Hypoxia reduces CD80 expression on monocytes but enhances their LPS-stimulated TNF-alpha secretion

Monocytes/macrophages in ischemic tissues are involved in inflammation and suppression of adaptive immunity via secretion of proinflammatory cytokines and reduced ability to trigger T cells, respectively. We subjected human mononuclear cells and mouse macrophages to hypoxia and reoxygenation, the main constituents of ischemia and reperfusion, and added lipopolysaccharide (LPS) to simulate bacterial translocation, which frequently accompanies ischemia. We monitored the secretion of tumor necrosis factor alpha (TNF-alpha) and the surface expression of human leukocyte antigen-DR and the costimulatory molecules CD80 and CD86 on monocytes/macrophages. Hypoxia selectively reduced the surface expression of CD80 (P<0.01), and synergistically with LPS, it enhanced TNF-alpha secretion (P<0.003). Reoxygenation reversed both phenomena. In the mouse macrophage cell line RAW 264.7, hypoxia reduced the surface expression of CD80 and increased its concentrations in the supernatants (P<0.01). Down-regulation of the mRNA coding for the membrane-anchored CD80 was observed, suggesting that hypoxia triggers alternative splicing to generate soluble CD80. Cumulatively, these results suggest that hypoxia simultaneously affects monocytes/macrophages to enhance inflammation and reduce their ability to initiate adaptive-immunity responses associated with ischemic injury.     

CD28/CTLA-4--CD80/CD86 and ICOS--B7RP-1 costimulatory pathway in bronchial asthma

Costimulatory molecules are cell surface glycoproteins that can direct, modulate and fine-tune T-cell receptor signals. The B7-1/B7-2--CD28/CTLA-4 and ICOS-B7RP-1 pathway provides key second signals that can regulate the activation, inhibition and fine-tuning of T-lymphocyte responses. The expression of B7-1/B7-2--CD28/CTLA-4 molecules on clinical samples from patients with asthma have been well studied, and the results indicate that different extents of these molecules are expressed on the surface of various cells, and that the concentrations of soluble form of these molecules are elevated in the sera of patients with asthma. There is a burst of papers describing an important role for B7-1/B7-2--CD28/CTLA-4 pathway in the Th1/Th2 balance. Similarly, ICOS stimulates both Th1 and Th2 cytokine production but may have a preferential role in Th2 cell development. Moreover, The B7-1/B7-2-CD28/CTLA-4 and ICOS-B7RP-1 pathway has been suggested of being involved in the development of airway inflammation and airway hyperresponsiveness. Further study of the functions of the pathways within the CD28/CTLA-4--CD80/CD86 and ICOS--B7RP-1 superfamily individually and their interplay should provide insights into the pathogenesis of asthma, and has great therapeutic potential for treatment of asthma.     

Intracellular IL-5 and T-lymphocyte subsets in atopic and nonatopic bronchial asthma

BACKGROUND: Allergen-stimulated IL-5 production by CD4+ T cells is the key issue in atopic asthma. On the other hand, virus-specific CD8+ T cells produce IL-5 and might play an important role in the pathogenesis of nonatopic asthma. OBJECTIVES: We sought to compare the IL-5-producing T-lymphocyte subsets in the peripheral blood of atopic and nonatopic asthmatic subjects, especially the contribution of IL-5-producing CD8(+) T cells in nonatopic asthma. METHODS: Heparinized blood samples were obtained from subjects with atopic asthma (n = 10), subjects with nonatopic asthma (n = 10), and healthy subjects (n = 10) and stimulated with ionomycin and phorbol myristate acetate in the presence of brefeldin A. Two-color flow cytometric analysis with mAbs to cell-surface antigens and intracellular IL-5 was used to detect the IL-5-producing T-cell subsets. RESULTS: A higher percentage of IL-5-producing CD3(+) T cells was detected in subjects with atopic and nonatopic asthma than that seen in the healthy subjects. The percentage of IL-5-producing CD4(+) T cells was significantly higher in subjects with atopic asthma than in the healthy subjects. The percentage of IL-5-producing CD8(+) T cells was significantly higher in subjects with nonatopic asthma than in the healthy subjects. The percentage of IL-5-producing CD8(+) T cells was higher in subjects with nonatopic asthma than in those with atopic asthma, but the difference was not statistically significant. CONCLUSIONS: CD4(+) T cells are the major source of IL-5 among CD3(+) lymphocytes in subjects with atopic asthma. On the other hand, increased IL-5 production by CD8(+) T cells, as well as by CD4(+) T cells, is a characteristic feature of nonatopic asthma.     

