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.     

IL-17 and IL-17F modulate GM-CSF production by lung microvascular endothelial cells stimulated with IL-1beta and/or TNF-alpha

In this study, we investigated the roles of CD4 T cell cytokines IL-17 and IL-17F in GM-CSF production from lung microvascular endothelial cells (LMVECs). While a wide range of doses of IL-17 or IL-17F alone did not induce GM-CSF release from LMVECs, IL-17 had an enhancing effect on macrophage-derived IL-1beta- and TNF-alpha-induced GM-CSF mRNA expression and production, whereas IL-17F had an enhancing effect on IL-1beta-induced GM-CSF production, but a marked inhibitory effect on TNF-alpha-induced secretion. GM-CSF production was further enhanced with the combination of three cytokines IL-1beta, TNF-alpha and IL-17 or IL-17F. Additionally, when Th1 or Th2 cytokine was combined with IL-1beta or TNF-alpha, both Th1 and Th2 cytokines had a modest stimulatory effect on TNF-alpha-induced GM-CSF production, whereas IL-4 and IFN-gamma profoundly attenuated IL-1beta-induced secretion. Moreover, the regulation by IL-17 plus Th1 or Th2 cytokine of GM-CSF production from LMVECs treated with IL-1beta or TNF-alpha was dependent on the concentration of IL-17. Our findings indicate that IL-17 and IL-17F play a differential regulatory role in GM-CSF production by LMVECs stimulated with IL-1beta and/or TNF-alpha, which is sensitive to Th1 and Th2 cytokine modulation.     

Sustained elevated levels of VCAM-1 in cultured fibroblast-like synoviocytes can be achieved by TNF-alpha in combination with either IL-4 or IL-13 through increased mRNA stability

Rheumatoid arthritis is characterized by hyperplasia of the synovial lining and invasion of cartilage and bone by a subset of resident synovial cells named fibroblast-like synoviocytes. They are characterized by elevated expression of the vascular cell adhesion molecule-1 (VCAM-1). The intensity of VCAM-1 expression correlates with the degree of inflammation of the synovial joint. Differential VCAM-1 expression may determine inflammatory cell accumulation through its interaction with leukocytes that express the counterreceptor integrins alpha4beta1 and alpha4beta7. Elevated levels of VCAM-1 expression are thought to be a consequence of the presence of inflammatory mediators, in particular IL-1beta and TNF-alpha. Fibroblast-like synoviocytes rapidly up-regulate VCAM-1 expression in response to IL-1beta and TNF-alpha, but also to IL-4. However, we now show that the response to IL-1beta or TNF-alpha is of a brief transient nature, even when applied continuously over a period of 12 days, whereas the response to IL-4 or IL-13 is sustained. Great synergy is obtained by combining either IL-4 or IL-13 with TNF-alpha, which results in a highly elevated but also sustained expression of VCAM-1. The mechanism by which IL-4 or IL-13 prolongs VCAM-1 expression can be explained by a dramatic increase in the half-life of VCAM-1 mRNA.     

A synergistic relationship between TNF-alpha, IL-1 beta, and TGF-beta 1 on IL-6 secretion by the IEC-6 intestinal epithelial cell line.

Intestinal epithelial cells are known to secrete a variety of cytokines and may play a role in the immune response at the intestinal mucosa. However, the regulatory mechanisms that govern the secretion of these cytokines are largely unknown. In this report, we have focused on the cytokine interactions that regulate interleukin (IL)-6 secretion by the non-transformed rat small intestinal epithelial cell line IEC-6. Tumour necrosis factor-alpha (TNF-alpha) was found to enhance both IL-6 mRNA expression and protein secretion by the IEC-6 cells. Furthermore, TNF-alpha acted in synergy with either transforming growth factor-beta 1 (TGF-beta 1) or IL-1 beta to greatly enhance IEC-6 cell IL-6 secretion. Although the IEC-6 cells are known to produce TGF-beta, autocrine-secreted TGF-beta was found to have no effect on the elevated IL-6 secretion induced by both TNF-alpha plus IL-1 beta. However, the addition of activated TGF-beta 1 to IEC-6 cultures stimulated with both TNF-alpha and IL-1 beta resulted in greatly elevated levels of IL-6 secretion. Therefore, activated TGF-beta 1 can augment IL-6 secretion stimulated by TNF-alpha and IL-1 beta, either alone or in combination, suggesting that intestinal epithelial cell IL-6 secretion may be under the control of a cytokine network at the intestinal mucosa.     

