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.     

Bradykinin stimulates interleukin-8 production by human lung fibroblasts.

Bradykinin (BK) is a potent inflammatory mediator that is generated from kininogens by the actions of plasma and tissue kallikreins. Lung fibroblasts have the potential to participate in the inflammatory responses by releasing proinflammatory cytokines in response to a variety of stimuli. We postulated that human lung fibroblasts might produce interleukin-8 (IL-8) in response to BK stimulation. The present study showed that BK stimulated human lung fibroblasts to produce IL-8 in a dose- and time-dependent manner. Furthermore, Northern blot analysis showed that BK increased IL-8 mRNA expression. The stimulatory effect of BK on IL-8 production was detected at the concentration of 10 nm, and the maximal stimulation was achieved with 100 to 1000 nm. Phorbol 12-myristate 13-acetate pretreatment diminished the ability of BK to stimulate IL-8 production. In addition, GF109203X, a selective protein kinase C inhibitor, blocked BK-induced IL-8 production. These observations suggest that the stimulatory effect of BK on IL-8 production by lung fibroblasts is, at least partially, mediated through protein kinase C. These data suggest that BK may be involved in the inflammatory reaction leading to interstitial lung disorders through stimulating IL-8 production by lung fibroblasts.     

Thrombin-induced interleukin-8 production and its regulation by interferon-gamma and prostaglandin E2 in human monocytic U937 cells.

Blood coagulation and inflammation pathways are linked in many aspects. A number of serum factors involved in coagulation cascades affect directly or indirectly inflammatory responses, whereas proinflammatory cytokines influence blood coagulation pathways. In this work we demonstrated that thrombin is an effective stimulus in inducing interleukin (IL)-8 expression in human monocytic cell line U937. IL-8 induction was found at the mRNA and protein levels. The effect of thrombin on IL-8 production was mimicked by thrombin receptor-activating peptide indicating that thrombin effect was mediated by the specific receptor for thrombin. Moreover, thrombin-induced IL-8 production by U937 cells was differentially regulated by interferon (IFN)-gamma and prostaglandin (PG)E2. While IFN-gamma enhanced thrombin-induced IL-8 production, PGE2 acted as a negative regulator. Taken together, thrombin may play an important role in communication between blood coagulation and inflammation by inducing IL-8 production by monocytes and this role for thrombin may be further regulated by lymphokines and lipid mediators.     

Thrombin impairs alveolar fluid clearance by promoting endocytosis of Na+,K+-ATPase

Coagulation is an emerging area of interest in the pathogenesis and treatment of acute lung injury. Concentrations of the edemagenic coagulation protease thrombin are elevated in plasma and lavage fluids from afflicted patients. We explored the impact of thrombin on the formation and resolution of alveolar edema. Intravascularly applied thrombin inhibited active transepithelial 22Na transport in intact rabbit lungs, suppressing alveolar fluid clearance. Epithelial permeability was unaffected, whereas endothelial permeability was increased. In A549 human lung epithelial cells and in mouse primary alveolar type II cells, thrombin blocked ouabain-sensitive Na+,K+-ATPase-mediated 86Rb+ uptake, without altering amiloride-sensitive sodium currents. Furthermore, thrombin downregulated cell-surface expression of Na+,K+-ATPase, but not ENaC alpha and beta subunits. The endocytosis inhibitor phalloidin oleate blocked all thrombin-induced effects on sodium transport activity. Similarly, diphenyleneiodonium chloride, an inhibitor of reactive oxygen radical production, as well as a protein kinase C-zeta inhibitor, prevented these thrombin-induced effects. Thus, thrombin signaling via reactive oxygen species and protein kinase C-zeta promotes Na+,K+-ATPase endocytosis, resulting in loss of function. We propose here a dual role for thrombin in mediating disturbances to fluid balance in the lung: thrombin concomitantly provokes edema formation by increasing endothelial permeability, and inhibits alveolar edema resolution by blocking Na+,K+-ATPase function.     

Thrombin stimulates production of interleukin-8 in human umbilical vein endothelial cells.

