Immunomodulation of in vitro antigen presentation by cations

The ability of various cations to alter an antigen-specific (ovalbumin) T cell activation system in vitro has been assessed. This in vitro system includes analysis of antigen capture, processing, and presentation by antigen presenting cells (APC) (splenocytes, B cells or macrophages) in a major histocompatibility complex (MHC) restricted fashion and the evaluation of the activation of a T-cell hybridoma, DO-11.10, which is specific for "processed" ovalbumin in association with I-Ad. Activation is determined by production of IL2 which is quantitated in a bioassay with HT-2 cells. Numerous metals (10 and 100 microM) were screened in the coculture assay (APC and DO-11.10 plus ovalbumin). Metals with inhibitory effects were Cd greater than Cu greater than Pb greater than Zn. Co and Cr had no modulatory effect and Ni had an enhancing effect (increased IL2 production). The effects of the modulatory metals were further assessed for influences on the individual cellular components of this system. Cd was toxic to all cell types whereas Cu was toxic only to irradiated splenocytes and Do-11.10. Pb was the only metal which was not toxic to any cell type but still inhibited antigen presentation. To prevent influences of metals on DO-11.10 or carryover into the bioassay, APC were preincubated with metals and then assessed for presentation capability. After preincubation, only Cd, Pb, and Cu were inhibitory. The inhibition by Cd was due to toxicity. Cu inhibited only irradiated splenocyte presentation and this effect was due to toxicity. Pb inhibition was due to a mechanism other than toxicity and its biochemical influences on APC are discussed.     

Inhibitory effect of tannic acid sulfate and related sulfates on infectivity, cytopathic effect, and giant cell formation of human immunodeficiency virus.

The acquired immune deficiency syndrome (AIDS) is thought to result from infection of T cells by a pathogenic human retrovirus, human immunodeficiency virus [HIV (HTLV-III/LAV)]. In this report, we synthesized sulfated plant polyphenols such as tannic acid sulfate, rutin sulfate, ellagic acid sulfate, (-)-epicatechin sulfate, and (-)-epigallocatechin 3-gallate sulfate, and examined the in vitro inhibitory effect on HIV infection using human T-cell lymphotropic virus type-I-carrying MT-4 cells, which are extremely susceptible to HIV infection. Of the compounds tested, tannic acid sulfate was the most effective and had low cytotoxicity. Tannic acid sulfate completely inhibited the cytopathic effect of HIV and the HIV-specific antigen expression in MT-4 cells at the concentration of 6 micrograms/ml. In addition, this sulfate inhibited giant cell formation in coculture at the concentration of 5 micrograms/ml.     

A comparative study of the antigen-specific immune response induced by co-delivery of CpG ODN and antigen using fusion molecules or biodegradable microparticles

CpG ODN are toll-like receptor 9 (TLR9) agonists that can enhance antigen presentation by antigen presenting cells (APCs) such as dendritic cells (DCs). The most potent antigen-specific responses are seen when CpG ODN and the antigen are co-localized in the same APC. CpG ODN-antigen fusion molecules and biodegradable microparticles entrapping CpG ODN and antigen can ensure both components are delivered to the same APC. In this study, we compared the efficacy of the CpG-ODN fusion molecules against biodegradable microparticles entrapping antigen and CpG ODN. Microparticles were prepared using a double emulsion solvent evaporation methodology. CpG ODN-OVA fusion molecules were prepared by mixing maleimide-activated protein with thiolated CpG ODN. Both CpG ODN-OVA fusion molecules and microparticles co-entrapping CpG ODN and OVA generated stronger IgG2a and interferon-gamma (IFN-gamma) responses than delivery of soluble CpG ODN and OVA. The microparticles generated stronger IgG2a and IFN-gamma immune responses than did CpG ODN-antigen fusion molecules.     