Regulation of hematopoietic growth factor production by genetically modified human bone marrow stromal cells expressing interleukin-1beta antisense RNA.

Interleukin-1 (IL-1) plays a major role in the regulation of bone marrow stromal cell function and hematopoiesis. It is known to induce secretion of the hematopoietic growth factors granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), IL-6, and IL-8 as well as IL-1 itself in stromal cells. We investigated the role of IL-1beta-mediated growth factor production in the human stromal cell line L88/5. Using liposome-mediated DNA transfer, two stromal cell transfectants that constitutively express IL-1beta antisense (AS) RNA were generated. Expression of IL-1beta AS RNA and IL-1beta RNA was determined by RT-PCR. The stromal cell transfectants were strongly impaired in their endogenous IL-1beta production, and this effect was present even when strong IL-1beta inducers, such as IL-1alpha and tumor necrosis factor-alpha (TNF-alpha), were used. Reduced endogenous IL-1beta levels had no effect on the constitutive production of IL-6, IL-8, and GM-CSF measured by ELISA. In contrast to lipopolysaccharide (LPS) stimulation, IL-1alpha-mediated stimulation of GM-CSF production was significantly reduced in AS transfectants. TNF-alpha induced GM-CSF production was also reduced. IL-6 and IL-8 production was increased in transfectants, suggesting a negative regulatory role of IL-1beta in L88/5. This new approach using AS technology to specifically target constitutive RNA expression will allow further characterization of the bone marrow cytokine network in normal and malignant hematopoiesis.     

Blockade of alpha6 integrin inhibits IL-1beta- but not TNF-alpha-induced neutrophil transmigration in vivo

In vitro and in vivo evidence supports a functional role for the integrin alpha6beta1 in neutrophil migration through the perivascular basement membrane, a response that in vivo appears to be associated with platelet/endothelial cell adhesion molecule-1 (PECAM-1)-mediated up-regulation of alpha6beta1 on the cell surface of transmigrating leukocytes. As the involvement of PECAM-1 in leukocyte migration is cytokine-specific, the aim of the present study was to investigate whether alpha6beta1 exhibited a similar profile of stimulus specificity in this context. The cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha) were used to elicit neutrophil migration in two murine models of inflammation, migration through cremasteric venules, as observed by intravital microscopy, and migration into the peritoneal cavity. The role of alpha6beta1 was investigated using an alpha6 integrin-blocking monoclonal antibody GoH3. In both models, GoH3 significantly inhibited neutrophil transmigration induced by IL-1beta but not TNF-alpha. This cytokine-specific role of alpha6 integrin was associated with enhanced cell-surface expression of alpha6beta1 on transmigrated neutrophils (as compared with blood cells) in response to IL-1beta but not TNF-alpha. Using lipopolysaccharide as an inflammatory stimulus in the cremaster muscle model, the study also provides evidence for the involvement of alpha6 integrin in leukocyte transmigration as mediated by endogenously generated IL-1beta. Collectively, the findings demonstrate that alpha6beta1 blockade inhibits neutrophil migration induced by exogenous and endogenous IL-1beta but not TNF-alpha, observations that are associated with increased expression of the integrin on transmigrated leukocytes.     

Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes.

The regulation by Cryptococcus neoformans encapsulation of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-alpha secretion induced by LPS or acapsular C. neoformans. In contrast, no regulator effect on IL-1 beta was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-alpha does not affect IL-1 beta production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococal polysaccharide could be devised.     

Effect of lisofylline and pentoxifylline on the bacterial-stimulated production of TNF-alpha, IL-1 beta IL-10 by human leucocytes.

The present study concerns the effect of the xanthine derivates lisofylline (LSF) and pentoxifylline (PTX) on the production of pro-inflammatory cytokines tumour-necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) and the de-activating cytokine interleukin-10 (IL-10) by human leucocytes during stimulation with lipopolysaccharide (LPS), heat-killed Gram-negative bacteria (GNB) or Gram-positive bacteria (GPB). The production of TNF-alpha and IL-1 beta by leucocytes stimulated with LPS, Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae was inhibited by both drugs. The production of IL-10 by leucocytes stimulated with LPS and Hib was inhibited by both xanthine derivates only at 48 hr. However, incubation of leucocytes with S. pneumoniae in the presence of LSF or PTX stimulated the production of IL-10 about four- and twofold at 24 hr and 48 hr, respectively. In all instances, the extent of inhibition or enhancement of cytokine production by LSF or PTX was equal. The divergent effects of xanthine derivates on the IL-10 production indicate the existence of distinct intracellular pathways depending on whether leucocytes are stimulated by GPB or GNB.     