Interleukin-8 (IL-8) is regarded as an important mediator of inflammation because of its potent and specific chemotactic activity on neutrophils. In the present investigation, human umbilical vein endothelial cells (HUVEC) stimulated with thrombin were found to produce IL-8, in a dose- and time-dependent manner. After stimulation with 10 U/ml thrombin for 24 hr, the level of IL-8 in the conditioned medium was 14 ng/ml, or enough to elicit PMN chemotaxis in vitro. Northern blot analysis revealed that thrombin as well as IL-1 beta elevates the level of IL-8 mRNA preceding the formation of IL-8 protein. A synthetic peptide SFLLRN [human thrombin receptor-activating peptide (TRAP)] was found to mimic the action of thrombin. Preincubation with anti-thrombin compounds such as hirudin and antithrombin-III-heparin almost completely suppressed the action of thrombin without affecting the actions of other stimuli including IL-1 beta, phorbol 12-myristate 13-acetate (PMA) and TRAP. Diisopropylfluorophosphate-treated thrombin did not stimulate IL-8 production. Calphostin-C, a protein kinase C (PKC) inhibitor, attenuated the production of IL-8 by thrombin, TRAP and PMA, but left the action of IL-1 beta unchanged. These results strongly suggest that catalytic activation of thrombin receptor by thrombin results in PKC-dependent IL-8 production accompanied by an increase in IL-8 mRNA level.     

IL-8 production and AP-1 transactivation induced by UVA in human keratinocytes: roles of D-alpha-tocopherol

Identification of individual response-signal pathway induced by UVA-irradiation is necessary for understanding photo-biological and -pathological mechanisms with respect to the prevention of UV-irradiated skin damage and aging. Here, we investigated the role of D-alpha-tocopherol in the regulation of IL-8 production and AP-1 binding activity in UVA-irradiated human keratinocytes. UVA dramatically upregulated IL-8 mRNA expression and protein secretion and enhanced the AP-1-DNA binding activity. These effects of UVA irradiation were effectively reduced by D-alpha-tocopherol in a dose-dependent manner. The human keratinocytes expressed various NAD(P)H oxidase components, gp91phox homologues Nox1, and p22phox, p47phox, p67phox, as well as NOXO1, suggesting that cellular stress induced by UVA included the activation of non-phagocytic NADPH oxidase system, leading to AP-1 transactivation and IL-8 expression. D-alpha-tocopherol significantly inhibited the NADPH oxidase activity and the formation of malondialdehyde-thiobarbituric acid under UVA exposure. These results demonstrated that D-alpha-tocopherol may be able to prevent the IL-8 upregulation and the increase in AP-1 activation induced by UVA irradiation through down-modulating cellular oxidative stress.     

Type-specific induction of interleukin-8 by adenovirus.

Infection with adenovirus (Ad) causes acute pneumonia in a type-specific fashion because type 7 but not type 5 Ad has been isolated as a causative agent. We postulated that the type specificity of induction of pneumonia may be related to type-specific cytokine induction in lung cells. To test this hypothesis, we infected human fetal lung fibroblasts and the lung epithelial cell line A549 with live type 5 and type 7 Ad. Virus inactivated by irradiation was used as a control. Type 7 but not type 5 Ad induced interleukin (IL)-8 protein production in both cell types in a dose- and time-dependent manner. Inactivated virus had no effect on the production of IL-8 protein. Type 7 but not type 5 virus also stimulated IL-8-specific messenger RNA (mRNA) production in these cells. Because half-life of IL-8 mRNA was prolonged in both type 5- and type 7-infected A549 cells, induction likely involves enhancement of message stability as well as other effects. Virus early gene expression did not consistently correlate with IL-8 message induction and followed induction in fibroblasts. These results suggest that there is type-specific induction of IL-8 production during infection of lung cells with Ad. Induction involves message stabilization and may not require viral gene expression. Because IL-8 is one of the important mediators of lung inflammation, type-specific induction of this and other cytokines may account for the different consequences of lung infection with different types of Ad.     

