Splenocyte Culture Supernatants Research Articles

Effects of β-Lapachone on the Production of Th1 and Th2 Cytokines in C57BL/6 Mice

In this study, we investigated the effects of β-lapachone (β-lap) on the production of cytokines in C57BL/6 mice. The culture supernatants of splenocytes exposed to β-lap plus lipopolysaccharide or concanavalin A (Con A) were harvested to determine Th1 (tumor necrosis factor-α, interferon-γ, interleukin [IL]-1β, IL-2, IL-17A, and IL-12) and Th2 (IL-4, IL-5, IL-6, and IL-10) cytokines. IL-2, a Th1 cytokine, was expressed at higher levels in splenocytes treated with β-lap and with lipopolysaccharide plus β-lap. Expression of Th2 cytokines, including IL-4, IL-5, IL-6, and IL-10, was lower in the culture supernatants of mouse splenocytes exposed to Con A plus β-lap than in supernatants from cells that were exposed to Con A alone. These findings demonstrate that β-lap downregulates the immediate hypersensitivity reaction induced by Con A. Therefore, Th2 responses involve the downregulation of IL-4, IL-5, IL-6, and IL-10, preventing the occurrence of allergies and various manifestations of allergic inflammation. These results suggest that β-lap may have potential preventive or adjunct anti-inflammatory therapy applications because of its function in modulating the production of cytokines. However, further in vivo investigations of this process are necessary to elucidate the mechanisms involved.

Effects of lysed Enterococcus faecalis FK-23 on experimental allergic rhinitis in a murine model

In the current study, we sought to investigate whether lysed Enterococcus faecalis FK-23 (LFK), a heat-killed probiotic preparation, attenuated eosinophil influx into the upper airway and had immunomodulatory activity in a murine allergic rhinitis model. Eighteen BALB/c mice were divided into three groups; the ovalbumin (OVA)-sensitized/challenged group, which received saline orally for 6 weeks (OVA group), the OVA-sensitized/challenged group, which received LFK orally for 6 weeks (LFK-fed group), and the non-sensitized group, which received saline for 6 weeks (saline control group). Nasal rubbing and sneezing were monitored during the study. After the final challenge, interleukin (IL)-4, interferon (IFN)-γ, and OVA-specific IgE levels in the sera and splenocyte culture supernatants were determined, eosinophilic infiltrate into the upper airway was quantified, and splenic CD4+CD25+ regulatory T cells (Tregs) were examined by flow cytometry. We found that nasal rubbing was significantly reduced in LFK-fed mice compared to the OVA group on d 27 and 35, and sneezing was significantly inhibited by LFK administration for 35 d. LFK-fed mice had significantly less eosinophil influx into the nasal mucosa than the OVA group. There were no significant differences between the LFK-fed group and OVA group in the serum and splenocyte culture supernatant levels of IL-4, IFN-γ, and OVA-specific IgE. Interestingly, the LFK-fed mice had a significantly greater percentage of splenic CD4+CD25+ Tregs than OVA group. Our results indicate that oral administration of LFK may alleviate nasal symptoms, reduce nasal eosinophilia, and increase the percentage of CD4+CD25+ Tregs in experimental allergic rhinitis.

T Helper Type 1-Bias in a Regulatory T Cell-Deficient Mouse Model for Immune Thrombocytopenia

