IL-10 augments CD23 expression on U937 cells and down-regulates IL-4-driven CD23 expression on cultured human blood monocytes: effects of IL-10 and other cytokines on cell phenotype and phagocytosis.

The effects of human recombinant interleukin-10 (IL-14) on the expression of several markers on U937 and human peripheral blood monocytes was studied by immunofluorescence and fluorescence-activated cell sorter (FACS) analysis. IL-10 augmented Fc IgE receptor (Fc epsilon RII/CD23) further enhanced by cotreatment with IL-4 or interferon-gamma (IFN-gamma). In contrast, the basal level of Fc epsilon RII expression on blood monocytes appeared to fall in response to IL-10, and this effect became more evident on IL-4-treated cells. Furthermore, the constitutive and IFN-gamma-triggered Fc gamma RI/CD64 expression was augmented on both monocytes and U937 cells. Thus the expression of Fc gamma RII/CD32, Fc gamma/RIII/CD16, Fc alpha R/CD89, the receptor for complement components (CR1/CD35, CD3/CD11b, CR4/CD11c) and the receptor for transferrin/CD71 was not significantly influenced on IL-10-treated cells. IL-10 modestly triggered CD14 antigen expression on monocytes but not U937. The expression of intercellular adhesion molecule-1 (ICAM-1)/CD54 on monocytes was significantly inhibited by IL-10. As expected, a marked reduction of the constitutive as well as of the IFN-gamma or IL-4-driven expression on HLA-DR, HLA-DP and HLA-DQ was observed on IL-10-cultured monocytes. On the other hand, the expression of major histocompatibility complex (MHC) class I molecules was slightly and dose-dependently induced on IL-10-treated monocytes. The ability of blood monocytes to phagocytose IgG-sensitized ox erythrocytes, and to bind and ingest opsonized Escherichia coli or latex particles, was amplified by IL-10. Our data demonstrate that IL-10 modulates the expression of a wide variety of structures on human mononuclear phagocytes, and augments their phagocytic capacity.     

Beta-2-adrenoceptor agonists up-regulate the in vitro Fc epsilon receptor II/CD23 expression on, and release from, the promonocytic cell line U937 and human blood monocytes.

The possible regulatory role of beta 2-adrenoceptor agonists in the modulation of CD23 on the human promonocytic cell line U937 and on human monocytes was investigated. Incubation for 48 h in the presence of interleukin-4 (IL-4; 30 U/ml) induced optimal expression and release of CD23 on both U937 cells and human monocytes. When a beta 2-adrenoceptor agonist, either salbutamol or fenoterol, was added to U937 cells or monocytes both the IL-4-induced CD23 expression and release were markedly up-regulated. This effect was dose-dependent, starting at 10 nM and reaching a maximum at 1 microM final concentration of either salbutamol or fenoterol. The potentiating effect of salbutamol and fenoterol on CD23 expression and release was not observed when a beta-adrenoceptor antagonist, either D,L-propranolol (1 microM) or butoxamine (1 microM), was added to the incubation medium. The alpha-adrenoceptor agonist norepinephrine (1 microM) was ineffective in enhancing the IL-4-induced expression and release of CD23 from U937 cells or human monocytes, demonstrating the specificity of the beta 2-adrenoceptor-mediated effect. In the absence of IL-4, a moderate but significant increase in the CD23 expression on U937 cells and human monocytes by these drugs was observed, as compared to the basal values. These results indicate that, besides their bronchodilator effect, beta 2-adrenoceptor agonists may regulate IgE-dependent processes.     

Synergism of interleukin 6 and 1 alpha,25-dihydroxyvitamin D3 in induction of myeloid differentiation of human leukemic cell lines.