Human pulmonary fibroblasts exhibit altered interleukin-4 and interleukin-13 receptor subunit expression in idiopathic interstitial pneumonia

Abnormal proliferation of pulmonary fibroblasts is a prominent feature of chronic pulmonary fibrotic diseases such as idiopathic interstitial pneumonia (IIP), but it is not presently clear how this proliferative response by lung fibroblasts can be therapeutically modulated. In the present study, we examined whether it was possible to selectively target primary human pulmonary fibroblasts grown out of surgical lung biopsies (SLBs) from IIP patients based on their expression of interleukin-4 receptor (IL-4R) and IL-13R subunits. Pulmonary fibroblast lines cultured from patients with the severest form of IIP, namely usual interstitial pneumonia, exhibited the greatest gene and protein expression of IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2 compared with primary pulmonary fibroblast lines grown from other IIP SLBs and normal SLBs. When exposed to increasing concentrations of a chimeric protein comprised of human IL-13 and a truncated version of Pseudomonas exotoxin (IL13-PE), the proliferation of primary usual interstitial pneumonia fibroblasts was inhibited to a much greater extent compared with fibroblast lines from nonspecific interstitial pneumonia and respiratory bronchiolitis/interstitial lung disease patient groups. Fibroblasts from normal patients exhibited minimal susceptibility to the cytotoxic effect of IL13-PE. IL13-PE-mediated targeting of IIP fibroblasts was dependent on their expression of IL-4Ralpha and IL-13Ralpha2. Thus, these data suggest that the abnormal proliferative properties of human lung fibroblasts from certain IIP patient groups can be modulated in a manner that is dependent on the IL-4 and IL-13 receptor subunit expression by these cells.     

Interleukin-8 is a major neutrophil chemotactic factor derived from cultured human gingival fibroblasts stimulated with interleukin-1 beta or tumor necrosis factor alpha.

Inflammatory mediators produced by cells in the gingiva have been implicated in the initiation and progression of periodontal disease, a common infectious disease. In this study, we examined the biological activity of neutrophil chemotactic factors and the kinetics of expression of interleukin-8 (IL-8) mRNA derived from normal gingival fibroblasts in response to inflammatory mediators in an in vitro model. Gingival fibroblasts stimulated by either recombinant human interleukin-1 beta or recombinant human tumor necrosis factor alpha produced neutrophil chemotactic factors after 4 h, whereas expression of cell-derived IL-8 mRNA was detected within 1 h after stimulation. Furthermore, in a neutralization assay, rabbit anti-recombinant human IL-8 antiserum inhibited neutrophil chemotactic activity to basal levels. These results provide evidence that gingival fibroblasts synthesize potent chemotactic factors such as IL-8 in the presence of the inflammatory mediators interleukin-1 beta and tumor necrosis factor alpha. The activity of these factors may contribute to neutrophil-mediated processes in the pathogenesis of periodontal disease.     

Interferon-gamma inhibits interleukin-8 production by human polymorphonuclear leucocytes.

Stimulation of human polymorphonuclear leucocytes (PMN) with phagocytosable particles [yeast-IgG (Y-IgG)], lipopolysaccharide (LPS), tumour necrosis factor (TNF) or formyl-methionyl-leucyl-phenyl-alanine (FMLP) results in an increase of the interleukin-8 (IL-8) mRNA accumulation and a subsequent release of the protein. Here, we report that interferon-gamma (IFN-gamma) down-regulates the constitutive IL-8 mRNA levels expressed by resting PMN. As shown by Northern analysis, this down-modulation occurred rapidly, was not dependent on new protein synthesis, and was not caused by an increased rate of degradation of IL-8 mRNA. Preincubation of PMN with IFN-gamma significantly inhibited their ability to release IL-8 upon stimulation with TNF, LPS, FMLP and Y-IgG, but enhanced the respiratory burst capability in response to FMLP and TNF. TNF-, LPS- and FMLP-induced expression of IL-8 mRNA was also selectively inhibited by IFN-gamma. Taken together these findings suggest that IFN-gamma has important regulatory effects on acute inflammatory response because of its capacity to modulate negatively IL-8 gene expression and secretion by human PMN. Further observations revealed that, in human PMN, degradation of IL-8 mRNA is finely regulated, and that cycloheximide (CHX), an inhibitor of protein synthesis, super-induces the mRNA accumulation for IL-8 in a dose- and time-dependent manner.     

