Human hemoglobin shares bioactivities ascribed to human tumor necrosis factor-alpha

Hemoglobin mediated cytotoxicity and apoptosis has been evaluated in Tumor necrosis factor-alpha (TNF-alpha) sensitive cell line, U937 and compared with TNF-alpha. Both species of hemoglobin, Hemoglobin A2 and Hemoglobin A0 induced apoptosis and cytotoxicity in U937 cell as measured by flow cytometry and 3-(4,5-dihydro-6-(4-(3,4-dimethoxybenzooyl)-1-piperazinyl)-2(1H)-quinoline (MTT) assay respectively. Different concentration of Hemoglobin A0 (4 ng/mL to 4000 ng/mL) induced apoptosis ranging from 9% to 16% in U937 cells. 4000 ng/mL hemoglobin A0 showed maximal apoptotic cells. TNF-alpha showed 87% apoptotic U937 cells at concentration of 1 pg/mL. HbA0 displayed cytotoxicity in U937 cell line at higher concentration in comparison to TNF-alpha. 4000 ng/mL of hemoglobin A0 showed optimal cytotoxic response in U937 cells. A dose response curve was also observed with varying doses of hemoglobin A0. U937 cells pretreated with serum activated LPS for 1 hr and incubated with different concentration of hemoglobin or human TNF-alpha for 24 h reduced the cytotoxic effect on U937. Dexamethasone treatment of U937 cells helped in protecting the HbA0 and HbA2 mediated cytotoxicity and anti-TNF-alpha antibody neutralized the hemoglobin mediated apoptosis and cytotoxicity. It is therefore apparent that human hemoglobin shares some of the bioactivities previously ascribed to TNF-alpha. Sharing of bioactivities of TNF-alpha by hemoglobin is interesting and suggests that cell free hemoglobin can mimic TNF-alpha functionally.     

Dual role of TNF-alpha in NK/LAK cell-mediated lysis of chronically HIV-infected U1 cells. Concomitant enhancement of HIV expression and sensitization of cell-mediated lysis

The U937-derived chronically HIV-infected U1 cell line and uninfected U937 cell clones were efficiently lysed by both unstimulated (NK) and IL-2-stimulated (lymphokine-activated killer; LAK) peripheral blood mononuclear cells (PBMC) of healthy HIV-seronegative donors. Pretreatment of target cells with IFN-gamma down-modulated killing of both U1 cells and two U937 cell clones, and up-regulated MHC class I expression. In contrast, TNF-alpha enhanced the sensitivity of infected U1 cells, but not of U937 cell clones to NK / LAK cell lysis. Co-cultivation of IL-2-stimulated PBMC with U1 cells triggered expression and replication of HIV by cell-cell contact, and this effect was inhibited by anti-TNF-alpha antibodies (Ab); virus production was partially inhibited by zidovudine. Of interest, anti-TNF-alpha Ab protected U1 cells from LAK cell activity. Thus, TNF-alpha can induce HIV expression from chronically infected U1 cells, but also plays an important role in sensitizing these cells to lysis.     