Interleukin 6 (IL-6) is a multifunctional cytokine with an important role in immunity. We analyzed the effect of recombinant human IL-6 in combination with 1 alpha,25-dihydroxyvitamin D3 (Vit. D3) on differentiation of the human myeloid leukemic cell lines U937 and HL-60 with respect to alterations in antigen expression and functional activity. Of a panel of antigens analyzed, only CD11b (the alpha chain of CR3), and CD14 (a cell surface protein recognizing the lipopolysaccharide-binding protein-lipopolysaccharide complex) had significantly increased expression. Expression of ICAM-1 (CD54), a ligand for LFA-1, was also found to be enhanced with a concomitant increase of ICAM-1 mRNA levels. Enhanced nonspecific esterase levels and induction of respiratory burst activity confirmed that cell differentiation was induced. Furthermore, IL-6 and Vit. D3 had a profound effect on functional activities, as shown by enhancement of rosetting between sheep erythrocytes, sensitized with C3bi (EAC), and either U937 or HL-60 cells. Also, phorbol myristate acetate-induced homotypic adhesion of U937, which is ICAM-1 dependent, was markedly induced by these agents. These results indicate an important role of IL-6 and Vit. D3 in myeloid cell function and development.     

Biochemical and functional alterations induced by CD23 ligation in the human promonocytic cell line U937.

The early events triggered in interleukin-4 (IL-4)-stimulated U937 cells by ligation of CD23/Fc epsilon RII with specific monoclonal antibodies (mAb) were analysed, as a model of the action of this molecule on the differentiation of promonocytic cells. As well as IL-4-activated human monocytes, addition of anti-CD23 mAb to IL-4-treated U937 cells triggered cAMP accumulation but did not evoke significant polyphosphoinositide hydrolysis. However, by a microspectrofluorometric technique allowing single cell analysis, anti-CD23 mAb was found to elicit calcium mobilization in these cells. In addition, the treatment induced phenotypic alterations in these cells, as evidenced by the acquisition of the monocyte marker CD14 and the increase of the alpha-chain (CD11a) and of the common beta-chain (CD18) of the leucocyte function-associated antigen 1 (LFA-1) family antigens. Although weaker than in monocytes, CD23 ligation evoked a small secretion of the pro-inflammatory mediators IL-6 and thromboxane B2. These data suggest that a significant maturation of promonocytic cells towards a more mature monocytic phenotype can be achieved through successive exposure to IL-4 and CD23 ligation.     

Effects of 1 alpha,25-dihydroxyvitamin D3 and cytokines on the expression of MHC antigens, complement receptors and other antigens on human blood monocytes and U937 cells: role in cell differentiation, activation and phagocytosis.

The effect of calcitriol/1 alpha,25-dihydroxyvitamin D3, alone and in combination with cytokines, on the expression of various antigens (Ag) on human peripheral blood monocytes and U937 cells was studied by flow cytometry. Both constitutive and interferon-gamma (IFN-gamma), interleukin-4 (IL-4), IL-6 and tumour necrosis factor-alpha (TNF-alpha)-induced human leucocyte antigen (HLA)-DR, HLA-DP and HLA-DQ Ag expression on monocytes was significantly down-regulated by calcitriol, IL-10 and transforming growth factor-beta (TGF-beta). The effects of calcitriol were concentration dependent and reached maximal inhibitory levels after 3-5 days. Modulation of HLA-DR by calcitriol and IFN-gamma at the protein level correlated with the amount of mRNA specific for the HLA-DR alpha-chain, as judged by Northern blot analysis. The basal as well as IL-4, IL-6, IFN-gamma, TNF-alpha and TGF-beta-driven levels of HLA-ABC Ag were significantly diminished by calcitriol. On U937 cells calcitriol markedly induced CD11a and CD11b expression and weakly up-regulated CD11c whereas on monocytes, constitutive CD11a, CD11b and CD11c expression was significantly down-regulated by calcitriol. The expression of CD14 Ag was strongly induced on U937 cells but only modestly on monocytes. Both the basal level of CD71 and IL-4, IFN-gamma or TNF-alpha-driven expression was diminished on calcitriol-treated U937 cells. In addition, calcitriol suppressed the expression of CD71 Ag on monocytes. The ability of monocytes to phagocytize opsonized Escherichia coli was diminished by calcitriol. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates expression of MHC, CD11b, CD11c, CD14 and CD71 Ag on both monocytes and U937 cells, and impairs the phagocytic property of monocytes.     

