Regulation and production of IL-8 by human proximal tubular epithelial cells in vitro

A number of inflammatory kidney diseases are associated with interstitial nephritis and influx of leucocytes in the renal interstitium. Potentially the influx of neutrophils in the interstitium may be induced by the chemotactic cytokine IL-8. In the present study we have analysed the production of IL-8 by cultured human proximal tubular epithelial cells (PTEC) in response to a number of proinflammatory cytokines. Primary cell lines of proximal tubular epithelium obtained from ten different kidneys, and cultured under serum-free conditions, were found to produce IL-8 to different degrees from not detectable levels up to 10·8±1·5 ng IL-8 per 1×10⁵ cells in 72 h. Gel filtration chromatography of PTEC supernatant indicated that the size of IL-8 of PTEC is 15·1 and 8·1 kD, and is chemotactically active for polymorphonuclear neutrophils (PMN). Addition of 0·5 ng/ml rIL-1α or 1000 U/ml recombinant tumour necrosis factor-alpha (TNF-α) to the culture media of PTEC induced an up-regulation of IL-8 production up to 6·3-fold and 3·0-fold, respectively. The up-regulation by IL-1α and TNF-α was dose- and time-dependent. In contrast, 500 U/ml recombinant interferon-gamma (rIFN-γ) down-regulated the production of IL-8 3·4-fold. Northern blot analysis showed that IL-1α and TNF-α increased the expression of IL-8 mRNA, whereas IFN-γ reduced IL-8 mRNA expression. Taken together, these experiments indicate that human PTEC are a potential source of IL-8 in the kidney, and that IL-8 produced in the proximal tubule can be induced by various proinflammatory cytokines.     

Histamine induces IL-6 production by human endothelial cells.

Histamine is one of the major mediators implicated in the physiopathology of allergy. On vascular endothelium, histamine mainly induces early effects: an increase in vasopermeability leading to oedema, a release of lipid mediators and a transient expression of P-selectin. The aim of this study was to evaluate the effects of histamine on adhesion molecule expression and IL-6 production by human endothelial cells. Histamine did not modulate the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin, but induced a transient expression of P-selectin as previously reported. In addition, histamine increased in a dose- (from 10(-5) to 10(-3) M) and time- (from 4 h to 24 h) dependent fashion the IL-6 synthesis by endothelial cells. Tumour necrosis factor-alpha (TNF-alpha)-induced IL-6 production was also potentiated in a dose-dependent manner by histamine, without modification of the time course of IL-6 secretion. Moreover, this increase of IL-6 production induced by histamine was inhibited in a dose-dependent manner by H1 and H2 histamine receptor antagonists (50% inhibition of IL-6 production at 5 x 10(-4) M and 4 x 10(-5) M, respectively). So, histamine induces, besides already well known effects, a late stimulation of endothelial cells, i.e. the production of IL-6.     

Diesel exhaust particles stimulate human airway epithelial cells to produce cytokines relevant to airway inflammation in vitro

Background: Epidemiologic and experimental studies suggest that air pollution such as diesel exhaust particles (DEPs), one of the important air pollutants, may play a role in the increasing prevalence of allergic airway diseases. Objective: We studied the effect of suspended particulate matter (SPM) and its main component, DEPs, on the production of IL-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) by human airway epithelial cells in vitro. Methods: SPM obtained from high-volume air samplers and DEPs were added to cultured human nasal polyp–derived upper airway, normal bronchial, and transformed bronchial epithelial cells. Production of GM-CSF and IL-8 by airway epithelial cells was evaluated. Results: Nontoxic doses of DEPs showed a significant stimulatory effect on IL-8 and GM-CSF production by these three kinds of epithelial cells in a dose- and time-dependent fashion. SPM had a stimulatory effect on GM-CSF, but not IL-8, production. These effects were abrogated by treatment with a protein synthesis inhibitor, cycloheximide, suggesting that the process required a de novo protein synthesis. On the double-chamber plates, airway epithelial cells responded to DEPs only when they were stimulated from the apical sides, which can be a model for in vivo environments. Neither charcoal nor graphite showed such stimulatory effects, indicating that the activity of DEPs did not derive from their particulate nature. Benzo(a)pyrene, one of the main aromatic hydrocarbons contained in DEPs, showed a stimulatory effect on the release of the cytokines, and this organic substance might have a causative effect on of the potency of DEPs. Conclusion: We conclude that SPM and DEPs, its main component, might be important air pollutants in the activation of airway epithelial cells for the release of cytokines relevant to allergic airway inflammation. (J Allergy Clin Immunol 1998;101:778-785.)     

