Dust mite proteolytic allergens induce cytokine release from cultured airway epithelium

Endogenous proteolytic enzymes have been shown to be potential sources of airway inflammation inducing proinflammatory cytokine release from respiratory epithelial cells; however, whether any of the exogenous proteases from important allergen sources such as the house dust mite present in our environment behave in a similar fashion is unclear. To this end, we have investigated whether the mite cysteine and serine proteolytic allergens, Der p 1 and Der p 9, respectively, induced cytokine production from primary human bronchial epithelial cells and from the epithelial cell line BEAS-2B. Cells were exposed to mite proteases, and cells or supernatants were assayed for cytokine release, cytokine mRNA expression, and modulation of intracellular calcium ion concentration. Both proteases induced concentration- and time-dependent increases in the release of granulocyte-macrophage (GM)-CSF, IL-6, and IL-8 as well as an increase in the expression of IL-6 mRNA. Cytokine release and mRNA expression were first observed at 8 h and 2 h after protease exposure, respectively. The minimum concentration of each protease that was required to stimulate GM-CSF, IL-6, and IL-8 release was approximately 10 ng/ml. Cytokine release was initiated by 1 to 2 h of protease exposure, although maximum concentrations were detected only after a 24-h incubation. IL-6, but not IL-8 and GM-CSF, was shown to be degraded by both proteases at concentrations of > 2 microg/ml. The proteases also stimulated changes in the intracellular calcium ion concentration. All mite protease-induced phenomena were inhibited using appropriate protease inhibitors. These results suggest that the proteolytic activity of an allergen may stimulate the release of proinflammatory cytokines from human bronchial epithelium.     

Individual cell analysis of the cytokine repertoire in human immunodeficiency virus-1-infected monocytes/macrophages by a combination of immunocytochemistry and in situ hybridization

The expression of many cytokines is dysregulated in individuals infected with the human immunodeficiency virus-1 (HIV-1). To determine the effects of HIV-1 infection on cytokine expression in individual cells (at the single cell level), we investigated the intracellular levels of proinflammatory cytokines (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, IL-6, and IL-8) and hematopoietic growth factors (granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage colony-stimulating factor [GM-CSF]) in monocyte-derived macrophages, mock-infected, or infected with HIV-1 by immunocytochemical staining for cytokine protein and compared this with secreted cytokine levels as determined by specific enzyme-linked immunosorbent assay (ELISA). No difference in the frequency or intensity of cell-associated immunocytochemical cytokine staining could be observed between HIV-1 and mock-infected cells even though the level of secreted proinflammatory cytokines increased and the hematopoietic growth factors decreased in HIV-1-infected cultures. Furthermore, equal expression of cytokine mRNA was observed in all cells in the culture regardless of whether the cells were productively infected with HIV-1 as determined by double-labelling immunocytochemical staining for HIV-1 p24 antigen and in situ hybridization for cytokine mRNA expression. These results indicate that HIV-1 infection results in dysregulation of intracellular cytokine mRNA expression and cytokine secretion not only in HIV-1-infected cells, but also through an indirect way(s) affecting cells not producing virus.     

The relationships between seasonal variations in the concentration of urea in bulk milk and the production and fertility of dairy herds

The retinal pigment epithelium (RPE) is clinically involved in diverse ocular inflammatory diseases. Because perturbed RPE cells produce a variety of inflammatory substances, RPE cells may play an integral part in these diseases. Interleukin-1 (IL-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are pleiotropic cytokines with the ability to trigger numerous inflammatory responses. This report shows that cultured human RPE cells synthesize interleukin-1 beta (IL-1 beta) and GM-CSF in response to the potentially inflammatory cytokine, IL-1 alpha, but not to E. coli endotoxin. Control RPE cells made little or no mRNA or protein for either IL-1 beta or GM-CSF. Upon stimulation of the cells by IL-1 alpha, both IL-1 beta and GM-CSF mRNAs were readily apparent by 3 hours, persisted for over 24 hours, and were translated into immunologically detectable proteins. GM-CSF protein was secreted into the culture medium, whereas IL-1 beta protein remained cell associated. The IL-1 alpha-induced mRNA and protein production were inhibited by dexamethasone. These observations provide additional evidence that RPE cells are capable of playing a pivotal role during ocular inflammation.     