Structure-immune response relationships of hapten-modified collagen II peptides in a T-cell model of allergic contact dermatitis

Allergic contact dermatitis (ACD) is mediated by T cells that specifically recognize hapten-modified peptides. T cells are known to recognize antigens as short processed peptides bound to major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). It has previously been demonstrated that T cells can specifically recognize carbohydrates on the lysine at position 264 of the immunodominant (256-273) sequence from type II collagen (CII) and that such recognition is critical for the development of arthritis in mice and may play a role in rheumatoid arthritis in humans. In the present study, we have used this approach in modeling ACD, but instead of the carbohydrate, the strong sensitizer 2,4-dinitrofluorobenzene (DNFB) is bound to the epsilon-amine of the lysine at position 264. Specific T-cell hybridomas of this antigenic peptide, with dinitrophenyl (Dnp) on the epsilon-amine of lysine at position 264 (CIILysDnp 3), were established from mice immunized with CIILysDnp 3. In an immune response assay, these T-cell hybridomas were tested with a series of new synthetic hapten-modified peptides, all chemically identical except for the stereochemimistry (D, L) and the length of the position-264 amino acid side chain bonding the hapten. The T-cell hybridomas recognized the CIILysDnp 3 peptide used for immunization; interestingly, they also recognized the CII peptide with a one-carbon-longer side chain (homolysine), CIIhLysDnp 6, and CIIAlaPipDnp 11, having a ring structure analogous to that of lysine with the same number of carbons in the bonding chain as in the CIILysDnp 3 peptide used for immunization. Dnp-modified CII peptides with a shorter bonding chain produced no immune response. These data demonstrate that the T-cell recognition of the Dnp-modified peptides is highly specific and moreover dependent on the length of the amino acid side chain that bonds the Dnp.     

Ozone exposure enhances antigen-presenting activity of interstitial lung cells in rats

Ozone (O(3)) as a major component of photochemical air pollutants can increase the levels of allergen-specific antibody and may aggravate allergic symptoms. Antigen presentation is one of the factors contributing to allergic symptoms. Our present study is designed to clarify whether O(3) may increase the antigen-presenting (AP) activity of whole lung cells and its mechanisms. Male Wistar rats were exposed to 1 ppm O(3) for 3 days. The AP activity of whole lung cells and dendritic cells (DC) was measured by proliferation of T-cells. The expression of Ia and costimulatory molecules (B7.1, B7.2, CD11b/c) in lung cells was measured by flow cytometry, and the number of Ia-bearing cells, DC, macrophages, and B-cells in lung interstitum was examined immunohistochemically. The results show that O(3) increases AP activity of whole lung cells and DC, the expression of molecules associated with antigen presentation, and the number of AP cells (APC) in lung. Our results suggest that O(3) may enhance AP activity of lung cells caused by increases in the expression of cell-surface molecules and the number of APC in lung. The increase in the AP activity might contribute to subsequent antibody production, airway hyperresponsiveness and aggravation of allergic responses.     

Anti-CD40 Treatment of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-exposed C57Bl/6 mice induces activation of antigen presenting cells yet fails to overcome TCDD-induced suppression of allograft immunity

We have previously demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the induction of the costimulatory molecule CD86 (B7-2) on B220+ and Mac-1+ spleen cells following the injection of allogeneic P815 tumor cells. In this study, TCDD exposure was shown to suppress CD54 and major histocompatibility complex (MHC) class II expression on B220+, Mac-1+, and CD11c+ splenic antigen presenting cells (APC). Furthermore, interleukin-12 (IL-12) production by spleen cells from P815-immunized mice was significantly decreased following exposure to TCDD. To determine if exogenous costimulation could enhance the activation of APC, vehicle- and TCDD-treated mice were injected with an agonistic antibody to murine CD40. Stimulation with anti-CD40 increased the expression of CD86, CD54, and MHC class II on splenic APC and greatly enhanced the production of interleukin-12. TCDD treatment had minimal effects on the anti-CD40-induced expression of accessory molecules on splenic APC. TCDD exposure had no effect on anti-CD40-induced IL-12 in the plasma but suppressed its production from cultured spleen cells. Surprisingly, although stimulation via CD40 increased the activation of APC, allograft effector functions were not restored in TCDD-treated mice, perhaps due to persistent defects in antigen processing and presentation, cytokine production, T cell function, or CD40-independent pathways of APC activation.     

Class II-targeted antigen is superior to CD40-targeted antigen at stimulating humoral responses in vivo

We examined the efficacy of using monoclonal antibodies to target antigen (avidin) to different surface molecules expressed on antigen presenting cells (APC). In particular, we targeted CD40 to test whether the "adjuvant" properties of CD40 signaling combined with targeted antigen would result in enhanced serologic responses. We targeted avidin to class II as a positive control and to CD11c as a negative control. These surface proteins represent an ensemble of surface molecules that signal upon ligation and that are expressed on professional APC, in particular dendritic cells (DC). We observed that targeting class II molecules on APC was superior to targeting CD40, or CD11c. However, CD40 and CD11c could function as targets for antigen bound monoclonal antibodies under certain conditions. Interestingly, inclusion of anti-CD40 mAb with the targeting anti-class II-targeted antigens negatively affects humoral response, suggesting that CD40 signaling under certain conditions may suppress processing and/or presentation of targeted antigen.     