β2-Adrenoceptor stimulation augments the IL-4-induced CD23 expression and release and the expression of differentiation markers (CD14, CD18) by the human monocytic cell line, U 937

The effect of β_2-adrenoceptor agonists and interleukin-4 (IL-4) on the CD23 expression on, and release from, the human promonocytic cell line, U 937, was investigated. As assessed by flow cytometry, incubation of U 937 cells in the presence of salbutamol, fenoterol or IL-4 induced a concentration- and time-dependent increase in CD23 expression, that was maximal after 48 hr and followed by a decrease thereafter. In addition, salbutamol potentiated the effect of IL-4, the optimal concentration of the drug being a function of the concentration of this cytokine. This synergy between IL-4 and β_2-adrenoceptor agonists was also observed for the release of the soluble form of CD23. The effect on CD23 expression of salbutamol and fenoterol, but not of IL-4, was blocked in the Wesence of d, l-propranolol (1 μm) or butoxamine (1 μm). The α-adrenoceptor agonist, norepinephrine (1 μm), was ineffective in inducing CD23 expression or potentiating the one evoked by IL-4. Salbutamol down-regulated the expression of Fc_γRI (CD64) and Fc, RII (CD32) whereas IL-4 was ineffective. Only when added together at the onset of the culture did salbutamol and IL-4 induce, after 48 hr, the expression of the monocyte marker, CD 14. The expression of CD 18 was up-regulated in response to salbutamol either alone or in combination with IL-4, this cytokine alone being inefficient. These data suggest that IL-4 and β_2-adrenoceptor agonists induce differentiation of U 937 cells into monocyte-like cells.     

维生素D3诱导的U937细胞中CD14表达的实验研究

目的: 探讨维生素D3诱导U937细胞上CD14蛋白的表达及其对内毒素 (LPS)刺激的反应性。方法: 用0. 1μmol/LVitD3与U937细胞共同培养 24h诱导CD14基因的表达, 并观察U937细胞对不同浓度的LPS刺激不同时间的反应性。结果: VitD3能稳定诱导U937细胞表达CD14mRNA和CD14蛋白。经VitD3诱导的U937细胞对LPS刺激的敏感性显著增强, 表现为低浓度LPS刺激即能诱导该细胞核中NF -κB激活, 促进TNF- αmRNA的转录和表达, 并将表达的TNF α释放入培养上清中。结论: VitD3能诱导U937细胞中CD14基因和蛋白的表达, 并增加其对LPS刺激的反应性。     

Influence of IL-2 and IL-4 on the IgE synthesis and the IgE-binding factor (sCD23) production by human lymphocytes in vitro.

The influence of IL-2 and IL-4 on the mitogen-induced immunoglobulin E and IgG production in vitro was analysed. Furthermore the expression of Fc epsilon RII (CD23 antigen), as well as the release of its soluble products, the isotype-specific IgE binding factors (IgE-BF), was determined. Recombinant IL-2 (rIL-2) exerted opposite effects on the synthesis of IgE by human lymphocytes that were stimulated either by pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I (SAC). rIL-2 induced a dose-dependent suppression of IgE and IgG synthesis in the presence of PWM. This effect was accompanied by a significant decrease of IgE-binding factor (BF), whereas the expression of Fc epsilon RII was not significantly modulated by rIL-2. A marked increase of IgE production was observed when lymphocytes, prestimulated with SAC for 48 hr, were further incubated with increasing amounts of rIL-2 for 6 days. In contrast, IL-4 in concentrations ranging from 500 to 4.9 U/ml did not lead to an enhancement of IgE synthesis in lymphocytes that were prestimulated with SAC. However, SAC-induced IgG secretion was significantly enhanced by 2.3 U/ml of rIL-4. A dose-dependent enhancement of IgE-BF was observed in SAC-prestimulated lymphocyte cultures in the presence of rIL-2 as well as rIL-4. These results demonstrate that the mitogen used for lymphocyte activation, T-cell-derived lymphokines such as IL-2 and IL-4, and IgE-specific binding factors (soluble CD23), are responsible for the induction of human IgE antibody production in vitro.     