Immunohistochemical staining of IL-1 alpha and IL-1 receptor antagonist but not IL-1 beta in cultures of sertoli cells

The interleukin-1 (IL-1) system has been suggested to be involved in the cell cell cross talk within the testis. To identify a testicular cell source of IL-1 alpha, IL-1 beta and IL-1 receptor antagonist (IL-1ra), immature mouse Sertoli cells were isolated, purified, cultured and examined for the cellular compartment localization of these cytokines by immunohistochemical staining. Our results show that both Germ cells and Sertoli cells in unpurified Sertoli cell cultures (before hypotonic shock) and purified culture of Sertoli cells (after hypotonic shock) were stained for IL-1 alpha. The levels of this cytokine were increased in Sertoli cells when the purified cultures were stimulated with lipopolysaccharide (LPS) (5 microg/mL). However, we could not identify a positive staining for IL-1 beta when Sertoli cell cultures were stained for this cytokine, even after stimulation with various concentrations of LPS (0.1-10 microg/mL). On the other hand, immunohistochemical staining of isolated Sertoli cells without treatment with hypotonic shock (cultures containing Sertoli cells and Germ cells) for IL-1ra showed constitutive positive staining of both cell types (Sertoli cells and Germ cells). Our results, using immunohistochemical staining, may indicate the different expression of IL-1 alpha, IL-1 beta and IL-1ra in Sertoli cells. These results may suggest the involvement of IL-1 system in the autocrine and paracrine regulation of testicular cell functions.     

Comparative study of cytokine release by human peripheral blood mononuclear cells stimulated with Streptococcus pyogenes superantigenic erythrogenic toxins, heat-killed streptococci, and lipopolysaccharide.

The differences between toxic or septic shocks in humans during infections by streptococci and gram-negative bacteria remain to be fully characterized. For this purpose, a quantitative study of the cytokine-inducing capacity of Streptococcus pyogenes erythrogenic (pyrogenic) exotoxins (ETs) A and C, heat-killed S. pyogenes bacteria, and Neisseria meningitidis endotoxin (lipopolysaccharide [LPS]) on human peripheral blood mononuclear cells (PBMC) and monocytes has been undertaken. The levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-6, IL-8, tumor necrosis factor alpha (TNF-alpha), and TNF-beta induced by these bacterial products and bacteria were determined by using cell supernatants. The capacity of ETs to elicit the monocyte-derived cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha was found to depend on the presence of T lymphocytes, because of the failure of purified monocytes to produce significant amounts of these cytokines in response to ETs. PMBC elicited large amounts of these cytokines, as well as IL-8 and TNF-beta, with an optimal release after 48 to 96 h. The most abundant cytokine produced in response to ETA was IL-8. In contrast to the superantigens ETA and ETC, LPS and heat-killed streptococci stimulated the production of significant amounts of IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha, with optimal production after 24 to 48 h in monocytes, indicating no significant involvement of T cells in the process. ETs, but neither LPS nor streptococci, were potent inducers of TNF-beta in PBMC. This study outlines the differences in the pathophysiological features of shock evoked by endotoxins and superantigens during infection by gram-negative bacteria and group A streptococci, respectively. The production of TNF-alpha was a common pathway for LPS, streptococcal cells, and ETs, although cell requirements and kinetics of cytokine release were different.     

Enhanced interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha production by LPS stimulated human monocytes isolated from HIV+ patients

Periodontal disease and tooth loss is a common finding among advanced HIV+ patients. In addition to local oral lipopolysaccharide (LPS) stimulation, systemic up-regulation of monocyte pro-inflammatory cytokine secretion may also be involved in the pathogenesis of HIV disease. A study was undertaken to investigate IL-1beta, IL-6 and TNF-alpha production by resting and LPS stimulated monocytes isolated from HIV+ patients and also to investigate the relationship of the patient's HIV viral load status to the cytokine production. Whole blood samples in EDTA were collected from 39 HIV-1 infected patients and 20 age and sex matched uninfected controls. Plasma was separated by centrifugation. Viral load was determined using a quantitative RT-PCR. Monocytes were isolated by Ficoll-hypaque gradient separation followed by overnight plastic adherence. Cultured monocytes (1x10(6)/ml) were stimulated with LPS (1 microg/ml) of either P. gingivalis or F. nucleatum for 2, 8, 24 and 48 h and supernatant fluids were collected. IL-1beta, IL-6, and TNF-alpha levels in supernatant fluids were estimated by ELISA. Increased overall production of IL-1beta, IL-6 and TNF-alpha by LPS stimulated monocytes isolated from HIV-1 infected patients was observed when compared to HIV-1 uninfected controls. LPS stimulated monocytes from HIV-1 infected patients with high viral load (HVL) produced significant (p<0.05) elevations in these pro-inflammatory cytokines when compared to HIV-1 uninfected controls. Both LPS of P. gingivalis and F. nucleatum produced a comparable cytokine production by monocytes after 8 h of stimulation. These data suggest that enhanced IL-1beta, IL-6 and TNF-alpha is produced by monocytes/macrophages isolated from HVL HIV+ patients and may be involved in the overall pathogenesis of HIV-1 infection.     