The effect of IL-18 on IL-12-induced CD30 expression and IL-4 and IFN-gamma production by allergen and PPD specific T cells

CD30 is expressed on activated T cells that, as has been suggested, preferentially produce IFN-gamma. Interleukin 12 increases antigen-induced CD30 expression on T cells and IFN-gamma production. Synthesis of IFN-gamma can be augmented further by IL-18. The aim of our study was to investigate whether IL-18 affects the IL-12 induced CD30 expression and cytokine production by allergen or PPD specific T cells. Mononuclear cells of healthy or atopic volunteers were stimulated with PPD or allergen, respectively, to obtain specific T cell lines. T cells were restimulated with appropriate antigen and antigen-presenting cells in the presence of IL-12, IL-18 or a combination of these cytokines. After 3 days, expression of CD30 was investigated on CD4 and CD8 T cells and IFN-gamma and IL-4 cytokine production was estimated in the culture supernatants. Flow cytometric analyses showed no effect of IL-18 on CD30 expression during IL-12 co-stimulation. At the same time after the optimal stimulation for CD30 expression, the levels of IFN-gamma were high in PPD-stimulated cell lines but have not been up-regulated by IL-18. IFN-gamma levels were much lower in allergen-stimulated T cells and although they were up-regulated by IL-12 there was no additional or synergistic effect from IL-18. IL-18, however, increased production of IL-4 in allergen-stimulated cell lines. Our studies provide new information about IL-18 activity on human cells and question its exclusive role in Th1 mediated responses.     

Differential function of CD80- and CD86-transfected human melanoma cells in the presence of IL-12 and IFN-gamma

Introduction of co-stimulatory molecules like CD80 and CD86 represents a means to augment the immunogenicity of tumor cells and to induce immune responses directed at tumor antigens. Here we compared CD80- and CD86-transfected human melanoma cells to induce primary immune responses by their capacity to promote proliferation of human allogeneic resting T lymphocytes. CD80- and CD86-transfected SkMel63 melanoma cells induced T cell activation to a comparable degree, which was found to be independent of the cell surface density of these co- stimulatory molecules. Co-expression of CD80 and CD86 did not result in a synergistic increase in T cell proliferation. Both CD80 and CD86 transfectants induced the proliferation of isolated CD4+ or CD8+ T cells. Exogenous IL-2, IL-4 and tumor necrosis factor-alpha respectively enhanced primary T cell proliferation independent of CD80 or CD86 expression. Interestingly, differential activities of CD80 and CD86 were observed following stimulation of resting T cells in the presence of IL-12. Whereas IL-12 increased T cell proliferation in the presence of CD86-transfected melanoma cells, it exhibited an inhibitory function in the presence of CD80-expressing SkMel63 cells. Experimental evidence indicates that this inhibitory effect was mediated by IFN- gamma since (I) IFN-gamma secretion of stimulated T cells was augmented by IL-12, (II) exogenous IFN-gamma also inhibited T cell proliferation induced by CD80- but not CD86-transfected SkMel63 cells and (III) the inhibitory effect of IL-12 was blocked by an anti-IFN-gamma mAb.      

The effect of allergen immunotherapy on in vitro IL-4 and IFN-gamma production by peripheral mononuclear cells in house dust-sensitive Chinese patients with bronchial asthma

The IFN-gamma produced by Th1 cells and IL-4 produced by Th2 cells are two most important cytokines in the regulation of IgE production. House dust immunotherapy has been tried in the treatment of house dust-sensitive Chinese asthmatic patients with good results. We examined the influence of such treatment on in vitro IL-4 and IFN-gamma production by peripheral blood mononuclear cells in house dust-sensitive asthmatic patients. Allergen immunotherapy in house-dust sensitive asthmatic patients can significantly decrease IL-4 production from peripheral mononuclear cells (p<0.05). The production levels of IL-4 in patients without treatment had higher levels than those in patients with hyposensitization (p<0.01). Such therapy also have some effect on promotion of IFN-gamma production in asthmatic patients. In conclusion, immunotherapy with house dust may have the potential ability to shift the Th1/Th2 balance of immune response to allergens and to create a favorable cytokine microenvironment to suppress the allergic reaction in the asthmatic airway.     