Allergen-induced CD23 on CD4+ T lymphocytes and CD21 on B lymphocytes in Patients with allergic asthma: Evidence and regulation

Interaction of CD4⁺ T cells and B cells is necessary for IgE production. It has been recently demonstrated that cell surface antigen CD21 is a ligand for CD23 (Fc?RII) and that the pairing of these molecules may participate in the control of IgE production. In this study we investigated the effect of the Dermatophagoides pteronyssinus (Dpt) allergen and recombinant interleukin(rIL)-4 on the expression of CD21 and CD23 on T and B cells of asthmatic patients allergic to Dpt and of healthy controls. Peripheral blood mononuclear cells (PBMC) were incubated alone or with Dpt allergen (100 biological units/ml) and/or rIL-4 (100 U/ml) for up to 7 days. The flow-cytometric analysis of double-fluorescence staining revealed that Dpt allergen and/or rIL-4 induced CD23 on CD4⁺ T lymphocytes only in allergic patients. The allergen-induced CD23 on T cells is de novo synthesized antigen since no induction of CD23 on T cells was observed in cultures with 0.4 μg/ml actinomycin D. Moreover, 100 U/ml of interferon-γ inhibited the induction of CD23 on CD4⁺ T cells. T cells obtained from healthy donors did not express CD23 or CD21 antigen upon incubation with allergen and/or rIL-4. Although rIL-4 also induced CD23 in controls, the expression was only observed on CD20⁺ cells. The allergen alone induced a significant elevation of the mean fluorescence intensity of both CD21 and CD23 only in allergic in dividuals. When the cell proliferation was analyzed, a slightly increased stimulation index upon cultivation of PBMC was obtained from non-allergic donors as well, but less than in allergic patients. The co-expression of major histocompability complex class II molecules and CD23 on CD4⁺ T lymphocytes in allergic patients, as assessed by the three-color immunofluorescence analysis, indicates that these cells were activated. We conclude that CD4⁺ T lymphocytes possess a unique capability to express CD23 upon exposure to allergen. Moreover, the allergen-mediated induction of CD23 on T cells observed only in allergic patients may be the reason for the increase of IgE production. This would not occur in non-allergic individuals as there is no CD23 expression on T cells.     

Effect of recombinant human interleukin 2 (rIL-2) on normal peripheral blood B cells and B lymphoblastoid cell lines

The role of interleukin 2 (IL-2) for growth and differentiation of normal and malignant B cells still remains controversial. We assessed normal peripheral blood B cells and cell lines derived from patients with B non-Hodgkin's lymphomas (NHLs) with respect to their responsiveness to recombinant human IL-2 (rIL-2). The NHL cell lines used in our experiments expressed the Tac antigen (CD25)--a compound of the IL-2 receptor (IL-2R)--in a percentage ranging from 28 to 57%. As measured in a [3H]thymidine uptake assay, the normal peripheral blood B cells demonstrated a dose-dependent proliferative response to rIL-2, whereas the NHL cells did not show any responsiveness to rIL-2. In a clonogenic culture assay we evaluated the colony formation of the NHL cells and found a decrease of 28 to 41% on average in the presence of rIL-2 (10-50 U/ml). This moderate inhibitory effect on the clonal growth of the NHL cells was not due to a differentiation inducing effect of rIL-2, as studied by measuring the Ig production under increasing doses of rIL-2 (1 to 100 U/ml). Thus, malignant NHL B cells may express the CD25 compound of the IL-2 receptor on their surface, demonstrating a different functional responsiveness to rIL-2 compared to normal peripheral blood B cells.     

