantial amounts of eicosanoids. PGI2 was the principal product. Taken together these data demonstrate a potential feedback regulation of receptor-triggered arachidonic acid metabolism by eicosanoids acting at the level of Fc gamma 2b receptor expression.     

A Method for the Detection and Quantitation of Fc Receptor Sites on Cells

A new rosette technique for identification of Fc-receptor-bearing cells is based on the ability of sheep erythrocyte coated with protein A of Staphylococcus aureus (ES) to form rosettes with cell treated with monomeric IgG or aggregated IgG. The IgG is attached to lymphocytes through its Fc region and to a trypsin-resistant but pronase sensitive receptor (considered an Fc receptor) The ES rosette technique facilitates studies of the interaction of IgG with Fc receptor sites; the binding of any IgG preparation reacting with protein A of Staphylococcus aureus (SpA) can be studied by the technique. Inhibition of rosette formation by SpA was used for quantitation of IgG fixed to the cell surface (that is, the number of Fc reception/cell). The sensitivity of the method permits quantitation of less than 10⁵ IgG molecules bound to the Fc receptors. The relative affinity constant between Fc receptors and IgG ligands was estimated by plotting the percentage of ES rosettes as a function of IgG concentration and calculating the reciprocal of the IgG concentration giving half the maximal number of ES rosettes.