Differential expression and modulation of costimulatory molecules CD80 and CD86 on monocytes from patients with systemic lupus erythematosus

Patients with systemic lupus erythematosus (SLE) were recently shown to be defective in costimulatory molecule CD80 (B7-1) expression on antigen-presenting cells. This study was undertaken to further investigate the expression and cytokine regulation of both CD80 and CD86 (B7-2) on monocytes from patients with SLE. Freshly isolated and in vitro cytokine-stimulated peripheral blood mononuclear cells from 13 patients with SLE and 10 healthy subjects were analysed, cytometrically with dual-fluorescence staining, to detect expression of CD80 and CD86 in the CD14+ monocyte population. The results showed that, as in normal individuals, an overwhelming majority (95.62+/-3.54%) of monocytes from patients with SLE expressed the CD86 molecule, but only a few monocytes (5.54+/-4.36%) had detectable CD80 expression. The effects of interleukin-10 (IL-10) on the expression of CD80 and CD86 on monocytes from patients with SLE and normal controls were similar. IL-10 down-regulated the expression of CD86 while it slightly enhanced that of CD80. Interferon-gamma (IFN-gamma) increased both CD80 and CD86 expression on monocytes from both SLE patients and normal groups, albeit less significantly in the former than in the latter, i.e. CD80: 142.84+/-65.99% versus 226.08+/-78.90%, P<0.05; and CD86: 72.55+/-74.23% versus 153.99+/-94.14%, P<0.05, when expressed as percentage modulation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) showed a capacity for up-regulation of CD80 and CD86 expression on monocytes, of a magnitude that was similar both in patients with SLE and in normal subjects. We concluded that CD80 and CD86 were differentially expressed and modulated on monocytes and the defective IFN-gamma-induced up-regulation of CD80 and CD86 expression on SLE monocytes might be a factor in the pathogenesis of SLE.     

阻断CD28-CD86共刺激途径抑制小鼠气道炎症的研究

TH2淋巴细胞及其分泌的细胞因子在哮喘发病过程中起关键作用。T细胞激活需要由APC表面的共刺激分子与T细胞表面的共刺激分子受体结合形成的共刺激信号。已经公认T淋巴细胞表面的CD28／CTLA4与APC表面的B7-1(CDS0)、B7-2(CD86)是产生共刺激信号的主要分子。本实验旨在探讨抗CD86单克隆抗体对小鼠哮喘模型气道炎症的影响。     

M-CSF、IL-10对单核细胞IL-12、IL-18产生及HLA-DR和CD80表达的影响

目的 :探讨巨噬细胞集落刺激因子 (M CSF)和白细胞介素 10(IL 10 )对人外周血单核细胞分泌IL 12、IL 18及细胞表面HLA DR和CD80表达的影响。方法 :从健康献血员血液中分离单核细胞并进行体外培养 ,分别以M CSF和IL 10单独及共同作用 ,收集上清 ,用ELISA法检测单核细胞IL 12和IL 18的分泌 ,用流式细胞术检测细胞表面HLA DR及CD80的表达。结果 :①M CSF能诱导单核细胞分泌IL 18(P <0 .0 5 ) ;IL 10则能抑制IL 18的产生 (P <0 .0 5 ) ,并拮抗M CSF增强LPS诱生IL 18的作用 (P <0 .0 5 )。M CSF和IL 10均能抑制单核细胞分泌IL 12 p4 0 (P <0 .0 5 ) ,并具有协同效应 (P <0 .0 5 )。②M CSF能诱导单核细胞表面HLA DR的表达 (P <0 .0 5 ) ;IL 10则可抑制HLA DR的表达 ,并拮抗M CSF对HLA DR的诱导作用。M CSF对CD80表达的影响不大 ,IL 10能促进CD80的表达。结论 :M CSF和IL - 10可通过对单核细胞分泌IL 12、IL 18及HLA DR和CD80表达的调节 ,影响T细胞的活化、分化及其介导的免疫应答     

Effect of CD80 and CD86 blockade and anti-interleukin-12 treatment on mouse acute graftversus-host disease