Human intestinal epithelial cells down-regulate IL-8 expression in human intestinal microvascular endothelial cells; role of transforming growth factor-beta 1 (TGF-β1)

Cytokines produced from intestinal epithelial cells may function as signals to neighbouring immune cells. In the present study we analysed the effects of colonic epithelial cell lines (HT-29, Caco-2, HCT-116, Colo-320) and freshly isolated intestinal epithelial cells on IL-8 expression in the SV-40T transfected human microvascular endothelial cell line (HMEC-1). Epithelial cell-conditioned media and transwells preventing physical contact between epithelial and endothelial cells were used. TGF-β1 and IL-8 levels were determined by ELISA and Northern blot analysis. Increasing concentrations of IL-1β led to increasing production of IL-8. The addition of epithelial cell-conditioned medium or epithelial cells to HMEC-1 cells in a two-compartment co-culture system resulted in a strong decrease in IL-8 at the protein and mRNA level. Decrease of IL-8 was markedly stronger when epithelial cells were co-cultured in contact with HMEC-1 cells, indicating that not only soluble factor(s) play a role in the induction of IL-8 suppression in HMEC-1 cells. MoAbs against TGF-β1 partially inhibited down-regulation of endothelial IL-8 expression. In further studies, IL-8 expression in freshly isolated human intestinal microvascular endothelial cells (HIMEC) was also down-regulated by intestinal epithelial cells. Our results demonstrate that intestinal epithelial cells down-regulate IL-8 expression in HMEC-1 cells. TGF-β1 is a candidate factor of epithelial–endothelial communication in the colonic mucosa.     

Inhibitory activity of loratadine and descarboxyethoxyloratadine on histamine-induced activation of endothelial cells

Background The allergic inflammatory reaction is characterized by leucocyte adherence and infiltration processes which are controlled by the expression of adhesion molecules on the surface of vascular endothelium. One of the main mediators implicated in allergic reactions is represented by histamine. Histamine is a potent activator of endothelial cells (EC): it induces the expression of P-selectin on the surface of endothelium and the secretion of IL-6 and IL-8. Objectives Loratadine (L), a histamine H₁-antagonist, and one of its active metabolites, descarboxyethoxyloratadine (DCL), were studied at different concentrations for their ability to reduce the histamine-induced activation of human umbilical vein EC (HUVEC). Methods HUVEC were stimulated in the presence of histamine at 10^-6M, 10^-5M and 10^-4M. We assessed by ELISA the expression of P-selectin on EC surface, as well as cytokine production in EC supernatants of 24 h culture. Results Our results showed that for a 10^-4 M-histamine stimulation, L and DCL have a similar inhibitory effect on P-selectin expression (IC50= 13 × 10^-9 M and 23 × 10^-9 M, respectively). L and DCL inhibited significantly IL-6 and IL-8 secretion induced by histamine with a more powerful efficiency of the active metabolite. For the dose of 10^-4 M histamine, a 50% inhibition of IL-6 secretion was obtained for a dose of DCL equal to 2.6 × 10^-12 M whereas the same magnitude of effects were only reached for a higher concentration of L (0.3 × 10^-6 M). Similar results were obtained for IL-8 (IC50 = 0.2 × 10^-6M for L and 10^-9 M for DCL). Analysis of IL-8 mRNA expression by RT-PCR was in accordance with these data. Conclusions These results demonstrate that both L and DCL are active to reduce the histamine-induced activation of EC. Interestingly, DCL seems to be effective at lesser concentrations especially to inhibit cytokine secretion.     