Treatment with interleukin-4 prolongs allogeneic neonatal heart graft survival by inducing T helper 2 responses

BACKGROUND: Eosinophils are a prominent feature of chronic hyperplastic sinusitis with nasal polyposis (CHS/NP). Our previous studies showed that their presence was associated with the expression of GM-CSF and RANTES mRNA. In allergic NP, increased expression of IL-5 was also found. OBJECTIVE: We wished to examine cytokine immunoreactivity for IL-5, GM-CSF and RANTES mRNA in allergic and non-allergic NP and compare immunoreactivity with expression of cytokine mRNA by in situ hybridization. Methods NP were obtained from five allergic and eight non-allergic subjects with CHS/ NP. Middle turbinate tissue from eight normal subjects were used as controls. Cell-associated cytokine mRNA was detected by in situ hybridization (ISH). Cytokine immunoreactive cells were enumerated by immunostaining. Colocalization immunostaining was also performed to identify specific cell types producing IL-5. RESULTS: Immunostaining for GM-CSF, IL-5 and RANTES protein was increased in both allergic and non-allergic NP compared with control middle turbinates. Allergic polyps contained greater numbers of IL-5 immunoreactive cells (P = 0.01), whereas non-allergic polyps contained greater numbers of GM-CSF immunoreactive cells (P = 0.04). Immunostaining was primarily associated with inflammatory cells, but immunostaining for RANTES and, to a lesser extent GM-CSF, was also seen in the epithelium. The density of immunoreactive cells was variably correlated with cytokine mRNA+ cells (GM-CSF: R=0.56, P=0.05; IL-5: R=0.76, P=0.003; and RANTES: R=0.89, P=0.0005). Colocalization immunostaining revealed that the majority of IL-5 immunoreactive cells in both allergic and non-allergic NP were T lymphocytes. However, allergic NP contained greater numbers of IL-5+/CD3+ T lymphocytes and IL-5+ mast cells, whereas non-allergic NP contained greater numbers of IL-5+ eosinophils. CONCLUSION: We conclude that GM-CSF, IL-5 and RANTES are produced in increased amounts in both allergic and non-allergic NP. Distinguishing features of non-allergic NP include fewer numbers of CD3 T lymphocytes, fewer IL-5+/CD3+ T lymphocytes and greater numbers of IL-5+ eosinophils. These differences may suggest different mechanisms of eosinophil accumulation and activation in allergic vs non-allergic NP.     

Interleukin-17

The particular interest of IL-17, a homodimeric cytokine of about 32 kDa, is the strict requirement for an activation signal to induce its expression from a rather restricted set of cells, human memory T cells or mouse alpha beta TCR+CD4-CD8- thymocytes. In contrast with the tightly controlled expression pattern of this gene, the IL-17 receptor, a novel cytokine receptor, is ubiquitously distributed but apparently more abundant in spleen and kidney. In addition to its capture by the T lymphotropic Herpesvirus Saimiri (HVS), this cytokine is inducing the secretion of IL-6, IL-8, PGE2, MCP-1 and G-CSF by adherent cells like fibroblasts, keratinocytes, epithelial and endothelial cells. IL-17 is also able to induce ICAM-1 surface expression, proliferation of T cells, and growth and differentiation of CD34+ human progenitors into neutrophils when cocultured in presence of irradiated fibroblasts. In vitro, IL-17 synergizes with other proinflammatory signals like TNF alpha for GM-CSF induction, and with CD40-ligand for IL-6, IL-8, RANTES and MCP-1 secretion from kidney epithelial cells. In vivo, injection of IL-17 induces a neutrophilia, except in IL-6-KO mice. The involvement of IL-17 in rejection of kidney graft has also been demonstrated. The role of this T cell secreted factor in various inflammatory processes is presently investigated.     