Human Hemoglobin Shares Bioactivities Ascribed to Human Tumor Necrosis Factor‐α

Hemoglobin mediated cytotoxicity and apoptosis has been evaluated in Tumor necrosis factor‐α (TNF‐α) sensitive cell line, U937 and compared with TNF‐α. Both species of hemoglobin, Hemoglobin A₂ and Hemoglobin A₀ induced apoptosis and cytotoxicity in U937 cell as measured by flow cytometry and 3‐(4,5‐dihydro‐6‐(4‐(3,4‐dimethoxybenzooyl)‐1‐piperazinyl)‐2(1H)‐quinoline (MTT) assay respectively. Different concentration of Hemoglobin A₀ (4 ng/mL to 4000 ng/mL) induced apoptosis ranging from 9% to 16% in U937 cells. 4000 ng/mL hemoglobin A₀ showed maximal apoptotic cells. TNF‐α showed 87% apoptotic U937 cells at concentration of 1 pg/mL. HbA₀ displayed cytotoxicity in U937 cell line at higher concentration in comparison to TNF‐α. 4000 ng/mL of hemoglobin A₀ showed optimal cytotoxic response in U937 cells. A dose response curve was also observed with varying doses of hemoglobin A₀. U937 cells pretreated with serum activated LPS for 1 hr and incubated with different concentration of hemoglobin or human TNF‐α for 24 h reduced the cytotoxic effect on U937. Dexamethasone treatment of U937 cells helped in protecting the HbA₀ and HbA₂ mediated cytotoxicity and anti‐TNF‐α antibody neutralized the hemoglobin mediated apoptosis and cytotoxicity. It is therefore apparent that human hemoglobin shares some of the bioactivities previously ascribed to TNF‐α. Sharing of bioactivities of TNF‐α by hemoglobin is interesting and suggests that cell free hemoglobin can mimic TNF‐α functionally.     

Effects of methylprednisolone on intracellular bacterial growth.

Clinical studies have shown positive associations among sustained and intense inflammatory responses and the incidence of bacterial infections. Patients presenting with acute respiratory distress syndrome (ARDS) and high levels of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), and IL-6, have increased risk for developing nosocomial infections attributable to organisms such as Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter spp., compared to those patients with lower levels. Our previous in vitro studies have demonstrated that these bacterial strains exhibit enhanced growth extracellularly when supplemented with high concentrations of pure recombinant TNF-alpha, IL-1 beta, or IL-6. In addition, we have shown that the intracellular milieu of phagocytic cells that are exposed to supraoptimal concentrations of TNF-alpha, IL-1 beta, and IL-6 or lipopolysaccharide (LPS) favors survival and replication of ingested bacteria. Therefore, we hypothesized that under conditions of intense inflammation the host's micromilieu favors bacterial infections by exposing phagocytic cells to protracted high levels of inflammatory cytokines. Our clinical studies have shown that methylprednisolone is capable of reducing the levels of TNF-alpha, IL-1 beta, and IL-6 in ARDS patients. Hence, we designed a series of in vitro experiments to test whether human monocytic cells (U937 cells) that are activated with high concentrations of LPS, which upregulate the release of proinflammatory cytokines from these phagocytic cells, would effectively kill or restrict bacterial survival and replication after exposure to methylprednisolone. Fresh isolates of S. aureus, P. aeruginosa, and Acinetobacter were used in our studies. Our results indicate that, compared with the control, stimulation of U937 cells with 100-ng/ml, 1.0-microg/ml, 5.0-microg/ml, or 10.0-microg/ml concentrations of LPS enhanced the intracellular survival and replication of all three species of bacteria significantly (for all, P = 0.0001). Stimulation with < or =10.0 ng of LPS generally resulted in efficient killing of the ingested bacteria. Interestingly, when exposed to graded concentrations of methylprednisolone, U937 cells that had been stimulated with 10.0 microg of LPS were able to suppress bacterial replication efficiently in a concentration-dependent manner. Significant reduction in numbers of CFU was observed at > or =150 microg of methylprednisolone per ml (P values were 0.032, 0.008, and 0.009 for S. aureus, P. aeruginosa, and Acinetobacter, respectively). We have also shown that steady-state mRNA levels of TNF-alpha, IL-1 beta, and IL-6 in LPS-activated cells were reduced by treatment of such cells with methylprednisolone, in a concentration-dependent manner. The effective dose of methylprednisolone was 175 mg, a value that appeared to be independent of priming level of LPS and type of mRNA. We therefore postulate that a U-shaped relationship exists between the level of expression of TNF-alpha, IL-1 beta, and IL-6 within the phagocytic cells and their abilities to suppress active survival and replication of phagocytized bacteria.     

U937 cells stimulated with opsonised zymozan particles provide a convenient laboratory source of tumour necrosis factor alpha.