Abstract 524 Background:Immune thrombocytopenia (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. Recently, we have found that approximately one third of Treg-deficient mice spontaneously develop thrombocytopenia with increased platelet-associated IgG and proportion of reticulated platelets. Platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contain IgG antibodies capable of binding to intact platelets, which are not detected in non-thrombocytopenic mice. The main target of anti-platelet autoantibodies is GPIb, and some mice also produce anti-GPIIIa antibodies. However, detailed mechanisms that elicit ITP during immune reconstitution through homeostatic proliferation in the absence of Tregs remain uncertain. Purpose:To evaluate T-helper (Th) cell balance that promotes anti-platelet autoantibody response in a Treg-deficient mouse model for ITP. Methods:Treg-deficient mice were prepared by inoculation of Treg-depleted CD4+ T cells obtained from BALB/c mice into syngeneic T cell-deficient nude mice. Platelet count was determined using flow cytometry 4 weeks after inoculation, and Treg-deficient mice with platelet count < 0.33 × 106/ul were regarded as ITP mice. Treg-deficient mice without thrombocytopenia were also used as a control. To evaluate cytokine profiles of Th cells, proportions of Th subsets in the freshly prepared splenic CD4+ T cells were evaluated by intracellular staining for IFN-γ, IL-4, and IL-17 followed by flow cytometry. Th1, Th2, and Th0 cells were defined as IFN-γ+IL-4−, IFN-γ−IL-4+, IFN-γ+IL-4+ cells, respectively, and Th17 and Th1/17 cells were defined as IFN-γ−IL-17+ and IFN-γ+IL-17+, respectively. In addition, CD4+ T cells were isolated from splenocytes using magnetic activated cell sorting, and were stimulated with phorbol 1,2-myristate 1,3-acetate and ionomycin for 4 days. The culture supernatants were subjected to a cytokine bead array to measure levels of interleukin (IL)-2, IL-4, IL-6, IL-10, IL-17, interferon (IFN)-γ, and tumor necrosis factor (TNF). Finally, to determine IgG subclasses of anti-platelet autoantibodies, splenocyte culture supernatants were incubated with platelets derived from BALB/c mice, followed by incubation with fluorescence-conjugated antibodies to IgG1, IgG2a, IgG2b, or IgG3. Then, the antibodies bound to platelets was detected by flow cytometry. Results:Fourteen ITP mice and 8 control mice were used at 6–8 weeks after inoculation. The proportions of Th1, Th2, and Th0 cells did not differ significantly between ITP and control mice, while the Th1/Th2 ratio was significantly increased in ITP mice than in control mice (8.3 versus 3.2, p < 0.01). The proportions of Th17 and Th1/17 cells were comparable between ITP and control mice. There was no difference in the in vitro production levels of cytokines except IL-4, which was lower in ITP mice compared to control mice (140 versus 600 pg/ml, p = 0.02). Increase in the IFN-γ/IL-4 ratio was noted in the culture supernatants from ITP mice, compared to those from control mice (15.6 versus 9.2, p = 0.04). The Th1/Th2 ratio detected by flow cytometric measurement and the IFN-γ/IL-4 ratio in in vitro cultures were correlated with each other (r = 0.85, p < 0.01). IgG subclasses of anti-platelet autoantibodies were heterogeneous among individual ITP mice, but IgG2a was the predominant subclass in the majority of ITP mice. Interestingly, a high Th1/Th2 ratio was associated with production of IgG2b anti-platelet antibodies, while the mice with a low Th1/Th2 ratio produced IgG1 anti-platelet antibodies. Conclusions:These findings suggest that induction of IgG anti-platelet autoantibody response in Treg-deficient mice is associated with Th1 bias, which is analogous to the Th balance in patients with primary ITP. The Th1/Th2 balance may modulate the autoimmune responses during expansion of CD4+ T cells in the absence of Tregs. Disclosures:No relevant conflicts of interest to declare.

Mesenchymal Stem Cell-Mediated Immature Dendritic Cells Induce Regulatory T Cell-Based Immunosuppressive Effect

Immature dendritic cells (imDCs) are increasingly viewed as mediators of T-cell tolerance. We investigated factors enabling induction of regulatory T (Treg) cells through syngeneic imDC/mesenchymal stem cell (MSC) co-cultures in vitro and immunosuppressive effects of MSC-mediated imDCs (MSCs were excluded after 72 h co-culture) in vivo. In these experiments, we found that Foxp3+ Treg cell population remarkably increased after the T cell priming phase when splenocytes were co-cultured with both imDCs and MSCs, presumably inducing naïve T cells into Treg cells by MSCs and imDCs. In parallel, TGF-β secretion was markedly induced from the imDC+MSC+splenocyte culture supernatant to a significant level at 72-h co-culture, compared to the MSC or imDC+splenocyte co-culture. Based on these results, using a murine melanoma tumor model, we confirmed that the subcutaneous injection of B16 cells induced a perfect tumor incidence in allogeneic recipients when MSC-mediated DCs were coinjected. Consequently, these results suggested that immune tolerance with MSC-mediated DCs leads to immunosuppression induced by at least Foxp3-specific Treg cells. This tool may be useful in clinical trials due to the yet unknown side effects of stem cell therapy.

Evaluation of recombinant proteins of Haemophilus parasuis strain SH0165 as vaccine candidates in a mouse model

The recently completed genome sequence of Haemophilus parasuis strain SH0165 allowed us to screen putative OMPs for the development of recombinant vaccines. The objective of this study was to evaluate the immunogenicity and protective efficacy of three OMPs of H. parasuis. Three putative OMPs (SmpA, YgiW and FOG) were cloned, expressed and purified by Ni affinity chromatography using nitriloacetic acid resin. Mice were immunized either individually (individual protein, IP) or synergistically (synergistic protein, SP) with the recombinant proteins. A significant increase in IgG titer was detected in all protein-immunized mice. Isotyping studies revealed that the antibodies produced were predominantly IgG2a-type, indicating a predominant Th1 response. A significant increase was observed in IL-2, IL-4 and IFN-γ levels in the culture supernatants of splenocytes isolated from immunized mice. Furthermore, mice were challenged intraperitoneally with 6×109CFU (5×LD50) of highly virulent homologous serovar 5 strain (SH0165) or 7.0×109CFU (5×LD50) of the heterologous serovar 4 strain (MD0322) at fourteen days after the last immunization. All of the recombinant proteins enhanced survival and reduced histopathological lesions. Our results indicated that the three OMPs showed protection both individually and synergistically against infection with the highly virulent H. parasuis in mice.