Nitric oxide and T helper cell immunity

In this article, the controversial role of nitric oxide (NO) in T helper (Th) cell activation and T-cell-dependent immunity will be discussed with an emphasis on immunosuppression by NO. NO is generated by antigen-presenting cells (APC) during the process of antigen presentation to T cells. In mouse models, activation of the inducible NO synthase (iNOS) in APC is triggered by Th1-cell-derived IFN-gamma, in combination with other soluble or membrane-associated T-cell factors. The NO so-produced inhibits T-cell proliferation, while it does not inhibit T cell cytokine production. NO blocks T-cell proliferation during G1/S transition. In mouse models of T-cell-mediated autoimmunity such as myelin antigen-induced EAE, the disease is exacerbated by genetic deletion of iNOS, indicating that NO suppresses T-cell-mediated immunity in vivo. Recent studies reveal that interaction with superoxide diminishes the T-cell regulatory activity of NO. The role for NADPH oxidase as a source for NO-inhibiting superoxide is discussed. In conclusion, NO plays an important regulatory role in the induction phase of T-cell-mediated immunity. Superoxide may enhance T-cell-mediated immunity by preventing the immunosuppressive activity of NO.     

In vivo administration of 15-deoxyspergulin inhibits antigen-presenting cell stimulation of T cells and NF-kappaB activation

15-Deoxyspergulin (DSG), a synthetic derivative of spergulin, was initially characterized for its antibiotic and antitumor effects. Recent studies have described the immunosuppressive properties of this molecule, but its mechanism of action is not clearly understood. In the study reported here, mice were treated in vivo with DSG prior to the measurement of IL-2 production and proliferation in an in vitro antigen presentation assay. At suboptimal antigen concentrations, elicited peritoneal macrophages or percoll isolated B cells from DSG-treated mice showed a 50-96% reduction in their ability to present chicken ovalbumin (cOva), cOva peptide, or superantigen (SAg) to MHC class II-matched antigen-specific primary T cells. No significant changes could be found in the cell surface expression of CD80, CD86, MHC I, MHC II, CD18, CD11b, CD40, CD25, and CD54 in antigen-presenting cells (APC) from control or DSG-treated animals. Activation with SAg of macrophages or splenocytes from DSG-treated mice revealed that there was a significant reduction in nuclear NF-kappaB levels compared to cells from untreated animals. Additionally, analysis of cytokines showed that production of TNF-alpha and IL-1beta was inhibited in cultures where macrophages from DSG-mice were used to present cOva to T cells. These results indicate that the effects of DSG in mice are not simply due to altered antigen processing or from any marked changes in cell surface antigen expression. Rather, the immunosuppression may arise from alterations in the release of one or more soluble factor from DSG-treated APC, which prevents effective antigen presentation and T cell activation.     

Gallium arsenide exposure impairs splenic B cell accessory function

Gallium arsenide (GaAs) is utilized in industries for its semiconductor and optical properties. Chemical exposure of animals systemically suppresses several immune functions. The ability of splenic B cells to activate antigen-specific helper CD4(+) T cell hybridomas was assessed, and various aspects of antigen-presenting cell function were examined. GaAs-exposed murine B cells were impaired in processing intact soluble protein antigens, and the defect was antigen dependent. In contrast, B cells after exposure competently presented peptides to the T cells, which do not require processing. Cell surface expression of major histocompatibility complex (MHC) class II molecules and several costimulatory molecules on splenic B cells, which are critical for helper T cell activation, was not affected by chemical exposure. GaAs exposure also did not influence the stability of MHC class II heterodimers, suggesting that the defect may precede peptide exchange. GaAs-exposed B cells contained a normal level of aspartyl cathepsin activity; however, proteolytic activities of thiol cathepsins B and L were approximately half the control levels. Furthermore, two cleavage fragments of invariant chain, a molecular chaperone of MHC class II molecules, were increased in GaAs-exposed B cells, indicative of defective degradation. Thus, diminished thiol proteolytic activity in B cells may be responsible for their impaired antigen processing and invariant chain degradation, which may contribute to systemic immunosuppression caused by GaAs exposure.     