Human immunodeficiency virus infection of monocytic and T-lymphocytic cells: Receptor modulation and differentiation induced by phorbol ester

The monocytic leukemic cell line U937 can be infected with human immunodeficiency virus type 1 (HIV-1) to become permanently infected virus producers. Uninfected U937 cells express T4 (CD4) antigen and form syncytia when mixed with HIV-1 producing cells. Anti-T4 monoclonal antibodies block syncytium formation indicating that the HIV-1 receptors on U937 cells include T4 antigen. The promyelocytic leukemic cell line HL60, while expressing only low amounts of surface T4 and not forming syncytia on exposure to HIV-1, can be infected by HIV-1 at lower efficiency than U937 and T-cell lines. 12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment induces the differentiation of U937 cells into macrophages. HIV-infected U937 cells retain the ability to differentiate, though less efficiently, as shown by the appearance of monocyte/macrophage surface markers. T4 antigen on both U937 and T-cell lines is down regulated by TPA treatment. Functional receptors for HIV-1, assayed by syncytium induction and pseudotype plating, are lost concomitantly with T4 antigen following TPA treatment of U937 cells and T cells.     

Tumour necrosis factor-alpha augments the expression of Fc IgE receptor (Fc epsilon RII/CD23) on human monocytic cell lines and down-regulates interleukin-4-driven Fc epsilon RII expression on monocytes.

We investigated the expression of the low affinity Fc IgE receptor (Fc epsilon RII/CD23) on the human monocytic cell lines U937, THP-1, Mono-Mac-6, and cultured human peripheral blood monocytes under stimulation with human tumour necrosis factor-alpha (TNF-alpha) and other cytokines. Fc epsilon RII was demonstrated by flow cytometry analysis employing the anti-Fc epsilon RII monoclonal antibody 3-5. TNF-alpha alone had a weak but significant stimulating effect on the Fc epsilon RII expression on the cell lines U937 and THP-1, and very modestly on Mono-Mac-6 cells. TNF-alpha strongly synergized with interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). IFN-alpha per se was ineffectual, but was able to increase the TNF-alpha effect. Furthermore, the action of TNF-alpha was slightly augmented by human IL-6. Similar effects were noted with TNF-beta alone or in combination with other cytokines. Interestingly, on human monocytes TNF-alpha weakly reduced the basal level of Fc epsilon RII, and markedly diminished the IL-4-induced Fc epsilon RII expression. Our results indicate that several cytokines may interact in a cytokine network to modulate Fc epsilon RII expression on monocytic cell lines. On human blood monocytes, TNF-alpha, like IFN-gamma or IL-6, counteracts the IL-4-induced Fc epsilon RII expression. These data suggest different regulatory pathways of Fc epsilon RII expression on blood monocytes and myelomonocytic cell lines.     

Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes.

The mononuclear phagocyte plays an important role in the regulation of microbe-induced inflammation, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. To evaluate the effects of the microbial flora associated with chronic adult periodontitis on cytokine induction, lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis was used to stimulate naive and phorbol ester-primed U937 monocytic cells, as well as elutriated human peripheral blood monocytes. We assessed the effect of this LPS, in comparison to that of LPS from Escherichia coli, on cell proliferation, cytokine induction, and surface expression of the LPS receptor CD14. P. gingivalis LPS stimulated proliferation of U937 cells at concentrations of greater than 1 ng/ml, while E. coli LPS inhibited proliferation. Phorbol myristic acid (PMA)-treated U937 cells and elutriated monocytes responded to E. coli LPS activation by producing tumor necrosis factor alpha (TNF-alpha) mRNA and protein; however, P. gingivalis LPS induced greater numbers of TNF-alpha mRNA-positive cells and higher (P < 0.05) levels of protein than did E. coli LPS. Both cell types expressed interleukin-1 beta (IL-1beta) mRNA and protein in response to either LPS treatment. Compared with E. coli LPS, P. gingivalis LPS induced significantly (P < 0.05) higher numbers of IL-1 mRNA-positive U937 cells and elutriated monocytes, as well as production of significantly more (P < 0.05) IL-1 protein by the monocytes. The PMA-treated U937 cells and the monocytes produced high levels of IL-1 receptor antagonist mRNA and protein which were only marginally affected by the LPS preparations. While E. coli LPS induced expression of CD 14 on the surface of PMA-primed U937 cells and monocytes, P. gingivalis LPS exhibited a significantly (P < 0.05) greater ability to enhance receptor levels. Our results indicate that P. gingivalis LPS can activate the mononuclear phagocyte for proliferation, cytokine production, and CD14 expression, providing evidence for the potential of this bacterial component to act as a critical regulatory factor in the chronic inflammatory response associated with periodontitis.     