Emergence of polyclonal autoantibody responses to multiple platelet surface glycoproteins in regulatory T cell-deficient mice. (115.28)

Abstract Naturally occurring CD4+CD25+Foxp3+ regulatory T cells (Tregs) are known to play an important role in preventing pathogenic autoimmune responses. Recently, we have found that approximately one third of mice deficient of CD4+CD25+Foxp3+ Tregs spontaneously develop thrombocytopenia with increased platelet-associated IgG, analogous to patients with immune thrombocytopenia. To identify platelet autoantigens in Treg-deficient mice, plasma samples from thrombocytopenic and non-thrombocytopenic Treg-deficient mice were subjected to immunoblots using platelets as an antigen in reduced and non-reduced conditions. IgG antibodies to platelet surface glycoproteins, such as GPIb, GPIIb, and GPIIIa, were detected specifically in thrombocytopenic mice in various combinations. IgG antibodies to native platelets were further assessed by flow cytometry using platelet eluates and splenocyte culture supernatants. IgG antibodies capable of binding to native platelets were detected in thrombocytopenic, but not in non-thrombocytopenic mice. Antibody reactivity was cancelled in the majority of samples when platelets derived from GPIb-deficient or GPIIIa-deficient mice were used instead of wild-type platelets as an antigen. In summary, Treg deficiency alone leads to emergence of polyclonal IgG autoantibody responses to native and denatured platelet surface glycoproteins, supporting a critical role of CD4+CD25+Foxp3+ Tregs in suppressing induction of autoimmunity.

Adeno-associated virus-mediated CCL11 shRNA reduced lung inflammatory responses in a mite allergen-sensitized mouse model (107.13)

Abstract Asthma is considered as a chronic pulmonary inflammatory disease, characterized by eosinophilia. Lung epithelium cells secrete CCL11, the major chemokine for the recruitment of eosinophils. Hybrid adeno-associated virus serotype 2/9 might achieve long-term gene delivery specifically to lung epithelium cells. Thus, this study tested whether hybrid AAV2/9 virus carrying shRNA specific for CCL11, sh47 and sh137, could reduce eosinophilia in a mite allergen-sensitized mouse model. Both sh47 and sh137 viruses significantly reduced CCL11 level in CCL11-transformed NIH 3T3 cells, compared with others. Both viruses were delivered into Dp2 (group 2 allergen of Dermatophagoides pteronyssinus) sensitized mice before first challenge. The results showed that sh47 virus significantly reduced airway resistance, airway hyperresponsiveness, CCL11 levels and eosinophilia in the lungs of Dp2-sensitized mice. Th2 cytokines were also significantly reduced in the bronchoalveolar lavage fluid and Dp2-stimulated mediastinal lymphocytes of sh47 virus-treated mice. However, serum levels of Dp2-specific IgG1 and IgE as well as Th2 cytokine levels in Dp2-stimulated splenocytes culture supernatants were at the comparable levels to the untreated group. The results suggest that sh47 virus relieved local instead of systemic inflammatory responses. Therefore, the hybrid AAV2/9 viral vector, which is preferable to target lung epithelia cells, might be applied as a novel therapeutic approach for asthma.

Preventive effect of IL-18 gene modified mature dendritic cells vaccine on airway inflammation in mouse asthma model

To investigate the preventive effect of interleukin-18 (IL-18) gene modified mature dendritic cells (mDC) vaccine on airway inflammation in mouse asthma model. The asthma model was induced by injection of ovalbumin (OVA) in BALB/c mice. IL-18 gene modified mouse mature dendritic cells (mDC) were detected by flow cytometry and its capacity of inducing allogeneic T cell responses was examined by mixed lymphocyte reaction (MLR). The OVA-induced asthmatic mice were randomly divided into 6 groups: PBS group, DXM group, mDC group, Ad-LacZ-mDC group, Ad-IL-18-mDC group and control group. The pathological changes in lung tissues were assayed by HE and AB-PAS staining. The numbers of inflammatory cells and percentage of eosinophils (EOS) in bronchoalveolar lavage fluid (BALF) were counted. The levels of IFN-γ IL-4 and IL-13 in culture supernatant of splenocytes were measured by ELISA method. The percentage of CD4(+)CD25(+)Foxp3(+) Treg was assessed by flow cytometry analysis. The vaccine was effective in decreasing the infiltration of EOS and accumulation of airway goblet cells in lung tissues, the numbers of inflammatory cells and percentage of EOS in BALF, and the levels of IL-4 and IL-13 in culture supernatant of splenocytes, and in increasing the levels of IFN-γ in culture supernatant of splenocytes and the percentage of CD4(+)CD25(+)foxP3(+) reg. IL-18 gene modified mDC vaccine has a preventive effect on airway inflammation in OVA-induced asthmatic mice.

