Induction of Interleukin-4 and Interleukin-5 Expression in Mast Cells Is Inhibited by Glucocorticoids

Inflammation in asthma and other allergic diseases is characterized by excessive production of immunoglobulin E (IgE) and the influx of leukocytes, especially eosinophils. Interleukin 4 (IL-4) and IL-5 are essential for IgE production and eosinophilia, respectively, and are produced by mast cells in allergic conditions, for which glucocorticoids are widely used therapeutically. We assessed the effect of glucocorticoids on IL-4 and IL-5 mRNA production by the RBL-2H3 cell line, an analog of mucosal mast cells. IL-4 and IL-5 mRNAs were induced by an antigen that is used to cross-link receptor bound IgE, by calcium ionophore, or by ionophore with phorbol ester and were markedly inhibited by dexamethasone. In cells activated with ionophore and phorbol ester, 10⁻⁶ M dexamethasone reduced the IL-4 and IL-5 mRNA levels to only 12.8 and 5.7%, respectively, of those in cells without dexamethasone, and 10⁻⁹ M dexamethasone caused reductions to 27 and 56%, respectively. Hydrocortisone at 10⁻⁶ and 10⁻⁷ M almost completely inhibited IL-4 and IL-5 mRNA production. Dexamethasone was markedly inhibitory even if it was added after the cells were activated, provided that it was present in the cultures for at least 1.5 h. These studies indicate that the expression of IL-4 and IL-5 mRNAs by mast cells is highly sensitive to glucocorticoids. The data suggest that these inhibitory effects may contribute to the clinical efficacy of glucocorticoids in the therapy of allergic diseases.     

Membrane-Anchored CD14 Is Important for Induction of Interleukin-8 by Lipopolysaccharide and Peptidoglycan in Uroepithelial Cells

We investigated the induction of interleukin-8 (IL-8) by bacterial lipopolysaccharide (LPS) and peptidoglycan (PGN) in the bladder cancer cell lines T24, 5637, UM-UC-3, and HT1197. T24 and 5637 cells strongly induced IL-8 after stimulation with LPS or PGN in a dose- and time-dependent manner, whereas UM-UC-3 and HT1197 cells did so very weakly. The expression of CD14 at the mRNA, total cellular protein, and cell surface protein levels differed among these cell lines, but the expression levels of Toll-like receptors 2 and 4 (TLR2 and TLR4) were not significantly different. The CD14 expression levels were found to correlate with the inducibility of IL-8 by LPS or PGN. Treatment of T24 and 5637 cells with phosphatidylinositol-specific phospholipase C to eliminate CD14 from the cell surface dramatically suppressed the induction of IL-8. On the other hand, UM-UC-3 cells transfected with CD14 cDNA expressed membrane-anchored CD14 and showed more efficent induction of IL-8 by LPS stimulation than untransfected controls. These results suggest that the presence of the membrane-anchored, but not the soluble, form of CD14 is a strong factor in IL-8 induction in bladder epithelial cells in response to bacterial components. The presence of the membrane-anchored form of CD14 may thus be a determinant for the inflammatory response of uroepithelial cells.     

Interleukin-6 and Interleukin-6 Receptor are Expressed by Cultured Glomerular Epithelial Cells

Interleukin-6 (IL-6) has been extensively studied in mesangial cells but little is known about the expression of this cytokine and its receptor in glomerular epithelial cells (GEC). IL-6 was detected in the culture supernatants of human GEC and its production was enhanced in time and dose dependent manner by lipopolysaccharide (LPS), interleukin-1β (IL-Iβ) and tumour necrosis alpha (TNF-α). Quiescent, serum-starved GEC did not express clearly IL-6 mRNA. Stimulation of cells with LPS, TNF-α or IL-1β resulted in an increase of detectable IL-6 mRNA. Interestingly, it was found that IL-6 induced its own mRNA attesting that this cytokine was secreted in autocrine fashion by GEC. GEC expressed IL-6 receptor (IL-6R) as demonstrated directly by the existence of IL-6R mRNA detected by northern blotting. Stimulation of GEC by pro-inflammatory mediators such as LPS increased the expression of IL-6R mRNA. The soluble form of IL-6 receptor (sIL-6R) was not detectable in the culture supernatants harvested from untreated or cytokine-treated cells. We investigated further, whether IL-6 may influence growth of cultured GEC. Incubation of GEC with recombinant (r) IL-6 resulted in a dose dependent increase in ³H thymidine incorporation indicating that IL-6 acts as an autocrine growth factor for GEC. We conclude that GEC are a potent source of IL-6, the local excessive expression of IL-6 and its receptor may play a substantive role in the regulation of processes which appear critical to the initiation of progressive glomerular disease such as cell proliferation.     