结核分支杆菌培养上清诱导呼吸道上皮细胞IL-8表达

目的：观察呼吸道上皮细胞对结核杆菌产物产生的炎症反应，探讨炎症反应信号传递的机制。方法：制备结核分枝杆菌强毒力H37RV株培养上清，用培养上清刺激肺上皮细胞株SPC-A-1和A549，酶联免疫吸附实验检测细胞IL-8表达量。用突变MyD88表达重组质粒转染SPC-A-1细胞，观察MyD88与结核杆菌培养上清诱导上皮细胞炎症反应信号传递的关系。结果：结核分枝杆菌强毒力H37RV株培养上清明显增加SPC-A-1和A549细胞IL-8的表达。用突变MyD88表达重组质粒pcDNA3．1-MyD88转染SPC-A-1。结果显著抑制了结核杆菌培养上清对SPC-A-l细胞的炎症刺激作用。结论：结核杆菌培养上清能够诱导呼吸道上皮细胞IL-8表达，，Toll／IL-1受体信号通路及MyD88分子与细胞炎症反应的信号传递相关，提示在肺结核病发病过程中。结核杆菌的代谢产物可能刺激肺上皮细胞产生炎症细胞因子，参与肺结核炎症病变的形成。     

Genotoxic effects of carbon black particles, diesel exhaust particles, and urban air particulates and their extracts on a human alveolar epithelial cell line (A549) and a human monocytic cell line (THP-1)

The possible genotoxicity of small particulate matter has been under investigation for the last 10 years. Diesel exhaust particles (DEP) are considered as "probably carcinogenic" (IARC group 2A) and a number of studies show genotoxic effects of urban particulate matter (UPM). Carbon black (CB) is carcinogenic in rats. In this study the cytotoxic and genotoxic potency of these three particle types was investigated by exposing human cells (A549 and THP-1 cell lines) in vitro to CB, DEP (SRM 1650, NIST), and UPM (SRM 1648, NIST) for 48 hr. Cytotoxicity was assessed using the Alamar Blue assay, whereas genotoxicity was assessed using the single-cell gel electrophoresis (comet assay). The particles were characterized with regard to their mean diameter in tissue culture medium (CB 100 nm, DEP 400 nm, UPM 2 microm), their total carbon content (CB 99%, DEP 85%, UPM 15%), and their acid-soluble metal composition (UPM > CB approximately DEP). The concentrations ranged from 16 ng/ml to 16 microg/ml for cytotoxicity tests and from 16 ng/ml to 1.6 microg/ml for genotoxicity tests. In both assays, paraquat was used as a reference chemical. The CB, DEP, and UPM particles showed no significant cytotoxicity. However, all three particles were able to cause significant DNA damage, although to a different extent in the two cell lines. The genotoxicity of washed particles and dichloromethane extracts was also investigated. In THP-1 cells CB washed particles and DEP extracts caused significant DNA damage. This difference in effect may be related to differences in size, structure, and composition of the particles. These results suggest that CB, DEP, and UPM are able to cause DNA damage and, therefore, may contribute to the causation of lung cancer. More detailed studies on influence of size, structure, and composition of the particles are needed.     

p38 MAP kinase regulates TNFα-, IL-1α- and PAF-induced RANTES and GM-CSF production by human bronchial epithelial cells

BACKGROUND: RANTES and granulocyte macrophage-colony stimulating factor (GM-CSF) play an important role in the production of allergic inflammation of the airway through their chemotactic activity for eosinophils. Recent studies have indicated that p38 mitogen-activated protein (MAP) kinase regulates cytokine expression in various cells; however, the role of p38 MAP kinase in RANTES and GM-CSF production in human bronchial epithelial cells (BECs) has not yet been determined. OBJECTIVE: In the present study, we examined serine phosphorylation of MKK3 and MKK6 which is the upstream regulator of p38 MAP kinase and p38 MAP kinase activation in tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha and platelet-activating factor (PAF)-stimulated BECs and the effect of SB 203580 as the specific inhibitor for p38 MAP kinase activity on RANTES and GM-CSF expression in order to clarify the intracellular signal regulating RANTES and GM-CSF production by human BECs. RESULTS: The results showed that TNF alpha, IL-1 alpha and PAF induced serine phosphorylation of MKK3 and MKK6, and p38 MAP kinase activation in BECs. SB 203580 inhibited p38 MAP kinase activity and RANTES and GM-CSF production by TNF alpha-, IL-1 alpha- or PAF-stimulated human BECs. CONCLUSIONS: These results indicate that p38 MAP kinase plays an important role in TNF alpha-, IL-1 alpha- or PAF-activated signalling pathway which regulates RANTES and GM-CSF production by BECs and that the specific inhibitor for p38 MAP kinase activity might be useful for the treatment of allergic inflammation of the airway.     