Syntheses of biotinylated alpha-galactosylceramides and their effects on the immune system and CD1 molecules.

A representative alpha-galactosylceramide (alpha-GalCer), KRN7000, has strong immunostimulatory and antitumor activity. Recent studies demonstrated that KRN7000-pulsed antigen-presenting cells (APC) can activate natural killer T (NKT) cells, a novel T-cell lineage, and CD1d molecules on APC play an important role in the activation of NKT cells. However, it remains unclear whether alpha-GalCers actually bind to CD1d molecules. To address this question, we synthesized three kinds of biotinylated alpha-GalCer and a biotinylated beta-GalCer and found that the biotinylated alpha-GalCers significantly stimulate the proliferation of murine spleen cells, but the biotinylated beta-GalCer does not and that all biotinylated compounds bind to CD1d molecules.     

白细胞介素—10和天花粉蛋白抑制抗原提呈细胞表面分子表达及T—细？…

AIM: To study immunoinhibitory effects and preliminary mechanism of IL-10 and trichosanthin. METHODS: Surface molecule expression on antigen processing cells (APC) was stained with fluorescence and analyzed by FACScan. B7-1 mRNA expression was detected with nested RT-PCR. RESULTS: IL-10 2 mg.L-1 and trichosanthin 10 mg.L-1 inhibited B7-1 molecule expression. By contrast, they had not the same effects on ICAM-1. IL-10 and trichosanthin down-regulated LFA-1 expression, but had no regulatory effect on CD40. IL-10 and trichosanthin dramatically inhibited T-cell proliferation and IL-2 production. B7-1 mRNA expression was undetectable in APC treated with IL-10 and trichosanthin. CONCLUSION: IL-10 and trichosanthin inhibit surface molecule expression on APC. They exert multiple immunoinhibitory effects.     

Effect of polyphenols on enniatins-induced cytotoxic effects in mammalian cells

Enniatins (ENs) are fungal secondary metabolites produced by genus Fusarium. The ENs exert antimicrobial and insecticidal effect, and has also been demonstrated cytotoxic effects on several mammalian cell lines. On the other hands, it has been proved that natural polyphenols have antioxidant effect. In this study, cell effects at low levels of exposure of four ENs (A, A₁, B and B₁) and five polyphenols (quercetin, quercetin-3-β-D-glucoside, rutin, myricetin and t-pterostilbene) present in wine; and the cytoprotective effect of these polyphenols exposed simultaneously with ENs in Chinese Hamster Ovary (CHO-K1) cells, were studied. Cell effects were determined by the MTT test after 24?h of exposure. All ENs showed cytotoxic effect. The IC₅₀ obtained ranged from 4.5?±?1.2 to 11.0?±?2.7 µM. The concentration of polyphenols tested ranged from 5 to 50 µM. Polyphenols did not show cytotoxicity and the cytoprotective effect of polyphenols varies depending on the EN tested. The cytoprotective effect of polyphenols in CHO-K1 cells exposed to ENs was as follow: quercetin, from 24 to 84%; quercetin-3-β-D-glucoside, from 12 to 76%; rutin, from 17 to 83%; myricetin, from 16 to 92% and pterostilbene from 25 to 100%. All polyphenols protected CHO-K1 cells against EN A₁ exposure.     

Advances in cathepsin S inhibitor design

Cathepsin S is expressed in antigen-presenting cells and plays a role in invariant chain processing and major histocompatibility complex class II (MHCII) antigen presentation leading to CD4+ T-cell activation. An oral cathepsin S inhibitor that blocks MHCII antigen presentation could result in a T-cell-selective immunosuppressant agent with improved safety over the current standard of care for the treatment of rheumatoid arthritis, psoriasis, multiple sclerosis and other autoimmune-based inflammatory diseases. This review focuses on advances in cathepsin S inhibitor utility and design since January of 2004.     