Fast modulation of glutamate and GABA receptor--mediate electrophysiological responses by glucocorticoid]

Endothelin (ET) is a powerful vasoconstrictor and bronchoconstrictor peptide that may be involved in the pathogenesis of bronchial asthma. We have investigated the effect of ET on the secretion of IL-6, IL-8, GM-CSF and G-CSF in a bronchial epithelial cell line (BEAS-2B). Incubation of BEAS-2B cells with ET-1 (10(-13) to 10(-7) M) for 4 h caused dose-related increases in the release of IL-8 (68% increase above control, P < 0.001) and IL-6 (43% increase above control, P < 0.001), compared to untreated control cells. After 48 h incubation, ET-1 also increased the release of IL-8 by 35% (P < 0.001) and GM-CSF by 38% (P < 0.01). ET-1 had no significant effect on G-CSF release. ET-1 did not induce cell proliferation at 24 or 48 h. Since ET-immunoreactive materials are expressed in epithelial cells in asthma, it is possible that ET-1 of epithelial origin may act in a paracrine or autocrine fashion on airway epithelial ET receptors to stimulate IL-8, IL-N6 and GM-CSF release. Thus, ET-1 may play a role in the regulation of the cytokine responses involved in inflammation of the airway mucosa.     

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Smooth muscle cells represent a significant percentage of the total cells in the airway but their contribution to the inflammatory response seen in airway disease has not been studied. Hence, we have looked at the release of the cytokine granulocyte-macrophage colony stimulating factor (GM-CSF) in response to bacterial lipopolysaccharide (LPS) and the pro-inflammatory cytokines interleukin-1 beta (IL-1 beta), tumour necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma). Human airway smooth muscle (HASM) cells released GM-CSF under basal conditions (45.4 +/- 13.1 pg ml-1) that was significantly enhanced by IL-1 beta and TNF alpha with a maximal effect seen at 10 ng ml-1 (1.31 +/- 0.07 ng ml-1 and 0.72 +/- 0.16 ng ml-1, respectively). In contrast, neither LPS nor IFN gamma produced a significant increase in GM-CSF release. However, HASM cells exposed to IL-1 beta, TNF alpha and IFN gamma generated more GM-CSF than that evoked by any cytokine alone (2.2 +/- 0.15 ng ml-1). The release of GM-CSF elicited by the cytokine mixture was inhibited by cycloheximide and dexamethasone. These data suggest that HASM cells might play an active part in initiating and/or perpetuating airway inflammation in addition to controlling airway calibre.     

Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin-2, interleukin-7 and interleukin-12

Interleukin (IL)-2 IL-7 and IL-12 stimulate the generation of lymphokine-activated killer activity and proliferation in natural killer (NK) cells by different mechanisms. In this study, we have compared the ability of IL-2, IL-7 and IL-12 to induce expression of cytokines and cytokine receptors both at the gene and protein level. IL-2 and IL-12 stimulated the CD56+ NK cells to release significant amounts of soluble p55 and p75 tumor necrosis factor receptor (TNFR), whereas less amounts of soluble TNFR were detected in IL-7-stimulated cultures. The p55 and p75 TNFR mRNA were expressed in resting NK cells, and no further induction was observed after cytokine-stimulation. Compared to the effects of IL-2, IL-7 induced lower, but substantial levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA, and IL-7 was a more potent GM-CSF-inducing stimulus than IL-12. IL-12 induced higher levels of interferon-gamma (IFN-gamma) mRNA than did IL-2, and IL-7 only weakly influenced the IFN-gamma expression. In accordance with the mRNA studies, IL-7 induced the secretion of high amounts of GM-CSF and no or low levels of IFN-gamma, whereas high amounts of IFN-gamma and low levels of GM-CSF were detected in supernatants from IL-12-stimulated NK cells. In conclusion, IL-2, IL-7 and IL-12 differentially regulate expression of cytokines and cytokine receptors both at the gene and protein level.     

Cell-associated human retinal pigment epithelium interleukin-8 and monocyte chemotactic protein-1: immunochemical and in-situ hybridization analyses