Therapeutic Benefit of Bortezomib on Acute Graft-versus-Host Disease Is Tissue Specific and Is Associated with Interleukin-6 Levels

Bortezomib, a proteasome inhibitor capable of direct antitumor effects, has been shown to prevent acute graft-versus-host disease (GVHD) when administered in a short course immediately after bone marrow transplantation (BMT) in mice. However, when bortezomib is given continuously, CD4⁺ T cell–mediated gastrointestinal tract damage increases GVHD mortality. To investigate the protective effects of bortezomib on other organs, we used a CD8-dependent acute GVHD (aGVHD) model of C3H.SW donor T cells engrafted into irradiated C57BL/6 recipients (minor MHC mismatch), which lack significant gut GVHD. Our data in this model show that bortezomib can be given continuously to prevent and treat aGVHD mediated by CD8⁺ T cells, but this effect is organ specific, such that only skin, and not liver, protection was observed. Despite the lack of hepatic protection, bortezomib still significantly improved survival, primarily because of its skin protection. Reduced skin GVHD by bortezomib was correlated with reduced serum and skin IL-6 levels. Administration of a blocking IL-6 antibody in this model also resulted in similar cutaneous GVHD protection. These results indicate that bortezomib or blockade of IL-6 may prevent CD8⁺ T cell–mediated cutaneous acute GVHD.     

Promotion of Colonization and Virulence by Cholera Toxin Is Dependent on Neutrophils

The innate immune response to Vibrio cholerae infection is poorly understood, but this knowledge is critical for the design of safe, effective vaccines. Using an adult mouse intestinal infection model, this study examines the contribution of neutrophils to host immunity, as well as the effect of cholera toxin and other secreted factors on this response. Depletion of neutrophils from mice with anti-Ly6G IA8 monoclonal antibody led to similar survival rates of mice infected with low or moderate doses of toxigenic V. cholerae El Tor O1. At a high dose, neutropenic mice showed increased rates of survival compared to neutrophil-replete animals. Expression of cholera toxin was found to be protective to the neutropenic host, and this phenotype can be replicated by the administration of purified toxin. Neutrophils do not effectively clear colonizing bacteria from the small intestine, nor do they alter induction of early immune-modulating signals. In both neutropenic and neutrophil-replete animals, the local response to infection is characterized by expression of interleukin 6 (IL-6), IL-10, and macrophage inflammatory protein 2 alpha (MIP-2). Overall, these data indicate that the innate immune response to toxigenic V. cholerae infection differs dramatically from the host response to nontoxigenic infection or vaccination, where neutrophils are protective to the host. In the absence of neutrophils, cholera toxin induces immunomodulatory effects that increase host survival. In cholera toxin-producing strains, similar to nontoxigenic infection, accessory toxins are critical to virulence, indicating that cholera toxin and the other secreted toxins modulate the host response by different mechanisms, with both contributing to bacterial persistence and virulence.     

Interleukin-6 Is an Autocrine Growth Factor in Human Prostate Cancer

Prostate cancer is the most common cancer in American men and the second leading cause of cancer deaths in this group. We have found that interleukin (IL)-6 protein concentrations are increased approximately 18-fold in clinically localized prostate cancers when compared to normal prostate tissue. Normal and neoplastic prostatic epithelial cells in culture, with the exception of LNCaP cells, secrete IL-6. Addition of exogenous IL-6 to primary epithelial cells in culture or the LNCaP prostate cancer cell line leads to phosphorylation of Stat-3 and increases in net cell proliferation. The concentration of IL-6 receptor is increased eightfold in the prostate cancer tissues and is increased in the cancer cells by immunohistochemistry. The increased expression of IL-6 receptor is correlated with increased proliferation of prostate cancer cells in vivo as assessed by Ki67 immunohistochemistry. These findings strongly support the hypothesis that IL-6 acts as a significant autocrine growth factor in vivo for primary, androgen-dependent prostate cancers.     