Escherichia coli-induced expression of IL-1 alpha, IL-1 beta, IL-6 and IL-8 in normal human renal tubular epithelial cells

The aim of the present study was to investigate whether the IL-1 family cytokines, in addition to IL-6 and IL-8, could be induced in normal human cortical epithelial cells in response to bacterial stimuli. Human renal tissue was obtained from 9 patients undergoing elective tumour nephrectomy. Renal cortical epithelial cells of tubular origin were prepared from the unaffected tissue. The proximal tubular cells were stimulated for 2, 6 and 24 h with a heat-inactivated pyelonephritogenic Escherichia coli strain DS-17. Cultured unstimulated tubular cells served as controls. IL-1 alpha, IL-1 beta, IL-1 receptor antagonist, IL-6, IL-8, IL-10, TNF-alpha, G-CSF and GM-CSF were analysed using immunohistochemistry at the single cell level. The nonstimulated cells were found to express low levels of IL-6 and IL-8 (mean value < 3% of total cells). In contrast, E. coli exposure resulted in significantly increased incidences of IL-6 and IL-8 expressing cells (mean values approximately 18% of total cells) peaking within two hours of stimulation (P < 0.008 and P < 0.02 versus non-stimulated cells, respectively). A gradual decrease was thereafter observed at 6 and 24 h, respectively, although persistently higher compared to controls. A different kinetic response was found for IL-1 alpha, IL-1 beta and IL-1 receptor antagonist-expressing cells, which peaked 24 h after E. coli stimulation (mean values 3--10%) (P < 0.008, P < 0.02, P < 0.02 versus non-stimulated cells, respectively). Low levels of TNF-alpha and GM-CSF were found in 3 of the 9 donated epithelial cells, peaking at 2 h, and IL-10 and G-CSF producing cells in 1 patient each. In conclusion we found that heat-inactivated pyelonephritic E. coli induced a proinflammatory cytokine response in the normal human proximal tubular cells including the IL-1 family, IL-6 and IL-8.     

Detection of interleukin-6 and interleukin-1 production in human thyroid epithelial cells by non-radioactive in situ hybridization and immunohistochemical methods.

Human endocrine thyroid epithelial cells have been described to produce cytokines in vitro. In order to determine whether they do so in vivo during thyroiditis, parallel studies on mRNA expression with a non-radioactive in situ hybridization technique and immunohistochemical detection for the protein were performed on frozen sections of thyroid samples from autoimmune thyroiditis (Graves' disease and Hashimoto's thyroiditis), non-toxic goitre and normal thyroid tissue. cDNA probes were sulphonated and their hybridization with mRNA was detected with a sulphonyl-specific monoclonal antibody. This signal was amplified and visualized with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. The protein products were detected with immuno-purified rabbit F(ab')2 antibody fragments recognizing recombinant human cytokines, visualized by the immunoperoxidase technique. Each sample was studied at the two levels. Both interleukin-6 mRNA and protein were found in the endocrine cells. There was no obvious difference between autoimmune thyroiditis and non-toxic goitre. However, normal thyroid epithelial cells produced less interleukin-6. Interleukin-1 alpha mRNA and its protein were found in epithelial cells from Hashimoto's thyroiditis samples, but not in the others, except one Graves' disease sample, in which only mRNA was detected. Interleukin-1 beta was not detected in these cells, its mRNA was only found in one of the Graves' disease samples. These cytokines were also detected in some infiltrating cells.     

The MHC class I related Fc receptor, FcRn, is expressed in the epithelial cells of the human mammary gland

The major histocompatibility complex (MHC) class I related neonatal Fc receptor (FcRn) plays multiple roles, being involved in transporting immunoglobulin G (IgG) and protecting this antibody class from catabolism. The presence of this receptor was previously demonstrated in the lactating murine mammary gland. In the current study we have investigated FcRn expression in various histologic types of human breast carcinoma and lymph node metastases. We used immunohistochemical methods to demonstrate the presence of FcRn in epithelial cells, whereas this Fc receptor could not be detected in mammary gland endothelial cells. The presence of the receptor was also found in the metastasizing epithelial cells within the lymph nodes, and this provides a useful marker for their identification.     