Babassu aqueous extract (BAE) as an adjuvant for T helper (Th)1-dependent immune responses in mice of a Th2 immune response-prone strain

BackgroundThe aqueous extract of a Brazilian palm-tree fruit - the babassu - (BAE) exerts a clear immunostimulative activity in vivo. In the present work, the possibility that BAE can promote Th1 immune responses in mice of a Th2 immune response-prone strain - the BALB/c was investigated. BAE itself, and preparations consisting of Leishmania amazonensis promastigote extract (LE), adsorbed or not to Al(OH)3, and in the presence or not of BAE, were used as immunogens. LE and Al(OH)3 have been shown to preferentially elicit Th2 immune responses.ResultsThe addition of BAE to LE-containing immunogenic preparations, adsorbed or not to Al(OH)3, clearly promoted the in vitro production of interferon γ (IFN-γ), a major Th1-dependent cytokine, and not of interleukin (IL-)4 (a Th2-dependent cytokine), by LE-stimulated splenocytes of immunized BALB/c mice. It also promoted the in vivo formation of IgG2a anti-LE antibodies. However, immunization with LE by itself led to an increased production of IL-4 by LE-stimulated splenocytes, and this production, albeit not enhanced, was not reduced by the addition of BAE to the immunogen. On the other hand, the IL-4 production by LE-stimulated splenocytes was significantly lower in mice immunized with a preparation containing Al(OH)3-adsorbed LE and BAE than in mice immunized with the control preparation of Al(OH)3-adsorbed LE without BAE. Moreover, an increased production of IFN-γ, and not of IL-4, was observed in the culture supernatants of splenocytes, from BAE-immunized mice, which were in vitro stimulated with BAE or which received no specific in vitro stimulus. No differences in IL-10 (an immunoregulatory cytokine) levels in the supernatants of splenocytes from mice that were injected with BAE, in relation to splenocytes from control mice, were observed. The spontaneous ex vivo production of NO by splenocytes of mice that had been injected with BAE was significantly higher than the production of NO by splenocytes of control mice.ConclusionsBased on the results described above, BAE, or biologically active molecules purified from it, should be further investigated as a possible adjuvant, in association or not with aluminium compounds, for the preferential induction of Th1-dependent immune responses against different antigens in distinct murine strains and animal species.

식품 알레르기 마우스 모델에서 감태 물 추출물의 IgE 분비 억제 효과

OVA를 항원으로 한 식품알레르기 마우스 모델을 이용하여 감태 물 추출물의 알레르기 억제 효과를 확인하였다. 먼저, OVA로 면역한 마우스의 비장세포에 감태 물 추출물을 첨가하여 배양한 후, 배양 상층액의 IgE antibody와 IL-4 및 IFN-γ cytokine 분비량을 측정하였다. 그 결과, 10～100 ㎍/mL 농도의 감태 물 추출물 첨가로 비장세포의 IgE 분비량이 농도 의존적으로 감소하였고, IL-4 및 IFN-γ 분비량도 감태 물 추출물 첨가 농도가 증가할수록 감소하였다. 감태의 알레르기 억제능을 in vivo 실험을 통해 확인하기 위해 OVA로 알레르기를 유발한 마우스에 감태 물 추출물을 복강주사 한 후 혈청과 비장세포의 IgE 분비능을 확인하였다. 혈청의 항체 분비량 측정 결과, 1, 5 및 10 ㎎/㎏?BW 농도의 감태 물 추출물 투여군이 PBS 투여군에 비해 유의적으로 낮은 OVA-specific IgE 분비량을 보였다. 반면, IgG1과 IgG2a 분비량은 추출물 투여군과 PBS 투여군 사이에 유의적인 차이를 보이지 않았다. 또한 비장세포의 IgE 분비량이 5 및 10 ㎎/㎏?BW 농도의 감태 물 추출물의 복강주사에 의해 감소함을 확인하였다. 이상의 결과를 통해 감태 물추출물이 IgE 분비량의 유의적인 감소 효과를 통해 식품알레르기 반응을 효과적으로 억제할 수 있을 것으로 사료된다.