Endothelial Cell Injury Caused by Candida albicans Is Dependent on Iron

Although it is known that Candida albicans causes endothelial cell injury, in vitro and in vivo, the mechanism by which this process occurs remains unknown. Iron is critical for the induction of injury in many types of host cells. Therefore, we investigated the role of iron in Candida-induced endothelial cell injury. We found that pretreatment of endothelial cells with the iron chelators phenanthroline and deferoxamine protected them from candidal injury, even though the organisms germinated and grew normally. Loading endothelial cells with iron reversed the cytoprotective effects of iron chelation. Moreover, chelation of endothelial cell iron significantly reduced phagocytosis of C. albicans by these cells, while candidal adherence to chelator-treated endothelial cells was slightly enhanced. Since endothelial cell phagocytosis of C. albicans is required for endothelial cell injury to occur, inhibition of phagocytosis is likely the principal mechanism of the cytoprotective effects of iron chelation. The production of toxic reactive oxygen intermediates by host cells is known to be inhibited by iron chelation. Therefore, we investigated whether treating endothelial cells with antioxidants could mimic the cytoprotective effects of iron chelation. Neither extracellular nor membrane-permeative antioxidants reduced candidal injury of endothelial cells. Furthermore, depleting endothelial cells of the endogenous antioxidant glutathione did not render them more susceptible to damage by C. albicans. These results suggest that candidal injury of endothelial cells is independent of the production of reactive oxygen intermediates and that the cytoprotective effects of iron chelation are not due to inhibition of the synthesis of these toxic intermediates.During the process of hematogenous dissemination, Candida albicans must first cross the endothelial cell lining of the vasculature to invade the tissue parenchyma. One mechanism by which the organism may escape from the vascular compartment is by causing endothelial cell injury. This injury likely results in exposure of the subendothelial cell basement membrane, which may enhance candidal adherence and facilitate tissue invasion (23). We have been investigating the mechanisms by which C. albicans injures endothelial cells in vitro. Previously, we found that phagocytosis of the organism by endothelial cells is required for endothelial cell damage to occur (8, 9). This phagocytosis requires both intact endothelial cell microfilaments and microtubules. In addition, although endothelial cells are able to phagocytize killed organisms (35), only the phagocytosis of live, germinating organisms causes endothelial cell injury (8).After endothelial cells phagocytize C. albicans, the organism may injure the endothelial cells by several potential mechanisms. It is possible that phospholipases and/or proteinases secreted by C. albicans injure host cells (6, 18, 38). Another possibility is that C. albicans causes endothelial cell injury by an iron-dependent process. For example, endothelial cells are known to synthesize and release superoxide anions during phagocytic activity (11, 14). Iron is required for the assembly of enzymes, such as xanthine oxidase (33) and cytochromes (20), that catalyze the synthesis of these reactive oxygen intermediates. In addition, iron serves as a cofactor, which converts these anions to highly reactive hydroxyl radicals (21) which cause cellular damage (10, 24). Finally, iron is required for the function of nitric oxide synthase, an enzyme that can catalyze the synthesis of cytotoxic concentrations of nitric oxide in some cell types (31).In this study, we used the iron chelators phenanthroline and deferoxamine to examine the role of iron in endothelial cell injury caused by C. albicans. We found that chelation of endothelial cell iron protected these cells from injury by C. albicans. The cytoprotective effects of iron chelation were likely due to reduced phagocytosis of this fungus by endothelial cells. Furthermore, we found that C. albicans damages endothelial cells by a process that is likely independent of the production of reactive oxygen intermediates.(This work was presented in part at the 35th Interscience Conference on Antimicrobial Agents and Chemotherapy, September 1995, San Francisco, Calif.)     