Biological function of CD40 on human endothelial cells: costimulation with CD40 ligand and interleukin-4 selectively induces expression of vascular cell adhesion molecule-1 and P-selectin resulting in preferential adhesion of lymphocytes

The expression of adhesion molecules on vascular endothelial cells determines the pattern of migration and extravasation of leucocytes in inflammation and immunity. Here we show that costimulation with CD40 ligand (CD40L) and interleukin (IL)-4 (or IL-13) gives rise to a unique pattern of adhesion molecule expression by human umbilical vein endothelial cells (HUVEC). CD40 ligation alone enhanced expression of vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin whereas IL-4 and IL-13 increased expression of VCAM-1 and P-selectin but not ICAM-1 or E-selectin. When IL-4 and CD40L were combined there was an additional increase of both VCAM-1 and P-selectin, but ICAM-1 and E-selectin were both inhibited. The combined effects of IL-4 and CD40L signalling were not the result of altered response kinetics, enhanced sensitivity of the endothelium, or increased expression of CD40 or the IL-4 receptor. The rise in VCAM-1 expression induced by combined IL-4 and CD40L stimulation was slower and more sustained than with tumour necrosis factor-alpha (TNF-alpha) and occurred only on a subset (75-80%) of the endothelial cell population compared to 100% with TNF-alpha. Costimulation with IL-4 and CD40L increased adhesion of T cells and B cells above levels obtained with either signal alone, but decreased adhesion of neutrophils. Furthermore, CD40 and IL-4 synergistically increased IL-6 but decreased IL-8 production by HUVEC. These results show that interactions between IL-4 and CD40 on endothelial cells give rise to specific patterns of adhesion molecule expression and cytokine production that may have important implications for lymphocyte and neutrophil migration and function at sites of inflammation.     

Proteinase-activated receptor2 agonists upregulate granulocyte colony-stimulating factor, IL-8, and VCAM-1 expression in human bronchial fibroblasts

Proteinase-activated receptors (PARs) are a novel family of G-protein-coupled receptors. PAR2 has been implicated in inflammatory airways disease. Although fibroblasts are pathologically important in the airways, the proinflammatory role of PAR2 in these cells remains unknown. We assessed PAR expression and functionality in human primary bronchial fibroblasts (HPBFs) before assessing PAR2-mediated HPBF proliferation, cytokine production, and adhesion molecule expression. RT-PCR and flow cytometry demonstrated that HPBFs express hPAR1, hPAR2, and hPAR3, but not hPAR4. Intracellular calcium signaling in HPBFs in response to PAR agonists showed that only hPAR1 and hPAR2 were functional receptors. We used the MTT assay to assess HPBF proliferation. Of the PAR2 agonist proteinases or selective PAR2-activating peptides (PAR2-APs) tested, none stimulated HPBF proliferation, whereas thrombin was a HPBF growth factor. mRNA for IL-8 and granulocyte colony-stimulating factor (G-CSF) was upregulated after addition of SLIGKV-NH2 when assessed by RT-PCR. No significant increase in G-CSF or IL-8 protein was detected. Trypsin stimulated IL-8 and G-CSF release from HPBF in a time- and dose-dependent manner. Leupeptin and soya trypsin inhibitor abrogated trypsin-stimulated cytokine release, indicating a requirement for trypsin's proteolytic activity. Trypsin and SLIGKV-NH2 stimulated an increase in VCAM-1 expression at 12 h after treatment, which declined thereafter. PAR2-driven upregulation of VCAM-1 cell surface expression and the release of IL-8 and G-CSF from bronchial fibroblasts may be important in promoting neutrophilic airways inflammation.