A Critical Role of CD4+CD25+ Regulatory T Cells In Prevention of Murine Autoantibody-Mediated Thrombocytopenia

AbstractAbstract 381Immune thrombocytopenic purpura (ITP) is a T cell-mediated autoimmune disorder, in which IgG autoantibodies to platelet surface glycoproteins promote platelet clearance in the reticuloendthelial system. Since CD4+CD25+ regulatory T cells (Tregs) are known to play a crucial role in the maintenance of immune homeostasis to self-antigens, it has been believed that Treg dysfunction contributes to the development of a various forms of human autoimmune disorders. Several lines of recent evidence have shown that Tregs are decreased in number and are functionally impaired in patients with ITP. However, it remains unclear how Treg alteration is involved in the pathophysiology of ITP.Recently, we have found that a group of Treg-deficient mice develop autoantibody-mediated thrombocytopenia. For preparation of Treg-deficient mice, Treg-depleted T cells were prepared from BALB/c splenocytes by serial purification steps consisting of a positive selection of CD4+ T cells and a negative selection of CD25+ cells using magnetic bead-based cell sorting, and were transferred into syngeneic T cell-deficient nude mice via tail vein. Treg-depleted T cell fraction transferred contained >99% CD4+CD25− cells, and was confirmed to lack expression of Foxp3, a typical Treg marker. Three weeks after transfer, approximately one third of the recipient mice spontaneously developed thrombocytopenia, which sustained for > 20 weeks. Thrombocytopenic mice represented elevated platelet-associated IgG and increased proportion of reticulated platelets, but non-thrombocytopenic mice did not. In addition, platelets eluates and culture supernatants of splenocytes prepared from thrombocytopenic mice contained IgG antibodies capable of binding to intact platelets, which were not detected in non-thrombocytopenic mice. The presence of anti-platelet antibodies and increased platelet turnover observed in thrombocytopenic Treg-deficient mice are analogous to ITP patients. Treg-deficient mice prepared by transfer of a less number of Treg-depleted T cells resulted in reduced prevalence of thrombocytopenia, suggesting that onset of thrombocytopenia depends on the number of conventional T cells transferred. Treg-deficient mice are known to frequently develop autoimmune gastritis, another autoimmune disease mediated by IgG anti-parietal cell antibodies, but anti-parietal cell antibodies were almost equally detected in plasma from thrombocytopenic and non-thrombocytopenic mice (70% versus 60%). Transplantation of Tregs together with Treg-depleted T cells completely prevented the onset of thrombocytopenia, but Treg transplantation was not effective as a treatment once thrombocytopenia occurred. To further investigate how Tregs exert the regulatory function, Treg-depleted T cells and Tregs were simultaneously transferred in the presence of antibodies that blocked engagement of cytotoxic T lymphocyte-associated antigen 4 (CTLA4). This treatment cancelled Treg function and resulted in development of thrombocytopenia in recipient nude mice, while mock treatment with control antibodies had no effect.In summary, these results together indicate that CD4+CD25+Foxp3+ Tregs play a critical role in preventing the development of murine autoantibody-mediated thrombocytopenia, in part, through CTLA4 engagement. Disclosures:No relevant conflicts of interest to declare.

Mesenchymal stem cell-mediated immature dendritic cells induce regulatory T cell-based immunosuppressive effect

Immature dendritic cells (imDCs) are increasingly viewed as mediators of T-cell tolerance. We investigated factors enabling induction of regulatory T (Treg) cells through syngeneic imDC/mesenchymal stem cell (MSC) co-cultures in vitro and immunosuppressive effects of MSC-mediated imDCs (MSCs were excluded after 72 h co-culture) in vivo. In these experiments, we found that Foxp3(+) Treg cell population remarkably increased after the T cell priming phase when splenocytes were co-cultured with both imDCs and MSCs, presumably inducing naive T cells into Treg cells by MSCs and imDCs. In parallel, TGF-β secretion was markedly induced from the imDC+MSC+splenocyte culture supernatant to a significant level at 72-h co-culture, compared to the MSC or imDC+splenocyte co-culture. Based on these results, using a murine melanoma tumor model, we confirmed that the subcutaneous injection of B16 cells induced a perfect tumor incidence in allogeneic recipients when MSC-mediated DCs were coinjected. Consequently, these results suggested that immune tolerance with MSC-mediated DCs leads to immunosuppression induced by at least Foxp3-specific Treg cells. This tool may be useful in clinical trials due to the yet unknown side effects of stem cell therapy.