Interleukin-6 production and secretion by human parathyroids

Parathyroid hormone (PTH) stimulates osteoblasts to produce the proinflammatory cytokine interleukin-6 (IL-6), causing bone resorption. In patients with primary hyperparathyroidism, elevated serum levels of IL-6 normalize after resection of parathyroid tumours. Because IL-6 is also expressed in normal parathyroids and in other endocrine cells (adrenal and islet), we hypothesized that parathyroid tumours might contribute directly to the elevated serum IL-6 levels in patients with hyperparathyroidism. Immunohistochemistry identified IL-6, PTH, and chromogranin-A (an endocrine and neuroendocrine tumour marker) in normal, adenomatous and hyperplastic parathyroids. Using immunofluorescence and confocal microscopy, IL-6 co-localized with PTH and with chromogranin-A in parathyroid cells. All cultured parathyroid tumours secreted IL-6 at levels markedly higher than optimally stimulated peripheral blood mononuclear cells. Supernates from cultured parathyroids stimulated proliferation of an IL-6-dependent cell line, and anti-IL-6 MoAb abolished this stimulatory effect. IL-6 mRNA was documented in cultured parathyroid tumours, cultured normal parathyroids, fresh operative parathyroid tumours and fresh operative normal specimens. In conclusion, these data show that parathyroid tumours and normal parathyroids contain, produce and secrete IL-6. Our findings present a novel pathway by which human parathyroids may contribute markedly to IL-6 production and elevation of serum IL-6 levels in patients with hyperparathyroidism. The physiological relevance of IL-6 production by human parathyroids remains to be determined, but IL-6 secretion by parathyroid tumours may contribute to bone loss and to other multi-system complaints observed in these patients.     

Clearance of Staphylococcus aureus Nasal Carriage Is T Cell Dependent and Mediated through Interleukin-17A Expression and Neutrophil Influx

The anterior nares of humans are the major reservoir for Staphylococcus aureus colonization. Approximately 20% of the healthy human population is persistently and 80% is intermittently colonized with S. aureus in the nasal cavity. Previous studies have shown a strong causal connection between S. aureus nasal carriage and increased risk of nosocomial infection, as well as increased carriage due to immune dysfunction. However, the immune responses that permit persistence or mediate clearance of S. aureus on the nasal mucosa are fundamentally undefined. In this study, we developed a carriage model in C57BL/6J mice and showed that clearance begins 14 days postinoculation. In contrast, SCID mice that have a deficient adaptive immune response are unable to eliminate S. aureus even after 28 days postinoculation. Furthermore, decolonization was found to be T cell mediated but B cell independent by evaluating carriage clearance in T-cell receptor β/δ (TCR-β/δ) knockout (KO) and IgH-μ KO mice, respectively. Upregulation of the cytokines interleukin 1β (IL-1β), KC (also termed CXC ligand 1 [CXCL1]), and IL-17A occurred following inoculation with intranasal S. aureus. IL-17A production was crucial for clearance, since IL-17A-deficient mice were unable to effectively eliminate S. aureus carriage. Subsequently, cell differential counts were evaluated from nasal lavage fluid obtained from wild-type and IL-17A-deficient colonized mice. These counts displayed IL-17A-dependent neutrophil migration. Antibody-mediated depletion of neutrophils in colonized mice caused reduced clearance compared to that in isotype-treated controls. Our data suggest that the Th17-associated immune response is required for nasal decolonization. This response is T cell dependent and mediated via IL-17A production and neutrophil influx. Th17-associated immune responses may be targeted for strategies to mitigate distal infections originating from persistent S. aureus carriage in humans.     

CD4+ T Cell Hyporesponsiveness after Repeated Exposure to Schistosoma mansoni Larvae Is Dependent upon Interleukin-10

The effect that multiple percutaneous exposures to Schistosoma larvae has on the development of early CD4⁺ lymphocyte reactivity is unclear, yet it is important in the context of humans living in areas where schistosomiasis is endemic. In a murine model of multiple infections, we show that exposure of mice to repeated doses (4×) of Schistosoma mansoni cercariae, compared to a single dose (1×), results in CD4⁺ T cell hyporesponsiveness within the skin-draining lymph nodes (sdLN), manifested as reduced CD4⁺ cell proliferation and cytokine production. FoxP3⁺ CD4⁺ regulatory T cells were present in similar numbers in the sdLN of 4× and 1× mice and thus are unlikely to have a role in effecting hyporesponsiveness. Moreover, anergy of the CD4⁺ cell population from 4× mice was slight, as proliferation was only partly circumvented through the in vitro addition of exogenous interleukin-2 (IL-2), and the in vivo blockade of the regulatory molecule PD1 had a minimal effect on restoring responsiveness. In contrast, IL-10 was observed to be critical in mediating hyporesponsiveness, as CD4⁺ cells from the sdLN of 4× mice deficient for IL-10 were readily able to proliferate, unlike those from 4× wild-type cohorts. CD4⁺ cells from the sdLN of 4× mice exhibited higher levels of apoptosis and cell death, but in the absence of IL-10, there was significantly less cell death. Combined, our data show that IL-10 is a key factor in the development of CD4⁺ T cell hyporesponsiveness after repeated parasite exposure involving CD4⁺ cell apoptosis.     