The U937 cell line has been shown to generate tumour necrosis factor alpha (TNF-alpha) in response to soluble stimuli such as PMA and LPS, but only after treatment with GM-CSF. We report here the generation of TNF-alpha from U937 cells following phagocytosis of opsonised zymozan particles without the need for pre-treatment with GM-CSF. The release of TNF-alpha from U937 cells was demonstrated by a specific radioimmunoassay, L929 cell killing and neutrophil 'priming'. The biological activities in the cell supernatant were inhibited by TNF-alpha antiserum. Phagocytosis was required for TNF-alpha production. Non-opsonised zymozan or latex particles which were not phagocytosed or pretreatment with cytochalasin B, which inhibited phagocytosis of opsonised zymozan particles, all failed to trigger TNF-alpha production. Phagocytosis failed to trigger detectable IL-1 generation, and production of IL-6 was insufficient to produce biological effects on neutrophils. The U937 supernatant thus provides a source of human TNF-alpha which can be generated conveniently and cheaply for experimental investigations.     

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.     

Interleukin-8 fails to induce human immunodeficiency virus-1 expression in chronically infected promonocytic U1 cells but differentially modulates induction by proinflammatory cytokines

This study addresses the role of interleukin (IL)-8, a CXC-chemokine, the level of which is reported to be raised in the peripheral circulation of human immunodeficiency virus-1 (HIV-1)-infected individuals, during the induction of HIV-1 expression from latency and during cytokine-mediated HIV-1 up-regulation. IL-8 at the higher concentrations tested (> or = 100 ng/ml) was unable to induce HIV-1 expression in the chronically infected promonocytic U1 cell line, as measured by p24 antigen enzyme-linked immunosorbent assay (ELISA), whereas at lower concentrations of 1 and 10 ng/ml, constitutive HIV-1 expression was only marginally reduced. HIV-1 replication in acutely infected U937 cells was also significantly reduced by IL-8. The potent up-regulation of HIV-1 expression in U1 cells by tumour necrosis factor-alpha (TNF-alpha) remained unaffected by the addition of IL-8. HIV-1 induction by IL-1beta, IL-6 and TNF-beta, cytokines grouped here as intermediate HIV-1 inducers, was suppressed by IL-8 at concentrations of 1 and 10 ng/ml. However, IL-8 at 100 ng/ml did not significantly alter the effect of IL-1beta, synergized with IL-6 in enhancing, and marginally suppressed TNF-beta-induced HIV-1 expression. IL-8 suppressed granulocyte-macrophage colony-stimulating factor (GM-CSF) and enhanced interferon-gamma (IFN-gamma)-induced HIV-1 expression in a dose-dependent manner. Pretreatment of U1 cells with IL-8 did not alter the IL-8-mediated effects on cytokine-induced HIV-1 expression, suggesting that this chemokine exerts its effect at the time of HIV-1 induction or at a postinduction stage. Furthermore, IL-8 was itself induced by cytokines that up-regulate HIV-1 expression in U1 cells and the levels produced correlated directly with the levels of p24 antigen produced, suggesting common pathways for cytokine induction of both HIV-1 and IL-8. These results show that IL-8, typically a non-inducer, can differentially modulate HIV-1 expression in U1 cells and that this is dependent on the inducing cytokine and on the concentration of IL-8.     

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 IFN-alpha on the inflammatory response of swine leukocytes to bacterial endotoxin.

Because low-dose interferon-alpha (IFN-alpha) treatment had proved effective in several models of chronic inflammation and autoimmune disease, a possible role of IFN-alpha in modulating the response of swine leukocytes to bacterial endotoxin was investigated in this study. Exposure of swine peripheral blood mononuclear cells (PBMC) to low concentrations of human IFN-alpha caused a strong, dose-dependent decrease in CD14 expression, the lowest level being observed at 5 U/ml IFN-alpha. This result was confirmed if PBMC were later exposed to purified lipopolysaccharide (LPS). A 10-fold lower IFN-alpha concentration (0.5 U/ml) caused the largest reduction of tumor necrosis factor-alpha (TNF-alpha) accumulation in the medium of pulmonary alveolar macrophages (PAM), stimulated with bacterial LPS. At 0.5 U/ml, the expression of the TNF-alpha gene in PAM was also strongly reduced, as opposed to cells pretreated with 50 U/ml IFN-alpha. In contrast, expression of the interleukin-1beta (IL- 1beta) gene was stimulated and that of the IL-6 gene was not significantly affected at both IFN-alpha concentrations. Results point to an important role of IFN-alpha in control of the inflammatory response to bacterial endotoxin in pigs.     