Immunohematopoietic modulation by oral β-1,3-glucan in mice infected with Listeria monocytogenes

In this study we demonstrated that the oral administration of β-1,3-glucan (Imunoglucan®) protects mice from a lethal dose of Listeria monocytogenes (LM) when administered prophylactically for 10 days at the doses of 150 and 300 mg/kg, with survival rates up to 40%. These doses also prevented the myelosuppression and the splenomegaly caused by a sublethal infection with LM, due to increased numbers of granulocyte–macrophage progenitors (CFU-GM) in the bone marrow. Investigation of the production of colony-stimulating factors revealed an increased colony-stimulating activity (CSA) in the serum of infected mice pre-treated with Imunoglucan®. The treatment also restored the reduced ability of stromal cells to display myeloid progenitors in long-term bone marrow cultures (LTBMC) and up-regulated IL-6 and IL-1α production by these cells in the infected mice, which was consistent with higher number of non-adherent cells. Additional studies to investigate the levels of interferon-gamma (INF-γ) in the supernatant of splenocyte cultures demonstrated a further increase in the level of this cytokine in infected-treated mice, compared to infected controls. In all cases, no differences were observed between the responses of the two optimal biologically effective doses. In contrast, no significant changes were produced by the treatment with the 50 mg/kg dose. In addition, no changes were observed in normal mice treated with the three doses used. All together our results suggest that orally given Imunoglucan® indirectly modulates immune activity and probably disengages Listeria induced suppression of these responses by inducing a higher reserve of myeloid progenitors in the bone marrow in consequence of biologically active cytokine release (CSFs, IL-1α, IL-6, and INF-γ).

Expression of Interleukin-17 in Autoimmune Dacryoadenitis in MRL/lpr Mice

Purpose: Th-cytokines play important roles in dacryoadenitis associated with Sjögren’s syndrome (SS). The aims of this study were to (i) investigate whether interleukin (IL)-17, a newly identified Th-cytokine, participates in autoimmune dacryoadenitis of MRL/lpr mice and (ii) examine the relationships among IL-17, IL-4, and interferon (IFN)-γ in these mice.Methods: Three-month-old MRL/lpr mice with lacrimal gland inflammation and 3-month-old control BALB/c mice were used. Frozen sections of lacrimal glands and submandibular lymph nodes were stained with monoclonal antibodies to cytokines IL-17, IL-4, and IFN-γ. Concentrations of IL-17 in serum and 48-h splenocyte culture supernatants were detected by ELISA.Results: Marked lacrimal gland inflammation was observed in all MRL/lpr mice but not in any BALB/c control mice. In the lacrimal glands of MRL/lpr mice, the proportions of cells that stained were 5 ± 3% for IL-17, 32 ± 14% for IL-4, and 4 ± 2% for IFN-γ. In the submandibular lymph nodes of MRL/lpr mice, expression of IL-17 and IL-4 was significantly higher than that of IFN-γ, and all three cytokines were significantly higher in MRL/lpr mice than in BALB/c mice. Concentrations of IL-17 in both serum and splenocyte culture supernatants of MRL/lpr mice were elevated compared to BALB/c controls.Conclusions: Since expression of IL-17 is elevated in 3-month-old MRL/lpr mice, we suggest that it plays a role in the pathophysiology of autoimmune dacryoadenitis in these mice.