Pulmonary fibroblast expression of interleukin-8: a model for alveolar macrophage-derived cytokine networking

The pulmonary fibroblast's (PF) unique location allows it to communicate in a bidirectional fashion between the vascular compartment and alveolar airspace, placing it in a strategic position for the elicitation of inflammatory leukocytes into the lung. In this study, we demonstrate that PF may contribute to pulmonary inflammation through the production of a potent neutrophil chemotactic factor, interleukin (IL)-8. PF-derived IL-8 expression was dependent upon stimulation by either tumor necrosis factor (TNF) or IL-1 but not lipopolysaccharide (LPS). Both TNF and IL-1 stimulation of PF resulted in a time- and dose-dependent expression of steady-state levels of mRNA, antigen, and specific chemotactic activity consistent with IL-8. Because it was apparent that cytokine networking may exist in the lung between alveolar macrophage (AM)-derived cytokines and the production of PF-derived IL-8, we next examined an in vitro model of cellular communication within the lung. We determined that LPS-stimulated AM-conditioned media induced significant levels of PF-derived IL-8 mRNA, which was inhibited by preincubation with specific neutralizing TNF and IL-1 beta antibodies. Furthermore, when AM were directly co-cultured with PF and stimulated with LPS, the kinetic analysis of PF-derived antigenic expression of IL-8 was shifted toward the right. This suggested that PF-derived IL-8 expression in co-culture was first dependent upon activation of the AM by LPS and subsequent elaboration of macrophage inflammatory mediators. These data provide evidence that cytokine networking between AM and PF may be operative in the lung, culminating in the generation of IL-8 and elicitation of inflammatory leukocytes.     

Lipopolysaccharides of Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis induce interleukin-8 gene expression in human gingival fibroblast cultures.

Lipopolysaccharides (LPS) prepared from Bacteroides intermedius (Prevotella intermedia) and Bacteroides (Porphyromonas) gingivalis by hot phenol-water extraction induced interleukin-8 (IL-8) mRNA in normal human gingival fibroblast cultures, as demonstrated by Northern (RNA) blot analysis. IL-8 mRNA levels began to increase after a 2-h exposure, reached a maximum after 12 h, and then dropped to the unstimulated level at 48 h. IL-8 mRNA levels were also enhanced in a dose-dependent manner. By contrast, LPS specimens from various Salmonella species with S and R chemotypes and bacterial [corrected] and synthetic lipid A preparations did not increase IL-8 mRNA levels in fibroblasts. Although recombinant human IL-1 alpha induced IL-8 mRNA expression in fibroblast cultures, an antiserum to recombinant human IL-1 alpha did not decrease the IL-8 mRNA accumulation induced by B. intermedius LPS. Fibroblasts primed with natural human gamma interferon (IFN-gamma) expressed higher IL-8 mRNA levels upon stimulation with B. intermedius LPS, but not with Salmonella LPS, compared with nontreated cells. Natural human IFN-beta exhibited a similar priming effect on the fibroblasts, and antiserum to IFN-beta added to the cultures together with B. intermedius LPS decreased the IL-8 mRNA levels. Therefore, endogenous IFN-beta enhanced IL-8 mRNA production in response to B. intermedius LPS in fibroblasts.     

Thrombin and TNF-alpha/IL-1beta synergistically induce fibroblast-mediated collagen gel degradation

Degradation of preexisting and newly synthesized extracellular matrix is thought to play an important role in tissue remodeling. The current study evaluated whether thrombin and TNF-alpha/IL-1beta could collaboratively induce collagen degradation by human fetal lung fibroblasts (HFL-1) and adult bronchial fibroblasts cultured in three-dimensional collagen gels. TNF-alpha/IL-1beta alone induced production of matrix metalloproteinases (MMPs)-1, -3, and -9, which were released in latent form. With the addition of thrombin, the latent MMPs were converted into active forms and this resulted in collagen gel degradation. Part of the activation of MMPs by thrombin resulted from direct activation of MMP-1, MMP-2, MMP-3, and MMP-9 in the absence of cells. In addition, tissue inhibitor of metalloproteinase-1 production was inhibited by the combination of thrombin and TNF-alpha/IL-1beta. These results suggest that thrombin and TNF-alpha/IL-1beta synergize to induce degradation of three-dimensional collagen gels through increasing the production and activation of MMPs, and that this effect is mediated through both direct activation of MMPs by thrombin and indirectly by thrombin activation of fibroblasts. Through such mechanisms, thrombin could contribute to many chronic lung disorders characterized by tissue remodeling.