Human retinal pigment epithelial (RPE) cells secrete chemokines, interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in response to pro-inflammatory cytokines. In this study we (1) examined the efficiency of human RPE IL-8 and MCP-1 secretion, (2) determined the amount of neutrophil and monocyte chemotactic activity in human RPE cell conditioned media and cell extracts that is attributable to IL-8 and MCP-1, respectively, and (3) assessed the sensitivity of immunohistochemistry and in situ hybridization for detecting chemokine production by cytokine-stimulated human RPE cells. Conditioned media and extracts from human RPE cells stimulated with various physiologic concentrations of interleukin-1 beta (IL-1 beta) (0.2-20 ng ml-1), tumor necrosis factor (TNF-alpha) (0.2-20 ng ml-1) or interferon-gamma (IFN-gamma) (10-1000 U ml-1) were examined to compare secreted and cell associated levels of IL-8 and MCP-1 at various time points up to 24 hr. ELISA demonstrated that IL-8 and MCP-1 are both efficiently secreted by pro-inflammatory cytokine treated human RPE cells. Substantial dose- and time-dependent RPE secretion of IL-8 was observed following stimulation with IL-1 beta or TNF-alpha, but cell associated IL-8 was detectable only after high dose (20 ng ml-1) IL-1 beta stimulation and comprised less than 1% of the total IL-8 induced. Dose- and time-dependent RPE cell MCP-1 secretion was also observed following IL-1 beta > TNF-alpha > IFN-gamma stimulation, with an average of 4% of the total MCP-1 retained within RPE. Bioassays demonstrated neutrophil and monocyte chemotactic activity in conditioned media from stimulated RPE cells, but not in human RPE cell extracts. Inhibition of conditioned media-induced chemotaxis by specific anti-IL-8 or anti-MCP-1 antibodies demonstrated that IL-8 and MCP-1 were responsible for the majority of HRPE-derived neutrophil (> 60%) and monocyte (53-57%) chemotactic activity, respectively. Using in situ hybridization IL-8 mRNA was readily detected within IL-1 beta > TNF-alpha stimulated RPE cells and MCP-1 mRNA easily visualized within IL-1 beta > TNF-alpha > or IFN-gamma stimulated cells. Immunohistochemistry to detect IL-8 was positive only in RPE cells exposed to high dose IL-1 beta (20 ng ml-1) for 8 or 24 hr and was weak. Immunohistochemical staining for MCP-1 in RPE cells was more intense and was visualized within RPE cells stimulated with IL-beta, TNF-alpha, or IFN-gamma. This study demonstrates that: (1) RPE cells efficiently secrete IL-8 and MCP-1 upon stimulation with pro-inflammatory cytokines; (2) secreted IL-8 and MCP-1 account for the majority of human RPE neutrophil and monocyte chemotactic activity; (3) in situ hybridization readily detects IL-8 and MCP-1 mRNA in cytokine stimulated RPE cells; and (4) immunohistochemistry demonstrates cell-associated MCP-1 in cytokine stimulated RPE cells, but only minimal cell-associated IL-8.     

Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells

Pseudomonas aeruginosa, an opportunistic human pathogen, causes acute pneumonia in patients with hospital-acquired infections and is commonly associated with chronic lung disease in individuals with cystic fibrosis (CF). Evidence suggests that the pathophysiological effects of P. aeruginosa are mediated in part by virulence factors secreted by the bacterium. Among these factors is pyocyanin, a redox active compound that increases intracellular oxidant stress. We find that pyocyanin increases release of interleukin-8 (IL-8) by both normal and CF airway epithelial cell lines and by primary airway epithelial cells. Moreover, pyocyanin synergizes with the inflammatory cytokines tumor necrosis factor alpha and IL-1alpha. RNase protection assays indicate that increased IL-8 release is accompanied by increased levels of IL-8 mRNA. The antioxidant n-acetyl cysteine, general inhibitors of protein tyrosine kinases, and specific inhibitors of mitogen-activated protein kinases diminish pyocyanin-dependent increases in IL-8 release. Conversely, inhibitors of protein kinases C (PKC) and PKA have no effect. In contrast to its effects on IL-8 expression, pyocyanin inhibits cytokine-dependent expression of the monocyte/macrophage/T-cell chemokine RANTES. Increased release of IL-8, a potent neutrophil chemoattractant, in response to pyocyanin could contribute to the marked infiltration of neutrophils and subsequent neutrophil-mediated tissue damage that are observed in Pseudomonas-associated lung disease.     