Maturation of human myelomonocytic leukemia cells following ligation of the low affinity receptor for IgE (Fc{varepsilon}RII/CD23)

The regulation of the low affinity receptor for IgE (FcRII/CD23)expression and its role were investigated in U937 cell lineand in leukemic cells from a patient (Amb) with acute myeloblasticleukemia. Both cell populations were CD23^– but could acquireCD23 expression following treatment with IL-4. CD23⁺ cells,however, remained blastic and did not show any significant phenotypicaland functional modifications. Following ligation of the CD23on U937 and Amb cells by anti-CD23 mAb, these leukemic cellsdifferentiated into mature monocyte/macrophage-like cells. CD23ligation promoted the expression of the monocyte marker, CD14,increased the expression of the common ß chain ofthe LFA-1 family (CD18), and down-regulated the expression ofthe promonocytic marker CD33. Morphological and phenotyplcalchanges were associated with functional modifications as CD23ligation allowed the acquisition of the oxidative metabolismin leukemic cells as revealed by luminol-dependent chemllumlnescence.As In mature monocytes, CD23 ligation induced an accumulationof intracellular cAMP in leukemic cells. These data indicatethat ligation of CD23 may induce the maturation of myelomonocyticcells into monocytic-like cells.     

Influence of Tumour Necrosis Factor-α on the Expression of Fc IgG and IgA Receptors, and Other Markers by Cultured Human Blood Monocytes and U937 Cells

The expression of Fc receptors for IgG (FcγR) and IgA (FcαR) and of various other antigens on the human monocytic cell line U937 and peripheral blood monocytes, under stimulation with human recombinant tumour necrosis factor-α (TNF-α) and other cytokines, was investigated by flow cytomelry. TNF-α, as well as interferon-γ (IFN-γ) or interleukin-6 (IL-6) had a significant up-regulating effect on U937 expression of FcγRI/CD64. Furthermore, the action of TNF-α was augmented by IL-6, and more evidently by IFN-γ. IFN-α alone had only a marginal effect, but was able to increase the TNF-α-driven FcγRI expression. In contrast to U937 cells, TNF-α did not enhance significantly FcγRI expression on human monocytes. Interestingly, on both U937 cells and monocytes. FcαR was augmented markedly by TNF-α. Furthermore, TNF-α induced the expression of HLA-DR and HLA-DP antigens on monocytes and U937 cells. The expression of FcγRII/CD32, FcγRIII/CD16. CD14, complement receptor type 1 (CR1/CD35). CR4 (CD11c/CD18), and MHC class-I antigens, was not influenced significantly by TNF-α. The results of this study show that TNF-α may act on human mononuclear phagocytes, alone or in combination with other cytokines, by modulating the expression of various cell-surface antigens.     

Interferon and (2'-5')oligoadenylate enhance the expression of low affinity receptors for IgE (Fc epsilon R2/CD23) on the human monoblast cell line U937

The regulation of low affinity IgE receptor (Fc epsilon R2/CD23) expression on the human monoblast cell line U937 was examined by an anti-Fc epsilon R2/CD23 monoclonal antibody (H107) and the cDNA probe for Fc epsilon R2/CD23. Alpha interferon (IFN-alpha) and its intracellular mediator, (2'-5')oligoadenylate (2, 5-A), induced Fc epsilon R2/CD23 expression on U937 with no significant increase of the Fc epsilon R2/CD23 mRNA. PMA and IFN-gamma increased both surface Fc epsilon R2/CD23 expression and the Fc epsilon R2/CD23 mRNA levels. IFN-alpha effectively induced 2, 5-A synthetase activity in U937 cells, whereas IFN-gamma induced little. The results suggest that the mechanisms of enhancement of Fc epsilon R2/CD23 expression on U937 cells by IFN-alpha and IFN-gamma are different and that 2, 5-A may play an important role in the Fc epsilon R2/CD23 expression on U937 cells induced by IFN-alpha.     