Immunosuppressive activity of 4-O-methylascochlorin

A major strategy for suppressing immune responses is the elimination of antigen-reactive lymphocytes through apoptosis. 4-O-Methylascochlorin (MAC) is a methylated derivative of a prenyl-phenol antibiotic, ascochlorin. MAC induces apoptosis in various lymphocyte cell lines. We found that MAC strongly suppressed killer T-cell activity induced by allogenic skin grafts. MAC did not suppress the killer T-cell activity induced by intraperitoneal injection of live allogenic tumor cells bearing both class I and II MHC. MAC suppressed IL-2 production of splenocytes from allogenic skin-implanted mice when induced by specific spleen adherent cells, but not by antibodies for T-cell receptor epsilon. These results suggest that MAC suppresses the antigen presentation process of alloantigen that is mediated by professional antigen presenting cells. MAC significantly increased the survival time of allogenic skin implanted on the flank mice. These results suggest that MAC may be clinically useful as an immunosuppressant that targets the antigen presentation process.     

Elucidation of cellular targets responsible for tetrachlorodibenzo-p-dioxin (TCDD)-induced suppression of antibody responses: II. The role of the T-lymphocyte

Various immunological assays have been applied by our laboratory in an attempt to assess the role of the T-cell in the TCDD-induced suppression of the antibody response by murine B6C3F1 splenic lymphocytes. Animals were treated in vivo (via gavage) with 1.0 microgram/kg TCDD in corn oil for 5 days before in vitro analysis of splenocyte immunocompetence and T-cell function. To study the effects on T-helper cell function, alterations in the proliferative responses of T-cells following TCDD exposure were investigated. Results show no significant difference in [3H]thymidine uptake between vehicle- and TCDD-treated whole splenocytes 24 h after in vitro stimulation with the T-cell mitogen Con A. This is consistent with the finding that IL-2 production at either 24 or 48 h after Con A stimulation of TCDD-treated lymphocytes was not significantly different from that of vehicle-treated controls. The possibility of the induction of a suppressor T-cell by TCDD was also investigated. Titration of T-cells from TCDD-treated mice into naive splenocyte cultures did not suppress the humoral response to either a T-dependent (SRBC) or a T-independent (DNP-Ficoll) antigen. In contrast, titration of cells stimulated in vitro with Con A for 48 h (a positive control for the induction of a suppressor T-cell) inhibited humoral responses of naive cells to both types of antigen. Likewise, T-cells plus macrophages from TCDD-treated mice did not suppress the in vitro humoral responsiveness of naive B-cells plus macrophages to a T-independent antigen (DNP-Ficoll). These results would indicate that an alteration in T-cell function following TCDD exposure does not play a role in the suppression of the antibody response elicited by antigen stimulation of murine B6C3F1 splenocytes.     

Vaccination therapy with tumor-dendritic cell hybrids: a promising therapeutic approach?

Dendritic cells (DCs) are potent antigen-presenting cells currently being investigated as a tool for antitumor vaccination strategies. As an alternative to loading DCs with tumor antigen preparations, hybrid cells generated by fusing tumor cells with DCs have successfully been evaluated in preclinical cancer models. Hybrid cells express a large repertoire of tumor-associated antigens, high levels of major histocompatibility complex class I and II molecules, and adhesion/co-stimulatory molecules. Therefore, these cells possess properties of both parental cell types that are necessary for the induction of primary helper and cytotoxic T-cell responses. The results of early clinical trials suggest that hybrid cell vaccination is a safe and well-tolerated procedure capable of inducing T-cell responses. However, the few objective clinical responses observed indicate that further optimizations are required.     

Relationship between vasodilation capacity and phenolic content of Spanish wines

We aimed to determine: 1) the concentration of polyphenols in Spanish red wines, 2) the vasodilatory properties of those wines in relation with their polyphenol concentrations and 3) the vasodilation induced by some of these polyphenols in rat aortic rings. In the wines studied the concentration of rutin and kaempferol was high compared with other polyphenols. All wines relaxed precontracted rat aortic rings and this effect was directly related with the concentration of myricetin and kaempferol in the wines. Kaempferol and rutin also induced endothelium-dependent and independent relaxation, kaempferol was more potent. This relaxation was not inhibited by the estrogen receptor alpha antagonist ICI 182,760. Kaempferol also potentiated the endothelium-dependent relaxation induced by acetylcholine, which was reversed by Nw-nitro-l-arginine methyl ester (l-NAME). These findings show a good correlation between the concentration of polyphenols (especially kaempferol) of Spanish red wines and the vasodilatory effect, which may confer on them unique features in the prevention of cardiovascular disease.