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。     

Selective up-regulation of human granulocyte integrins and complement receptor 1 by cytokines.

The percentage of human granulocytes expressing the integrins CD11b and CD11c as well as complement receptor 1 (CD35) was increased by short-term incubation of whole blood with interleukin-2 (IL-2), interleukin-4 (IL-4) and tumour necrosis factors alpha and beta (TNF-alpha and TNF-beta). The mean fluorescence intensity of granulocyte CD18 was also increased by the above cytokines, whilst that of CD11b was only increased by TNF-alpha. Up-regulation of granulocyte CD18 expression was seen with 1 U/ml of IL-2, TNF-alpha or TNF-beta, in contrast to the effect of IL-4 which was only observed with 100 U/ml. Similarly, enhanced expression of CD35 was induced by 1 U/ml of IL-2 or TNF-alpha but not by concentrations of IL-4 or TNF-beta lower than 100 U/ml. Cytokine effects on the CD11/CD18 complex and CD35 molecules were not modified by cycloheximide, suggesting that their increased expression was not due simply to synthesis de novo. None of the granulocyte surface determinants investigated was altered upon short-term incubation of blood with either IL-1, IL-6 or interferon-gamma (IFN-gamma). The demonstration in vitro that cytokines selectively up-regulate granulocyte integrins and complement receptor 1, suggests that similar mechanisms may be operating during the control of granulocyte-mediated inflammatory processes.     

IL—4对治疗前后亚急性重型病毒性肝炎患者PBMCs TN …

OBJECTIVE: The aim of this study was to compare the influence of IL-4 on the expression of TNF-alpha and IL-1 alpha mRNA by PBMCs from patients with subfulminant viral hepatitis(SFH) before and after IL-4 treatment. METHODS: The expression levels of TNF-alpha and IL-1 alpha mRNA by PBMCs were assessed by semiquantitative RT-PCR. RESULTS: IL-4 suppressed the expression of both TNF-alpha and IL-1 alpha mRNA by PBMCs from SFH patients before and after treatment dose-dependently. However, their dose-response curves showed significant differences. The inhibitory effect could be observed at a concentration of 100 U/ml and was near to maximum at 1,000 U/ml in acute phase, whereas the same suppressive action of IL-4 was reached at 100 U/ml in recovery phase. When PBMCs were cultured in the presence of IL-4 at a concentration of 100 U/ml, IL-4 down-regulated the production of both TNF-alpha and IL-1 alpha mRNA by acute phase PBMCs only to 18.18-21.98% of control cells(in absence of IL-4), while the inhibitory rates were all near to 50% in recovery phases. Moreover, it was also found that the suppressive effect of IL-4 on the expression of PBMCs TNF-alpha and IL-1 alpha mRNA in the patients with endotoxinemia and HBeAg was significantly decreased in acute phase. CONCLUSIONS: The inhibitory effect of IL-4 on the expression of PBMCs TNF-alpha and IL-1 alpha mRNA is markedly decreased in acute phase as compared with that in recovery phase and this declined response may be related to endotoxinemia and viremia.     