Interleukin-6 and Interleukin-12 Participate in Induction of a Type 1 Protective T-Cell Response during Vaccination with a Tuberculosis Subunit Vaccine

We examined the role of cytokines in the development of gamma interferon (IFN-gamma)-secreting protective T cells following immunization with a culture filtrate subunit vaccine against Mycobacterium tuberculosis containing the adjuvant dimethyldioctadecylammonium bromide (DDA). Depletion of either interleukin-6 (IL-6) or IL-12 with specific neutralizing antibodies during vaccination reduced the priming of T cells for antigen-specific proliferation and IFN-gamma secretion. Such reduction was also observed in IL-6 gene-disrupted mice as compared to wild-type animals. IL-6 was found to play a role in the initial differentiation of Th1 cells but not in their expansion. The defect found after IL-6 depletion or in IL-6-knockout mice was compensated by the inclusion of recombinant mouse IL-12 in the vaccine. The induction of protective immunity against an intravenous or an aerosol challenge with live, virulent M. tuberculosis was markedly reduced by neutralizing either IL-6 or IL-12 during immunization with the vaccine. Likewise, the effects of IL-6 neutralization were partially reversed by including IL-12 in the vaccine. Our data point to an important role of IL-6 and IL-12 in the generation of cell-mediated immunity to tuberculosis.     

B Cell-Activating Factor Enhances Interleukin-6 and Interleukin-10 Production by ODN-Activated Human B Cells

We aim to investigate the additive value of B cell‐activating factor (BAFF) when added to oligodeoxynucleotides (ODN)‐activated B cells with respect to TLR‐9, CD69, MHC‐II expression, IL‐6 and IL‐10 secretion and B cell cycling. Therefore, B cells from healthy individuals were incubated under the following conditions: (1) B cells with medium, (2) B cells with ODN 0.5 μm , (3) B cells with BAFF 20 μm and (4) B cells with both ODN 0.5 μm and BAFF 20 μm . We found that addition of BAFF did not enhance the expression of TLR‐9, CD69 and MHC‐II in ODN‐activated B cells. Incubation of B cells with BAFF and ODN together leads to a marked elevation of IL‐6 and IL‐10 levels compared to ODN alone. Synthesis and mitosis were higher in B cells stimulated by BAFF than in B cells stimulated by ODN. These findings suggest that both BAFF and TLR‐9 contribute independently to B cell function.     

Osteoblast Elastic Modulus Measured by Atomic Force Microscopy Is Substrate Dependent

The actin and microtubule cytoskeleton have been found to contribute to the elastic modulus of cells, which may be modulated by adhesion to extracellular matrix (ECM) proteins and subsequent alterations in the cytoskeleton. In this study, the apparent elastic modulus (E_app) of osteoblast-like MC3T3-E1 cells adhered to fibronectin (FN), vitronectin (VN), type I collagen (COLI), fetal bovine serum (FBS), or poly-l-lysine (PLL), and bare glass were determined using an atomic force microscope (AFM). The E_app of osteoblasts adhered to ECM proteins (FN, VN, COLI, and FBS) that bind cells via integrins were higher compared to cells on glass and PLL, which adhere cells through nonspecific binding. Also, osteoblasts adhered to FN, VN, COLI, and FBS had F-actin stress fiber formation, while osteoblasts on glass and PLL showed few F-actin fibers. Disruption of the actin cytoskeleton decreased E_app of osteoblasts plated on FN to the level of osteoblasts plated on glass, while microtubule disruption had no significant effect. This suggests that the elevated modulus of osteoblasts adhered to FN was due to remodeling of the actin cytoskeleton upon adhesion to ECM proteins. Modulation of cell stiffness upon adhesion to various substrates may influence mechanosignal transduction in osteoblasts.     