Lipopolysaccharide induces H1 receptor expression and enhances histamine responsiveness in human coronary artery endothelial cells

Summary Histamine is a well-recognized modulator of vascular inflammation. We have shown that histamine, acting via H1 receptors (H1R), synergizes lipopolysaccharide (LPS)-induced production of prostaglandin I(2) (PGI(2)), PGE(2) and interleukin-6 (IL-6) by endothelial cells. The synergy between histamine and LPS was partly attributed to histamine -induced expression of Toll-like receptor 4 (TLR4). In this study, we examined whether LPS stimulates the H1R expression in human coronary artery endothelial cells (HCAEC) with resultant enhancement of histamine responsiveness. Incubation of HCAEC with LPS (10-1000 ng/ml) resulted in two-fold to fourfold increases in H1R mRNA expression in a time-dependent and concentration-dependent fashion. In contrast, LPS treatment did not affect H2R mRNA expression. The LPS-induced H1R mRNA expression peaked by 4 hr after LPS treatment and remained elevated above the basal level for 20-24 hr. Flow cytometric and Western blot analyses revealed increased expression of H1R protein in LPS-treated cells. The specific binding of [(3)H]pyrilamine to H1R in membrane proteins from LPS-treated HCAEC was threefold higher than the untreated cells. The LPS-induced H1R expression was mediated through TLR4 as gene silencing by TLR4-siRNA and treatment with a TLR4 antagonist inhibited the LPS effect. When HCAEC were pre-treated with LPS for 24 hr, washed and challenged with histamine, 17-, 10- and 15-fold increases in PGI(2), PGE(2) and IL-6 production, respectively, were noted. Histamine-induced enhancement of the synthesis of PGI(2), PGE(2) and IL-6 by LPS-primed HCAEC was completely blocked by an H1R antagonist. The results demonstrate that LPS, through TLR4 activation, up-regulates the expression and function of H1R and amplifies histamine-induced inflammatory responses in HCAEC.     

LPS对晶状体上皮细胞TLR4和CD14 mRNA表达的影响

目的:探讨脂多糖(LPS)对晶状体上皮细胞(LECs)Toll样受体4(TLR4)和CD14表达的影响。方法:采用不同浓度的LPS与体外培养的牛LECs共同孵育不同时间,再用一步法反转录多聚酶链反应(PCR)检测LECs中TLR4mRNA和CD14mRNA的表达。结果:50μg/LLPS组、100μg/LLPS组、200μg/LLPS组、500μg/LLPS组、1000μg/LLPS组的LECs中TLR4mRNA的表达均分别显著高于对照组(P0.01);100μg/LLPS作用24h、48h、72h后LECs的TLR4mRNA表达均显著高于对照组(P0.01);100μg/LLPS作用24h后LECs的CD14mRNA表达亦显著高于对照组(P0.05)。结论:LPS可促进LECs中TLR4mRNA和CD14mRNA的表达,提示白内障手术后眼内细胞反应和后发性白内障的发生发展可能与TLR4和CD14表达增加有关。     

IL-1ra and IL-1 production in human oral mucosal epithelial cells in culture: differential modulation by TGF-beta1 and IL-4

Regulatory cytokines mediate the participation of oral mucosal epithelial cells (OMEC) in local immune responses. The aim of this study was to characterize the isoforms of IL-1 receptor antagonist (IL-1ra) in cultured human primary OMECs and to compare its production with that of IL-1 alpha (IL-1alpha) and IL-1 beta (IL-1beta). Western blot analysis showed that IL-1ra was 22 kDa in size hence slightly smaller than monocyte IL-1ra (25 kDa). A minor form of 20 kDa was also found in unstimulated cell culture lysates. In culture supernatants, IL-1 bioactivity increased after IL-1ra neutralization, indicating that the baseline production of IL-1ra is biologically relevant. Immunohistochemistry showed a relation between IL-1ra and involucrin expressions, suggesting that intracytoplasmic IL-1ra may be involved in cell terminal differentiation. In unstimulated culture lysates, there was far more IL-1ra than IL-1alpha and IL-1beta. TGF-beta1 markedly increased the IL-1ra/IL-1beta ratio from 93.6 : 1 to 300 : 1. IL-4, which is generally described as an anti-inflammatory cytokine, increased IL-1 but not IL-1ra production. TNF-alpha increased intracellular production of the three IL-1 members. IL-1ra levels were lower in supernatants than in lysates of cultured cells. Our results show that human OMECs constitutively produce significant amounts of a biologically active form of IL-1ra. TGF-beta1 mu(p)-regulation points to a positive amplification loop and IL-4 to a down-regulation loop, both including Th2 cells and OMECs. They may be important in oral tolerance and IgA production, respectively.