We investigated the efficacy of a combination of anti-CD80 and CD86 (CD80 + 86) monoclonal antibodies (mAb), anti-interleukin (IL)-12 mAb, or both, for prophylaxis in a mouse acute graft-versus-host-disease (GVHD) model. The treatment with a combination of anti-CD80 + 86 mAb efficiently reduced the lethality of GVHD, whereas mAb against either CD80 or CD86 alone had an effect. A delay in lymphocyte reconstitution and GVHD-associated histological changes in organs was observed at 30 days post-bone marrow transplantation (BMT) even in the anti-CD80 + 86 mAb-treated mice, although these manifestations were resolved by 100 days. In vitro, host alloantigen-specific T cell proliferative responses and generation of CTL were significantly reduced by anti-CD80 + 86 treatment. Furthermore, anti-CD80 + 86 mAb preferentially inhibited the production of interferon (IFN)-γ, but not IL-4 and IL-10, when cultures were assayed at 21 days. Although the anti-IL-12 mAb treatment alone inhibited the generation of cytotoxic T lymphocytes and IFN-γ production in vitro, administration of anti-IL-12 mAb in vivo reversed the beneficial effects of anti-CD80 + 86 treatment on host survival post-BMT. The adverse effect of anti-IL-12 treatment seems to result from impairment of natural immunity and hematopoiesis, rather than as a consequence of an incomplete blockade of T helper (Th)1 responses. Our results suggest that the prevention of GVHD-induced death results from the efficient blockade of Th1 cell activation by the anti-CD80 + 86 treatment. However, further treatment is required for a complete prevention of GVHD, which seems to be partly mediated by Th2 cells.     

Dysregulation of B7.2 (CD86) expression on monocytes of HIV-infected individuals is associated with altered production of IL-2.

T helper (Th) responses are mediated in part by immunoregulatory cytokines and the signals delivered by the costimulatory CD28-B7 pathway. In this study, we have investigated the relationship between the regulation of B7 isoform expression on antigen-presenting cells from HIV+ individuals and the production of Th cytokines. The level of expression of both B7.1 and B7.2 isoforms as measured by mean channel fluorescence was significantly decreased on freshly isolated monocytes from HIV+ individuals compared with HIV- controls. However, the levels of expression of B7.1 and B7.2 on both B cells and monocytes increased significantly following culture in HIV+ individuals compared with HIV- controls. B7 expression is subject to regulation by immunoregulatory cytokines. Therefore, we analysed the regulation of B7 expression by cytokines, namely IL-10 and tumour necrosis factor-alpha (TNF-alpha), the production of which is enhanced in HIV infection and have similar inhibitory effects on B7 expression. Two groups of HIV+ individuals were distinguished on the basis of the inhibitory effect of IL-10 and TNF-alpha on monocyte B7.2 expression. IL-10 inhibited B7.2 expression on monocytes from some HIV+ individuals (termed responders) like the HIV- controls. However, in a subset of HIV+ individuals (non-responders) this inhibitory effect was lost. Loss of inhibition of B7.2 expression by IL-10 was associated with significantly reduced IL-2 production by phytohaemagglutinin (PHA)- stimulated peripheral blood mononuclear cells (PBMC). These observations showing an association of B7 dysregulation on monocytes and B cells with altered production of IL-2 may have implications in HIV immunopathogenesis.     

Role of caspase in CD80 expression of superantigen-stimulated monocytes

In this study, we demonstrated evidence for the induction of CD80+ monocytes by staphylococcal enterotoxin B (SEB) via caspase actions. Pre-treatment with pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of NF-kappaB, resulted in a significant reduction in the percentage of SEB- and interferon-gamma (IFN-gamma) (produced by SEB) -induced CD80+ monocytes. SEB and IFN-gamma activated NF-kappaB, which was inhibited by PDTC. SEB induced the activation of caspase-3 and -8, and pre-treatment with z-VAD-fmk, a broad-spectrum inhibitor of caspases, prevented the induction of apoptosis. Treating with z-VAD-fmk resulted in a reduction of the generation of CD80+ monocytes. These results indicated that CD80 driven by NF-kappaB is regulated by the enzymatic actions of caspases, which allows monocytes to participate in massive T-cell activation.     

The inhibitory effect of quercetin on IL-6 production by LPS-stimulated neutrophils

Quercetin is a herbal flavonoid derived from various foods of plant origin and plays a role in anti-inflammation. Although a number of researches in the field have been done, the mechanism of anti-inflammatory effect of quercetin should be further darified. In the present study, we investigated the effects of quercetin on IL-6 production by LPS-stimulated neutrophils in human. Neutrophils were were pre-treated with quercetin at the final concentrations of ranging from 0-80 ~M for 30 rain, or not treated, and then incubated in the presence or absence of lipopolysaccharide （LPS） at a final concentration of 100 ng/ml for indicated time. The secretion level of IL-6 in the culture supernatants was assayed by ELISA, the intracellular level of IL-6 was detected by flow cytometry and the expression of IL-6 mRNA was analyzed by RT-PCR. The experiment results showed that neutrophils cultured with medium or quercetin alone did not express IL-6, but LPS （100 ng/ml） induced IL-6 expression of neutrophils. However, after pre-treatment of neutrophils with quercetin （40 ~Vl） for 30 rain, the inducible effects of LPS on the increase of IL-6 secretion, intracellular IL-6 level and IL-6 mRNA expression by neutrophils were abrogated. IL-6 is one of the important pro-inflammatory factors, especially in early phage of inflammation. Thus, our data suggested that quercetin might exert its anti-inflammatory effect through negatively modulating pro-inflammatory factors, such as IL-6. The inhibitory effects of quercetin on IL-6 production by neutrophils may provide a theoretical basis on future therapy of inflammation. Cellular ＆ Molecular Immunology. 2005;2（6）：455-460.     