Interleukin-6 production by tumor necrosis factor and lipopolysaccharide-stimulated rat renal cells

Interleukin-6 (IL-6) is produced by various cell types, including monocytes, fibroblasts, and endothelial cells. IL-6 has also been detected in the urine of normal and renal transplant patients. Thus, the possible production of this cytokine by glomeruli and mesangial cells was investigated. Rat glomeruli were obtained by serial sieving of cortical homogenates of blood-free kidneys. Mesangial cells were obtained from the glomeruli and cultured under standard methods in RPMI 1640 medium containing 15% fetal calf serum. Glomeruli or confluent monolayers cells were then incubated in RPMI 1640 for 18 hr, in the presence or not of tumor necrosis factor-alpha (TNF alpha), lipopolysaccharide (LPS), or platelet-activating factor (PAF). IL-6 activity was measured using the IL-6-dependent cell line subclone (B 9-9) and expressed with respect to a standard curve established with recombinant IL-6. Glomeruli generate IL-6 upon TNF alpha (100 ng/ml) and LPS (1 microgram/ml), 11,500 +/- 3000 and 22,000 +/- 7500 U/ml, respectively. Nonstimulated mesangial cells produced 50 +/- 5 U/ml (mean +/- SEM, n = 4) of IL-6. TNF alpha (1 ng/ml) and LPS (1 microgram/ml) induced the production of 800 +/- 90 and 40,000 +/- 5000 U/ml, respectively (n = 4). In contrast, PAF (0.1 nM-1 microM) did not increase IL-6 production from glomeruli or mesangial cells. These results demonstrate that renal cells spontaneously generate minimal amounts of IL-6 and that this production is significantly increased by TNF alpha or LPS. A synergy between LPS and TNF alpha was induced in glomerular cells with 10 ng/ml of TNF alpha and graded concentrations of LPS. Thus, the production of IL-6 by glomerular cells and its modulation by other cytokines or endotoxins may play a role in the local immunological processes leading to immune glomerular diseases.     

甘露聚糖结合凝集素诱导人白血病细胞系U937细胞凋亡

目的研究甘露聚糖结合凝集素（MBL）对白血病细胞系U937凋亡的影响。方法不同浓度MBL处理U937细胞后,应用CCK-8法分析细胞增殖情况,Hoechst33258染色观察细胞核及染色质,AnnexinV/PI双染流式细胞术分析细胞凋亡率,real-timePCR和免疫印迹分析凋亡相关基因及蛋白表达。结果 30～50μg/mlMBL培养72h,U937细胞增殖明显受抑,出现不同程度核固缩、核碎裂;随MBL浓度升高或作用时间延长,凋亡细胞数逐渐增多;Fas、Caspase-3mRNA、Fas和FasL蛋白表达量升高,Caspase-3和多腺苷二磷酸多聚酶（PARP）蛋白被剪切激活或失活。结论 MBL可诱导白血病细胞U937细胞凋亡,其机制与上调Fas表达、剪切Caspase-3和PAPR有关。     

Dual regulation of soluble tumor necrosis factor-alpha induced activation of human monocytic cells via modulating transmembrane TNF-alpha-mediated 'reverse signaling'

Transmembrane tumor necrosis factor-alpha (mTNF-alpha) is known to be the precursor of soluble TNF-alpha (sTNF-alpha). mTNF-alpha can act as a ligand on the TNF receptor- (TNFR)- bearing cell through 'forward signaling' or as a receptor on the TNF producing cell through 'reverse signaling'. In the current study, we investigated the role of mTNF-alpha-mediated reverse signaling in regulating sTNF-alpha-induced activation of human monocytic U937 cells. We demonstrated that pretreatment with sTNFRI, for inducing reverse signaling through mTNF-alpha, sensitized U937 cells to sTNF-alpha stimulation, as evidenced by an increase in reactive oxygen production and mRNA levels of proinflammatory cytokines (TNF-alpha, IL-1beta, and IL-8) in these cells. Further experiments revealed that IkappaB-alpha degradation was increased in the monocytic cells primed with sTNFRI, implying that reverse signaling of mTNF-alpha sensitizes U937 cells via an NF-kappaB-dependent mechanism. On the other hand, binding of sTNFRI to mTNF-alpha after sTNF-alpha-induced activation of U937 cells reduced mRNA stability (half-life) of IL-1beta and IL-8. The involvement of reverse signaling in the process was verified by using a mutated form of mTNF-alpha lacking the majority of the cytoplasmic domain. Our results clearly showed that enhanced mRNA degradation of the cytokines occurred only in U937 cells transfected with a wild-type mTNF-alpha, but not in those cells transfected with the mutant mTNF-alpha. Taken together, these data suggest that reverse signaling through mTNF-alpha may exert a double role in modulating sTNF-alpha bioactivity. It is positive when reverse signaling occurs prior to sTNF-alpha stimulation, while it is negative when reverse signaling occurs after the sTNF-alpha signal. Thus, our findings strengthen a role of mTNF-alpha-mediated reverse signaling in the regulation of immune-inflammatory response and control of inflammatory reaction.     

