Toll-like receptor 9 mediates oral bacteria-induced IL-8 expression in gingival epithelial cells

Previously, we reported that various oral bacteria regulate interleukin (IL)-8 production differently in gingival epithelial cells. The aim of this study was to characterize the pattern recognition receptor(s) that mediate bacteria-induced IL-8 expression. Among ligands that mimic bacterial components, only a Toll-like receptor (TLR) 9 ligand enhanced IL-8 expression as determined by ELISA. Both normal and immortalized human gingival epithelial (HOK-16B) cells expressed TLR9 intracellularly and showed enhanced IL-8 expression in response to CpG-oligonucleotide. The ability of eight strains of four oral bacterial species to induce IL-8 expression in HOK-16B cells, and their invasion capacity were examined in the absence or presence of 2% human serum. The ability of purified bacterial DNA (bDNA) to induce IL-8 was also examined. Six out of eight strains increased IL-8 production in the absence of serum. Usage of an endosomal acidification blocker or a TLR9 antagonist inhibited the IL-8 induction by two potent strains. In the presence of serum, many strains lost the ability to induce IL-8 and presented substantially reduced invasion capacity. The IL-8-inducing ability of bacteria in the absence or presence of serum showed a strong positive correlation with their invasion index. The IL-8-inducing ability of bacteria in the absence of human serum was also correlated with the immunostimulatory activity of its bDNA. The observed immunostimulatory activity of the bDNA could not be linked to its CpG motif content. In conclusion, oral bacteria induce IL-8 in gingival epithelial cells through TLR9 and the IL-8-inducing ability depends on the invasive capacity and immunostimulating DNA.     

c-Src mediates thrombin-induced NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells

In this study, we examined the regulation of NF-kappaB activation and IL-8/CXCL8 expression by thrombin in human lung epithelial cells (EC). Thrombin caused a concentration-dependent increase in IL-8/CXCL8 release in a human lung EC line (A549) and primary normal human bronchial EC. In A549 cells, thrombin, SFLLRN-NH2 (a protease-activated receptor 1 (PAR1) agonist peptide), and GYPGQV-NH2 (a PAR4 agonist peptide), but not TFRGAP-NH2 (a PAR3 agonist peptide), induced an increase in IL-8/CXCL8-luciferase (Luc) activity. The thrombin-induced IL-8/CXCL8 release was attenuated by D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (a thrombin inhibitor), U73122 (a phosphoinositide-phospholipase C inhibitor), Ro-32-0432 (a protein kinsase C alpha (PKC alpha) inhibitor), an NF-kappaB inhibitor peptide, and Bay 117082 (an IkappaB phosphorylation inhibitor). Thrombin-induced increase in IL-8/CXCL8-Luc activity was inhibited by the dominant-negative mutant of c-Src and the cells transfected with the kappaB site mutation of the IL-8/CXCL8 construct. Thrombin caused time-dependent increases in phosphorylation of c-Src at tyrosine 416 and c-Src activity. Thrombin-elicited c-Src activity was inhibited by Ro-32-0432. Stimulation of cells with thrombin activated IkappaB kinase alphabeta (IKK alphabeta), IkappaB alpha phosphorylation, IkappaB alpha degradation, p50 and p65 translocation from the cytosol to the nucleus, NF-kappaB-specific DNA-protein complex formation, and kappaB-Luc activity. Pretreatment of A549 cells with Ro-32-4032 and the dominant-negative mutant of c-Src DN inhibited thrombin-induced IKK alphabeta activity, kappaB-Luc activity, and NF-kappaB-specific DNA-protein complex formation. Further studies revealed that thrombin induced PKC alpha, c-Src, and IKK alphabeta complex formation. These results show for the first time that thrombin, acting through PAR1 and PAR4, activates the phosphoinositide-phospholipase C/PKC alpha/c-Src/IKK alphabeta signaling pathway to induce NF-kappaB activation, which in turn induces IL-8/CXCL8 expression and release in human lung EC.     

IL-33 mediates inflammatory responses in human lung tissue cells

IL-33 is a member of the IL-1 family and mediates its biological effects via the ST2 receptor, which is selectively expressed on Th2 cells and mast cells. Although polymorphic variation in ST2 is strongly associated with asthma, it is currently unclear whether IL-33 acts directly on lung tissue cells at sites of airway remodeling. Therefore, we aimed to identify the IL-33-responsive cells among primary human lung tissue cells. ST2 mRNA was expressed in both endothelial and epithelial cells but not in fibroblasts or smooth muscle cells. Correspondingly, IL-33 promoted IL-8 production by both endothelial and epithelial cells but not by fibroblasts or smooth muscle cells. Transfection of ST2 small interference RNA into both endothelial and epithelial cells significantly reduced the IL-33-dependent upregulation of IL-8, suggesting that IL-33-mediated responses in these cells occur via the ST2 receptor. Importantly, Th2 cytokines, such as IL-4, further enhanced ST2 expression and function in both endothelial and epithelial cells. The IL-33-mediated production of IL-8 by epithelial cells was almost completely suppressed by corticosteroid treatment. In contrast, the effect of corticosteroid treatment on the IL-33-mediated responses of endothelial cells was only partial. IL-33 induced activation of both ERK and p38 MAPK in endothelial cells but only ERK in epithelial cells. p38 MAPK was required for the IL-33-mediated responses of endothelial cells, whereas ERK was required for IL-33-mediated IL-8 production by epithelial cells. Taken together, these findings suggest that IL-33-mediated inflammatory responses of lung tissue cells may be involved in the chronic allergic inflammation of the asthmatic airway.     