IL-5 expression in the sputum of patients with bronchial asthma

Expression of IL-5 mRNA and the content of IL-5 in the sputum of patients with asthma of different severity were studied before and after treatment. The expression of IL-5 mRNA in mild asthma differed from that in severe and moderate asthma before and after treatment. The level of IL-5 before therapy was different in patients with mild and severe disease. In patients with severe asthma the level of IL-5 differed before and after treatment.     

Differential role of CD80 and CD86 on alveolar macrophages in the presentation of allergen to T lymphocytes in asthma

In the asthmatic lung the altered expression of costimulatory molecules (CD80, CD86) by alveolar macrophages contributes to T lymphocyte activation and expansion. We hypothesized that CD80 and CD86 on alveolar macrophages could differentially support allergic inflammation in adult asthma. Here we studied 11 subjects with mild allergic asthma and 11 atopic non-asthmatics as controls. Dermatophagoides-specific T cell lines were derived from peripheral blood from each subject. Bronchoalveolar lavage with evaluation of lung inflammatory cells was performed in all individuals at baseline and 24 h after allergen challenge. The expression of CD80 and CD86 costimulatory molecules by alveolar macrophages was studied and, in parallel, the efficiency of antigen presentation was measured in terms of IL-4 and IL-5 production by allergen-stimulated autologous T cells. We found that in asthmatic subjects (i) the percent of CD80+, but not CD86+ alveolar macrophages was increased at baseline and did not change following allergen challenge; (ii) CD86, but not CD80, membrane expression was up-regulated following allergen challenge; (iii) both CD80 and CD86 were required to support Th2 cytokine production by allergen-specific T cells, with a prevalent role of CD86 after allergen challenge. Our data indicate that alveolar macrophages deliver costimulatory signals via CD80 and CD86, which support the production of Th2 cytokines by allergen-specific T cells. They also indicate that CD86 in vivo is up-regulated in the 24 h following allergen exposure and that this modulation is functionally relevant.     

The effect of immunotoxins directed against CD80 and CD86 on primary T-cell alloresponses

Abstract: Activation of primary resting T cells requires costimulation which can be delivered by the B7 molecules (CD80 and CD86) expressed on activated antigen-presenting cells (APC). In the present study, we examined in vitro effects of immunotoxins (ITs) composed of gelonin conjugated to mAbs against CD80 or CD86 (αCD80-IT and αCD86-IT). The specificity of both ITs was demonstrated using CD80 and CD86 transfected cell lines. In primary mixed lymphocyte cultures (MLCs), it was found that the average inhibitory capacity of αCD86-IT (72%) and αCD80-IT (30%) was significantly higher than αCD86 (54%) and αCD80 (11%). In reculture MLC experiments it was found that peripheral blood mononuclear cells pretreated with αCD86/αCD80 regained full stimulatory capacity whereas αCD86-IT/ αCD80-IT pretreatment induced >95% loss of stimulatory capacity. Our results therefore demonstrate that these αB7-ITs functionally block B7-CD28 costimulatory signaling and eliminate activated APC.     

支气管哮喘病人CD4+T细胞CD25、CD30表达状况

目的：通过观察哮喘病人外周血CD4⁺T细胞CD25、CD30表达水平，了解哮喘病人T细胞活化状态。方法：将分离出的CD4⁺T细胞分别用PPD、PHA刺激，最后用流式细胞仪检测抗原刺激前后细胞表面CD25、CD30表达水平。结果：①哮喘病人CD4⁺T细胞CD25、CD30自然表达比率均低于健康对照（P<0.05、P<0.05）。②用PHA刺激哮喘病人CD4⁺T细胞后，CD25表达水平明显高于健康对照（P<0.01），但CD30表达无差异。③PPD刺激组CD25、CD30表达与健康对照间无差异。结论：哮喘病人CD4⁺T细胞活化状态明显异常。哮喘病人的CD4⁺T细胞无刺激因素时，活化水平低下，但接受刺激后表现出高水平的活化状态。