Surfactant protein-A enhances uptake of respiratory syncytial virus by monocytes and U937 macrophages

Surfactant protein (SP)-A is a known opsonin for a variety of pulmonary pathogens. SP-A enhances ingestion of these pathogens by interaction with an SP-A receptor (SP-AR) found on phagocytic cells such as peripheral blood monocytes (PBMC) and alveolar macrophages. Respiratory syncytial virus (RSV) is the most important respiratory pathogen in children. Recent studies have indicated that SP-A levels may be decreased in RSV bronchiolitis and pneumonia. In this study we examined the role of SP-A in uptake of RSV by both PBMC and U937 macrophages, a human macrophage cell line known to express SP-ARs. In addition, we studied the effect of SP-A- mediated uptake of RSV on production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 by these cells because incomplete immunity to recurrent RSV infection has been partially attributed to abnormal cytokine responses by macrophages. SP-A enhanced binding and uptake of fluorescently labeled RSV (RSV-FITC) by PBMC in a dose-dependent manner, with a maximal effect seen with 10 to 15 microg/ml SP-A as measured by both percent fluorescent monocytes and linear mean fluorescence (lmf) of individual cells. SP-A also enhanced uptake of RSV-FITC by U937 macrophages, with a maximal effect seen with 20 microg/ml SP-A as measured by both percent fluorescent monocytes and lmf. With respect to TNF-alpha levels, RSV alone slightly enhanced TNF-alpha production by PBMC and decreased TNF-alpha production by U937 macrophages measured at 12 h after addition of RSV. SP-A-mediated uptake of RSV significantly enhanced TNF-alpha production by PBMC and reversed the RSV-induced depression of TNF-alpha by U937 macrophages. RSV significantly enhanced IL-10 production by both cell types, which was reversed by SP-A-mediated uptake. These findings suggest that SP-A is an important opsonin for RSV and that SP-A-mediated uptake of RSV may alter some of the unusual cytokine responses that are postulated to be involved in incomplete immunity to recurrent infection.     

The effect of cytokines on the expression and function of Fc receptors for IgG on human myeloid cells

We examined expression and cytotoxic triggering capability of the three Fc receptors for IgG (Fc gamma R) on human monocytes, PMNs and myeloid cell lines after in vitro culture with various cytokines. Fc gamma R expression was evaluated using specific anti-Fc gamma R monoclonal antibodies (mAb). The cytotoxic capability of each Fc gamma R was examined after the effector cells were treated with the recombinant cytokines IFN-gamma. TNF alpha, GM-CSF, G-CSF, M-CSF, IL-1, IL-2, IL-3, IL-4, or IL-6. Hybridoma cell lines (HC) bearing antibody directed to Fc gamma RI (HC 32), Fc gamma RII (HC IV.3) or Fc gamma RIII (HC 3G8) were used as targets, as were chicken erythrocytes (CE) sensitized with heteroantibodies composed of anti-Fc gamma R mAbs (32, IV.3, 3G8) linked to anti-CE antibody. Only IFN-gamma treatment significantly increased Fc gamma R expression and then only Fc gamma RI. IFN-gamma dramatically up-regulated Fc gamma RI expression on all cells tested. However, ADCC was enhanced by treatment with a number of cytokines other than IFN-gamma. GM-CSF, TNF, and IFN-gamma treatment enhanced killing of HC 32 and HC IV.3 by in vitro cultured monocytes. G-CSF treatment enabled PMNs to kill HC through Fc gamma RII, whereas PMN killing of HC through Fc gamma RIII could not be induced by any of the cytokines studied. Although only IFN-gamma treatment increased ADCC of CE by monocytes, GM-CSF treatment as well as IFN-gamma treatment augmented ADCC of CE by PMNs. In addition to IFN-gamma treatment, IL-6 treatment enabled U937 cells to lyse CE. Whereas IFN-gamma-treated U937 cells killed CE through both Fc gamma RI and Fc gamma RII, IL-6-treated U937 cells killed CE only through Fc gamma RI. In addition to IFN-gamma treatment, G-CSF treatment enabled HL-60 cells to lyse CE through both Fc gamma RI and Fc gamma RII. These results demonstrate that although IFN-gamma appears unique in regulating Fc gamma R expression on myeloid cells, cytokines other than IFN-gamma affect ADCC by these cells in a receptor-specific manner.     