베르베린이 마우스 전염증성 사이토카인의 생성에 미치는 영향

본 연구에서는 berberine이 전염증성사이토카인의 생성에 미치는 효과를 관찰하기 위하여 TNF-<TEX>$\alpha$</TEX>, IL-<TEX>$1{\beta}$</TEX>, 그리고 IL-6생성을 정량하였다. 마우스 비장세포에 berberine을 작용시켰을 때 TNF-<TEX>$\alpha$</TEX>의 생성이 억제되었다. 또한 마우스 생체 내에서 LPS에 의한 TNF-<TEX>$\alpha$</TEX>의 상승이 berberine에 의해서 억제됨을 알 수 있었다. IL-<TEX>$1{\beta}$</TEX>의 생성에 있어서도 berberine을 고농도(3.0 <TEX>${\mu}g/ml$</TEX>)로 작용시켰을 때 억제되었고 LPS에 의한 IL-<TEX>$1{\beta}$</TEX>의 상승이 고농도의 berberine에 의해서 억제되었다. IL-6의 생성은 berberine에 의해서 억제되었으며 낮은 berberine 농도(0.3 <TEX>${\mu}g/ml$</TEX>)에서 LPS에 의한IL-6의 생성이 억제되었다. 따라서 이러한 결과들은 berberine이 TNF-<TEX>$\alpha$</TEX>, IL-<TEX>$1{\beta}$</TEX>, 그리고 IL-6와 같은 전염증성사이토카인의 생성을 하향 조절할 가능성을 시사한다 하겠다. Berberine has shown a number of beneficial effects, including anti-tumor, anti-inflammation, and vasodilatory effects. In this work we investigated the effects of berberine on the production of proinflammatory cytokines such as TNF-<TEX>$\alpha$</TEX>, IL-<TEX>$1{\beta}$</TEX>, and IL-6 in mice. The supernatants of cultured splenocytes exposed with berberine or berberine plus LPS were harvested to assay TNF-<TEX>$\alpha$</TEX>, IL-<TEX>$1{\beta}$</TEX>, and IL-6. The sera from the mice injected with berberine or berberine plus LPS were then isolated to assay these cytokines. The TNF-<TEX>$\alpha$</TEX> production in mice splenocyte cultures exposed to berberine was inhibited compared to the PBS control. The sera from LPS plus berberine injected mice showed lower levels of TNF-<TEX>$\alpha$</TEX> compared to those of LPS only injected mice. The IL-<TEX>$1{\beta}$</TEX> production in mice splenocyte cultures exposed to berberine was inhibited at a high dose (3.0 <TEX>${\mu}g/ml$</TEX>) compared to the PBS control. Also, the increase of IL-<TEX>$1{\beta}$</TEX> by LPS exposure in splenocyte cultures was inhibited by a high dose of berberine. The IL-6 in splenocyte culture supernatants showed lower levels after berberine compared to the PBS control. Also, production of IL-6 after LPS exposure in splenocyte cultures was inhibited by a low dose of berberine (0.3 <TEX>${\mu}g/ml$</TEX>). These findings suggest the probability that berberine down-regulates the production of proinflammatory cytokines such as TNF-<TEX>$\alpha$</TEX>, IL-<TEX>$1{\beta}$</TEX>, and IL-6.

Combination of methylprednisolone and minocycline synergistically improves experimental autoimmune encephalomyelitis in C57 BL/6 mice

Combination therapies with existing or novel drugs for multiple sclerosis (MS) have great clinical potential to improve MS treatment outcomes. Our previous studies had confirmed that the combined treatment of minocycline and prednisone produced beneficial effects partially through preventing the reduction of brain-derived neurotrophic factor and nerve growth factor mRNA expression in the cerebral cortex of experimental autoimmune encephalomyelitis (EAE) mice. As high-dose methylprednisolone administered intravenously has more superior efficacy than oral prednisone and had been provided as a stable therapy for MS patients at the onset of an acute relapse, we further evaluated the effects of combined methylprednisolone and minocycline at suboptimal doses on EAE mice at the acute stage in this study. Interferon gamma (IFN-γ) and interleukin-4 (IL-4), the hallmark cytokines that direct Th1 and Th2 development and play an important role in the pathogenesis of MS as well as EAE, were also assayed. Obtained results showed that combined treatment could successfully attenuate severe clinical deficit and suppress histopathological events in EAE. In addition, reduced IFN-γ and increased IL-4 production/expression were found in the splenocytes culture supernatants and brains of EAE mice by the combined treatment. Our data indicate that the combination of methylprednisolone and minocycline may be a promising therapy for MS.

Immunostimulatory Properties of a Polysaccharide Isolated fromAstraeus hygrometricus

Mushrooms have been used as food and for therapeutic purpose for decades, and various compounds derived from these have potential biological activities. Application of such agents of natural origin is vital with relevance to an increase in diseases involving immune dysfunction, cancer, and autoimmune conditions in recent years. These compounds belong mainly to polysaccharides, especially beta-D-glucan, glycopeptide/protein complexes, proteoglycans, proteins, and triterpenoids. Among these compounds, polysaccharides and their peptide/protein derivates have extremely important roles in immunomodulating activities. The present study aims to explore the immunomodulatory properties of polysaccharide isolated from the mushroom Astraeus hygrometricus. The polysaccharide isolated by hot alkaline extraction and chromatography was designated as AE2 and studied for its immunostimulatory potential in vivo in a murine model. Macrophages from treated mice showed higher production of nitric oxide and interleukin-1 and also exhibited an increased phagocytic potential. It also enhanced natural killer cell activation and proliferation of splenocytes with an increase in the T-helper (Th) 1 cytokine level of splenocyte culture supernatant. The cytokine level in serum also indicated a Th1 skew. The observed activities were associated with no general toxicity to the organism. The findings will be helpful in considering A. hygrometricus as a potential source of an immunomodulator and in designing further studies to understand its mode of action on immune system.