The regulation and functional consequence of proinflammatory cytokine binding on human intestinal epithelial cells

Products of an activated immune system may affect cells within the immune system as well as nonlymphoid cells in the local environment. Given the immunologically activated state of the intestinal tract, it is conceivable that locally produced cytokines could regulate epithelial cell function. To assess whether epithelial cells are targets for particular cytokines, we initiated studies on the binding of a panel of proinflammatory cytokines in freshly isolated epithelial cells from normal and inflammatory bowel disease (IBD) patients as well as in cell lines. Isolated intestinal epithelial cells (IEC) were stained with phycoerythrin-conjugated or biotinylated cytokines to determine the expression and density of receptors for IL-1beta, IL-6, granulocyte-macrophage CSF (GM-CSF), and TNF-alpha. Receptors for IL-1beta, IL-6, and GM-CSF were readily detectable in all epithelial cell preparations at levels equal to (GM-CSFR) or lower than those seen on monocytes. However TNFalpha-R were not detectable on freshly isolated IECs. Receptor density was greater in surface vs crypt epithelial cells, but no significant differences were seen between normal and IBD epithelial cells. Expression of IL-1R and IL-6R was enhanced by LPS and IFN-gamma. Functionally, IL-1beta enhanced proliferation of the IEC cell line, DLD1, whereas GM-CSF treatment of de-differentiated crypt-like DLD1 and HT29 cells resulted in enhanced expression of ICAM-1. Furthermore, TNF-alpha treatment enhanced the secretion of IL-8 and GRO-alpha in HT29 cells, but not in freshly isolated IEC cultures. The differential binding and function of proinflammatory cytokines on IEC support the hypothesis that these cytokines may be involved in normal physiological processes as well as in regulating mucosal immune responses.     

Magnetic ordering in the three-dimensional site-disordered Heisenberg model

The role of oncostatin M (OM) in modulating production of cytokines by connective tissue cells is largely unexplored. We have examined the effects of stimulating fibroblast cultures derived from human synovium and from normal lung with OM alone or in combination with IL-1, IL-1 alpha (or IL-1 beta) at 1 or 5 ng/ml, stimulated production of high levels of granulocyte-macrophage CSF (GM-CSF), IL-8, and IL-6 protein. At various concentrations (0.1-50 ng/ml), OM alone failed to significantly enhance protein or mRNA levels of GM-CSF, IL-8, IL-6, or G-CSF after 18 h of stimulation. When combined with IL-1 alpha or -beta, OM caused a dose-dependent inhibition of the IL-1-induced level of IL-8 and GM-CSF protein and mRNA expression, whereas IL-6 production was simultaneously enhanced. In contrast, when IL-6 or leukemia inhibitory factor (two other cytokines that share gp130 receptor components with OM) were used in a similar fashion in combination with IL-1 alpha, neither cytokine consistently altered the IL-1-induced levels of IL-8, GM-CSF, or IL-6. In addition, only OM and not IL-6 or leukemia inhibitory factor was able to induce STAT-1 nuclear factor binding to DNA in stimulated fibroblast extracts as measured by electrophoretic mobility shift assay. These results suggest that OM can significantly alter cytokine profiles of stimulated fibroblasts and may play a unique role in modulating cytokine production by these cells at sites of inflammation.     

Focal adhesion kinase upregulated by granulocyte-macrophage colony-stimulating factor but not by interleukin-3 in differentiating myeloid cells

The involvement of focal adhesion kinase (FAK) in myeloid differentiation was investigated in primary murine bone marrow (BM) cells. In unstimulated BM, FAK mRNA was detected in myeloid and lymphoid cells, but not in erythroid precursors. When the BM cells were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), FAK expression showed a remarkable difference depending on the cytokine. Although FAK was upregulated in the cells stimulated by GM-CSF (GM-treated cells), the kinase was barely detectable in the cells cultured with IL-3 (IL-3-treated cells). Morphology and flow cytometry analysis showed GM-CSF promoted the growth and differentiation of monocyte/macrophage lineage stronger than IL-3. In addition, motility of the cytokine-differentiated cells showed an overt distinction between the cultures, which was closely correlated with FAK expression. After 7 days of stimulation, GM-treated cells showed active migration and chemoattractant-induced morphologic polarization. In contrast, IL-3-treated cells showed minimal migration and polarization. These results suggest an important role of GM-CSF in the terminal differentiation of monocytes/macrophages, and possible involvement of FAK in functional maturity of this lineage.