冬凌草甲素增强巨噬细胞对凋亡的U937细胞的吞噬作用

目的：研究具有诱导肿瘤细胞凋亡活性的冬凌草甲素，促进巨噬细胞对因凋亡的肿瘤细胞的吞噬作用。 方法： DNA凝胶电泳检测UV照射（2.4 J/cm2, 4 min）的人组织淋巴瘤U937细胞凋亡；Giemsa染色，Hoechst 33258染色，镜下检测计数吞噬作用。 结果： UV照射（2.4 J/cm2, 4 min）诱导U937细胞发生凋亡，琼脂糖凝胶电泳可见凋亡典型的DNA梯带。2.7 μmol·L^-1的冬凌草甲素具有增强U937分化的巨噬细胞对UV照射诱导凋亡的U937细胞的吞噬作用，并呈时间剂量依赖性，但对非特异性荧光颗粒的吞噬效果较弱。加入抗TNFα和抗IL-1β的抗体，培养12 h, 吞噬增强作用明显受抑制。冬凌草甲素在人外周血来源的巨噬细胞吞噬凋亡的U937细胞过程中同样发挥增强吞噬的效果。 结论： 冬凌草甲素可特异地增强巨噬细胞对凋亡的U937细胞的吞噬作用，其吞噬机制是通过诱导巨噬细胞TNFα和IL-1β的释放。     

Synergistic effect of IL-4 and TNF-alpha in the induction of monocytic differentiation of a mouse myeloid leukaemic cell line (WEHI-3B JCS).

We have previously shown that non-cytotoxic concentrations (600-1200 U/ml) of recombinant mouse tumour necrosis factor-alpha (TNF-alpha) can induce differentiation of a subclone (JCS) of the WEHI-3B myelomonocytic leukaemia cell line into mature cells with the characteristics of macrophages. In the present study, the effects of recombinant mouse interleukin-4 (IL-4), either alone or in combination with mouse TNF-alpha, on the growth and differentiation of JCS cells were examined. IL-4 alone (20-5000 U/ml) inhibited the growth of JCS cells in a dose-dependent manner but did not induce cell differentiation. However, combinations of IL-4 and TNF-alpha acted in synergy to inhibit cell proliferation and induce monocytic differentiation of JCS cells, as shown by increased expression of the macrophage differentiation antigens (F4/80, Mac-1), stimulation of phagocytic activity, induction of non-specific esterase and NBT-reducing activities, increased plastic adherence and morphological criteria. Similar synergistic interactions were also shown by human TNF-alpha and mouse IL-4, indicating that TNF-alpha might exert its effects through the low-affinity (p55) TNF receptors. Moreover, the clonogenicity of JCS cells in vitro and their tumorigenicity in vivo were significantly reduced by combined TNF-alpha and IL-4 treatment. Our results indicate that TNF-alpha can act as a differential signal for JCS cells and that its effects are modulated by IL-4. Therefore, the combination of TNF-alpha and IL-4 may be useful in the treatment of some forms of myelomonocytic leukaemia.