Production of biologically active chicken interleukin (IL)-12 and IL-18 synthesized by the recombinant fowlpox virus

The single chain chicken interleukin-12 (chIL-12)- or mature chIL-18-encoding region was cloned into the nonessential gene F11L of fowlpox virus (FPV) to generate the recombinant (r) FPV/chIL-12 (rFPV/chIL-12) or rFPV/chIL-18 for rchIL-12 or rchIL-18 production. Splenocytes of chickens were cultured with various dilutions of binary-ethylenimine (BEI)-inactivated rFPV/chIL-12 or rFPV/chIL-18-infected cell lysate for 48 h for interferon-γ (IFN-γ) determination. It was found that 1:10,000 chIL-12 or 1:10 chIL-18 cell lysate stimulated the highest levels of IFN-γ production. When chickens given BEI-treated 1:10 rchIL-12 or rchIL-18 cell lysate, intraperitoneally, or rFPV (0.2 × 10 5 TCID 50 ) by wing-web puncture, the highest level of IFN-γ was detected in sera on day 3 postinoculation (dpi). In the splenocyte culture supernatants, the highest level of IFN-γ was detected at 14 dpi or 21 dpi in responses to rchIL-12 or rchIL-18, respectively. The results indicated that rchIL-12 or rchIL-18 could induce IFN-γ production both in vitro and in vivo assays, suggesting that both are biologically active and may allow them to be used in the future as the biological adjuvant in the poultry vaccine development, particularly co-administering with vaccine antigens.

Effect of orally administered bovine colostrum on cytokine production in vivo and in vitro in immunosuppressed mice

Female ICR mice were administered with bovine colostrum and were treated with cyclophosphamide to establish immunosuppressed status. The cytokine levels in sera and splenocyte culture supernatants and splenic T cell subpopulations were determined. Bovine colostrum substantially restored the percentage of the total T cell population (CD3+) and effector CD4+ T lymphocyte, as well as influencing the CD4:CD8 ratio in the spleen. It was effective in increasing the concentrations of interleukin (IL)-2, interferon-γ, granulocyte-macrophage colony-stimulating factor and decreasing the levels of IL-6 in vivo and in vitro. The levels of IL-10 in splenocyte culture supernatants were increased, whereas the levels of IL-4 in splenocyte culture supernatants were dramatically decreased. Moreover, bovine colostrum was efficacious in enhancing the production of IL-1α in vitro. Thus, bovine colostrum could exhibit immunoregulatory effects via modulating splenic T lymphocytes subpopulations. Bovine colostrum may have pleiotropic effects on the production of cytokines in vivo and in vitro.

Bambusae Caulis in Taeniam extract reduces ovalbumin-induced airway inflammation and T helper 2 responses in mice

Ethnopharmacological relevance Bambusae Caulis in Taeniam (BC) was known as traditional herbal medicine with anti-inflammatory property in the Orient. Aim of the study Allergic asthma is inflammatory disease of airways associated with enhanced T helper (Th) 2 lymphocytes responses to allergens, leading to eosinophilic infiltration and elevated serum IgE levels. Although there were some studies that BC extract had an anti-inflammatory property, there was no study on asthma. In present study, we investigated the suitability of BC extract as a therapeutic candidate in the treatment of allergic airway disease in ovalbumin-induced asthma model. Materials and methods Balb/C mice (female, 6 weeks old) were treated by ovalbumin sensitization and nebulization, and used as asthma model. The number of eosinophil in bronchoalveolar lavage (BAL) fluid and the degree of eosinophila were investigated by hematoxylin and eosin stain and the infiltration of inflammatory cells into lung tissues was examined by staining by hematoxylin and eosin solution. The levels of interleukin (IL)-4 in BAL fluid, immunoglobulin E (IgE) in serum, interferon (IFN)-γ and IL-4 production in splenocyte culture from Balb/C mice (not treated, 6 weeks old) that incubated with or without BC extract for 48 h were determined by enzyme-linked immunosorbent assay. Results The level of eosinophils was decreased by treatment of the animals with BC extract (40 mg/kg) and correspondingly, a significantly lowered degree of eosinophila was also reported ( p < 0.01). In lung tissue, BC extract reduced the increased immune cell infiltration induced by OVA ( p < 0.05). Furthermore, the levels of IL-4 and IgE in BAL fluid or serum up-regulated by OVA was decreased by BC extract. Finally, IFN-γ production was significantly increased ( p < 0.01), while IL-4 production significantly decreased ( p < 0.01), after treatment of the culture supernatants of splenocytes with BC extract. Conclusions These results indicated that BC extract reduces OVA-induced airway inflammation and Th 2 response in mice, suggesting that BC extract can be a therapeutic candidate for allergic airway disease, including asthma.