IL-12 and IL-23 modulate plasticity of FoxP3+ regulatory T cells in human Leprosy

Leprosy is a bacterial disease caused by M. leprae. Its clinical spectrum reflects the host’s immune response to the M. leprae and provide an ideal model to investigate the host pathogen interaction and immunological dysregulation. Tregs are high in leprosy patients and responsible for immune suppression of the host by producing IL-10 and TGF-β cytokines. In leprosy, plasticity of Tregs remain unstudied. This is the first study describing the conversion of Tregs into Th1-like and Th17-like cells using in vitro cytokine therapy in leprosy patients. Peripheral blood mononuclear cells from leprosy patients were isolated and stimulated with M. leprae antigen (MLCwA), rIL-12 and rIL-23 for 48 h. Expression of FoxP3 in CD4⁺CD25⁺ Tregs, intracellular cytokines IFN-γ, TGF-β, IL-10 and IL-17 in Tregs cells were evaluated by flow cytometry (FACS) after stimulation. rIL-12 treatment increases the levels of pStat4 in Tregs and IFN-γ production. In the presence of rIL-23, pStat3⁺ and IL-17A⁺ cells increase. rIL-12 and r—IL-23 treatment downregulated the FoxP3 expression, IL-10 and TGF-β production by Tregs and enhances the expression of co-stimulatory molecules (CD80, CD86). In conclusion rIL-12 converts Tregs into IFN-γ producing cells through STAT-4 signaling while rIL-23 converts Tregs into IL-17 producing cells through STAT-3 signaling in leprosy patients. This study may helpful to provide a new avenue to overcome the immunosuprression in leprosy patients using in vitro cytokine.     

Impaired accessory cell function in a human dendritic cell line after human immunodeficiency virus infection

We generated human dendritic cell (DC) hybridoma cell lines by fusing HGPRT-deficient promonocytic U937 cells with immature DCs obtained by culturing peripheral blood monocytes with interleukin-4 (IL-4; 1,000 U/ml) and granulocyte-macrophage colony-stimulating factor (100 U/ml) for 7 days and mature DCs by treatment with tumor necrosis factor alpha (12.5 microg/ml) for 3 days. Only one fusion with immature DCs was successful and yielded four cell lines--HB-1, HB-2, HB-3, and HB-9--with an overall fusion efficiency of 0.0015%. The cell lines were stable in long-term culture, displayed morphological features typical of DCs, and expressed distinct class I and class II molecules not present on U937 (A*031012, B*51011, Cw*0701, DRB3*01011 52, and DR5*01011). A representative cell line, HB-2, that expressed DC markers including CD83, CD80 and CD86 could be induced to produce IL-12 through CD40 stimulation. After human immunodeficiency virus (HIV) infection, there was impairment of antigen-presenting cell (APC) function, which was manifested by an inability to stimulate allogeneic T-cell responses. There was no change in expression of major histocompatibility complex class I and class II antigens, CD83, CD40, CD4, CD11c, CD80, CD86, CD54, and CD58, or IL-12 production in the HIV-infected HB-2 cells. The HIV-infected HB-2 cells induced T-cell apoptosis in the cocultures. T-cell proliferation could be partially restored by using ddI, indinivir, and blocking anti-gp120 and anti-IL-10 antibodies. Our data suggest that there are multiple mechanisms that DCs use to inhibit T-cell responses in HIV-infected patients. The HB-2 cell line could be a useful model system to study APC function in HIV-infected DCs.