Differential Effects of Interleukin-2 and Interleukin-7 on the Induction of CD4 and CD8 Expression by Double-Negative Human Thymocytes*

Expression of ceil surface CD4 (in the absence of CD3/T cell receptor) characterizes an early stage of intrathymic T cell development. Here, we investigated the appearance of CD4 and CD8 expression on highly purified CD4^?8^? double-negative human thymocytes in response to interleukin (IL)-7 and IL-2. While IL-7 preferentially promoted the appearance of CD4 single-positive and CD4⁺8⁺ double-positive thymocytes, I1-2 primarily induced CD8 single-positive thymocytes. A significant fraction of CD4 single-positive cells generated from double-negative thymocytes via IL-7 lacked cell surface CD3 expression. In contrast, the majority of CD8 single-positive cells generated from double-negative thymocytes via IL-2 coexpressed CD3. We conclude that IL-7 and IL-2 exert differential effects on the differentiation of early human T cell progenitors.     

药物对C反应蛋白诱导脑微血管内皮细胞黏附分子表达的作用

目的探讨一些药物对C反应蛋白（CRP）诱导脑微血管内皮细胞黏附分子表达的作用，为缺血性脑损伤机制及防治策略的进一步研究提供理论依据。方法培养脑微血管内皮细胞（bEnd．3），经辛伐他汀、吡格列酮、Heroin和AG490预处理后与CRP共孵育，采用免疫蛋白印迹（Westernblot-ting）检测细胞黏附分子表达。结果CRP能显著诱导bEnd．3细胞表达细胞间黏附分子（ICAM-1）、血管细胞黏附分子（VCAM-1），信号转导子及转录激活子-3（STA33）酪氨酸残基快速磷酸化。辛伐他汀、吡格列酮和AG490均能显著减少ICAM-1、VCAM-1的表达。结论CRP上调黏附分子表达的作用可能与JAK／STAT信号转导通路有关。辛伐他汀、吡格列酮均能抑制CRP诱导的脑内皮细胞ICAM-1、VCAM-1表达，可能是它们对缺血性脑损伤的保护机制之一。     

Inflammatory Monocyte Recruitment Is Regulated by Interleukin-23 during Systemic Bacterial Infection

Listeria monocytogenes is a Gram-positive intracellular pathogen that causes meningitis and septicemia in immunocompromised individuals and spontaneous abortion in pregnant women. The innate immune response against L. monocytogenes is primarily mediated by neutrophils and monocytes. Interleukin-23 (IL-23) is an important proinflammatory cytokine well known for its role in neutrophil recruitment in various infectious and autoimmune diseases. We have previously shown that IL-23 is required for host resistance against L. monocytogenes and for neutrophil recruitment to the liver, but not the spleen, during infection. Despite efficient neutrophil recruitment to the spleen, IL-23p19 knockout (KO) mice have an increased bacterial burden in this organ, suggesting that IL-23 may regulate the recruitment/function of another cell type to the spleen. In this study, we show that specific depletion of neutrophils abrogated the differences in bacterial burdens in the livers but not the spleens of C57BL/6 (B6) and IL-23p19 KO mice. Interestingly, L. monocytogenes-infected IL-23p19 KO mice had fewer monocytes in the spleen than B6 mice, as well as a reduction in the monocyte-recruiting chemokines CCL2 and CCL7. Additionally, the overall concentrations of tumor necrosis factor alpha (TNF-α) and nitric oxide (NO^•), as well as the percentages and total numbers of monocytes producing TNF-α and NO^•, were reduced in IL-23p19 KO mice compared to levels in B6 mice, leading to increased bacterial burdens in the spleens of L. monocytogenes-infected IL-23p19 KO mice. Collectively, our data establish that IL-23 is required for the optimal recruitment of TNF-α- and NO^•-producing inflammatory monocytes, thus revealing a novel mechanism by which this proinflammatory cytokine provides protection against bacterial infection.     

ELISA法测定白细胞介素－6

建立了灵敏的双抗体夹心ＥＬＩＳＡ法测定人白细胞介素－６．主要步骤为以鼠抗重组白细胞介素－６单抗包被，加入待检抗原，再加多克隆兔抗重组白细胞介素－６血清作用，最后加酶标羊抗兔示踪并放大其反应。本法简便，快速，重复性好，灵敏度达１ｎｇ／ｍｌ，适用于临床与基础研究。