IL-10, but not IL-4, suppresses infection-stimulated bone resorption in vivo

Periapical bone resorption occurs following infection of the dental pulp and is mediated mainly by IL-1alpha in the murine model. The production and activity of IL-1alpha is modulated by a network of regulatory cytokines, including those produced by Th1 (pro-inflammatory) and Th2 (anti-inflammatory) subset T cells. This study was designed to assess the functional role of the Th2-type cytokines IL-4 and IL-10 in infection-stimulated bone resorption in vivo. The dental pulps of the first molars were exposed and infected with a mixture of four common endodontic pathogens, and bone destruction was determined by micro-computed tomography at sacrifice on day 21. The results demonstrate that IL-10(-/-) mice had significantly greater infection-stimulated bone resorption in vivo compared with wild-type mice (p < 0.001), whereas IL-4(-/-) exhibited no increased resorption. IL-10(-/-) had markedly elevated IL-1alpha production within periapical inflammatory tissues (>10-fold) compared with wild type (p < 0.01), whereas IL-4(-/-) exhibited decreased IL-1alpha production (p < 0.05). IL-10 also suppressed IL-1alpha production by macrophages in a dose-dependent fashion in vitro, whereas IL-4 had weak and variable effects. We conclude that IL-10, but not IL-4, is an important endogenous suppressor of infection-stimulated bone resorption in vivo, likely acting via inhibition of IL-1alpha expression.     

ZIP9 but not the androgen receptor mediates testosterone-induced migratory activity of metastatic prostate cancer cells

LNCaP cells are derived from a metastatic lesion of human prostate adenocarcinoma. They express the classical androgen receptor (AR) and ZIP9, a Zn²⁺ transporter that also binds testosterone and mediates signaling by interacting with G-proteins.Our results show that LNCaP cells respond to testosterone by mobilizing their migratory machinery. Their exposure to testosterone triggers the formation of lamellipodia, reorganization of the actin cytoskeleton, phosphorylation of focal adhesion kinase (FAK) at Tyr925 and of paxillin at Tyr118, expression of matrix metalloproteinase 2 (MMP-2), and cell migration.Silencing ZIP9 expression by means of siRNA does not affect the responsiveness of the classical AR to testosterone; however, it prevents all of the testosterone effects described above: formation of lamellipodia cannot be induced, stimulation of FAK or paxillin phosphorylation or MMP-2 expression is prevented, and cell migration does not take place in the absence of ZIP9.The data presented show that testosterone/ZIP9 interactions might have not only physiological but also pathophysiological relevance. The fact that the migratory machinery of a metastatic prostate cancer cell line is activated exclusively through testosterone/ZIP9 and not through testosterone/AR interactions suggests that targeting specific inhibition of testosterone/ZIP9-mediated events might help in developing new therapeutic strategies against androgen-induced progression of prostate cancer.     

IL-9 promotes but is not necessary for systemic anaphylaxis

Anaphylaxis represents an extreme form of allergic reaction, consisting of a sensitization phase during which allergen-specific IgE are produced and an acute effector phase triggered by allergen-induced degranulation of mast cells. We studied the role of IL-9, a Th2 cytokine implicated in asthma, in different models of murine anaphylaxis. Using a passive model of systemic anaphylaxis, in which anti-DNP IgE Abs were administered before challenge with DNP-BSA, we found that IL-9-transgenic mice or wild-type mice treated with IL-9 for 5 days were highly sensitive to fatal anaphylaxis. This effect was reproduced in both anaphylaxis-susceptible and -resistant backgrounds (FVB/N or [FVB/N x BALB/c] F(1) mice, respectively) and correlated with increased serum concentrations of mouse mast cell protease-1 level, a protein released upon mast cells degranulation. By contrast, IL-9 did not increase the susceptibility to passive cutaneous anaphylaxis. IL-9 expression also increased the susceptibility to fatal anaphylaxis when mice were sensitized by immunization against OVA before challenge with the same Ag. In this model, serum from sensitized, IL-9-transgenic mice was more potent in transferring susceptibility to OVA challenge into naive mice, indicating that IL-9 also promotes the sensitization stage. Finally, using IL-9R-deficient mice, we found that despite its anaphylaxis-promoting activity, IL-9 is dispensable for development of both passive and active anaphylaxis, at least in the C57BL/6 mouse background. Taken together, the data reported in this study indicate that IL-9 promotes systemic anaphylaxis reactions, acting at both the sensitization and effector stages, but is not absolutely required for this process.     

Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha

Synthesis of the antimicrobial protein neutrophil gelatinase-associated lipocalin (NGAL) increases dramatically in bronchial epithelial cells and alveolear type II pneumocytes during lung inflammation. IL-1beta induces a >10-fold up-regulation of NGAL expression in the type II pneumocyte-derived cell line A549 cells, whereas TNF-alpha, IL-6, and LPS had no effect. Similar IL-1beta selectivity was demonstrated in primary bronchial epithelial cells and epidermal keratinocytes and for an NGAL promoter fragment transfected into A549 cells. By deletion and substitution analysis of the NGAL promoter, a 40-bp region containing an NF-kappaB consensus site was found to control the IL-1beta-specific up-regulation. Involvement of the NF-kappaB site was demonstrated by site-directed mutagenesis, by transfection with a dominant-negative inhibitor of the NF-kappaB pathway, and by EMSA. TNF-alpha activation of NF-kappaB, in contrast, did not increase NGAL synthesis, even though induced binding of NF-kappaB to the NGAL promoter was observed in vitro. IL-1beta specificity was not contained within the NF-kappaB site of the NGAL promoter, as determined by exchanging the NGAL promoter's NF-kappaB-binding sequence with that of the IL-8 promoter or with the NF-kappaB consensus sequence and by testing the NF-kappaB-binding sequence of the NGAL promoter against the heterologous SV40 promoter. Selectivity for the IL-1 pathway was substantiated by demonstrating that NGAL promoter activity could be induced by LPS stimulation of A549 cells transiently expressing Toll-like receptor 4, which use the same intracellular signaling pathway as the IL-1R. Together, this demonstrates a selective up-regulation of NGAL by the IL-1 pathway.     

Increased lung expansion alters lung growth but not alveolar epithelial cell differentiation in newborn lambs

Although increased lung expansion markedly alters lung growth and epithelial cell differentiation during fetal life, the effect of increasing lung expansion after birth is unknown. We hypothesized that increased basal lung expansion, caused by ventilating newborn lambs with a positive end-expiratory pressure (PEEP), would stimulate lung growth and alter alveolar epithelial cell (AEC) proportions and decrease surfactant protein mRNA levels. Two groups of lambs were sedated and ventilated with either 0 cmH(2)O PEEP (controls, n = 5) or 10 cmH(2)O PEEP (n = 5) for 48 h beginning at 15 +/- 1 days after normal term birth. A further group of nonventilated 2-wk-old lambs was used for comparison. We determined wet and dry lung weights, DNA and protein content, a labeling index for proliferating cells, surfactant protein mRNA expression, and proportions of AECs using electron microscopy. Although ventilating lambs for 48 h with 10 cmH(2)O PEEP did not affect total lung DNA or protein, it significantly increased the proportion of proliferating cells in the lung when compared with nonventilated 2-wk-old controls and lambs ventilated with 0 cmH(2)O PEEP (control: 2.6 +/- 0.5%; 0 PEEP: 1.9 +/- 0.3%; 10 PEEP: 3.5 +/- 0.3%). In contrast, no differences were observed in AEC proportions or surfactant protein mRNA levels between either of the ventilated groups. This study demonstrates that increases in end-expiratory lung volumes, induced by the application of PEEP, lead to increased lung growth in mechanically ventilated 2-wk-old lambs but do not alter the proportions of AECs.     

Bleomycin initiates apoptosis of lung epithelial cells by ROS but not by Fas/FasL pathway

Epithelial cells are considered to be a main target of bleomycin-induced lung injury, which leads to fibrosis in vivo. We studied the characteristics of in vitro bleomycin-induced apoptosis in a mouse lung epithelial (MLE) cell line. Bleomycin caused an increase of reactive oxygen species (ROS) resulting in oxidative stress, mitochondrial leakage, and apoptosis. These were associated with elevated caspase-8 and resultant caspase-9 activity and with upregulation of Fas expression. Glutathione and inhibitors of caspase-8 or caspase-9, but not of FasL, inhibited these effects, suggesting their dependence on ROS, caspase-8 and -9, in a Fas/FasL-independent pathway. However, postbleomycin-exposed MLE cells were more sensitive to Fas-mediated apoptosis. These results demonstrate that the initial bleomycin-induced oxidative stress causes a direct apoptotic effect in lung epithelial cells involving a regulatory role of caspase-8 on caspase-9. Fas represents an amplification mechanism, and not a direct trigger of bleomycin-induced epithelial cell apoptosis.     

Microtubules are stabilized in confluent epithelial cells but not in fibroblasts

Rhodamine-tagged tubulin was microinjected into epithelial cells (MDCK) and fibroblasts (Vero) to characterize the dynamic properties of labeled microtubules in sparse and confluent cells. Fringe pattern fluorescence photobleaching revealed two components with distinct dynamic properties. About one-third of the injected tubulin diffused rapidly in the cytoplasm with a diffusion coefficient of 1.3-1.6 x 10(- 8) cm2/s. This pool of soluble cytoplasmic tubulin was increased to greater than 80% when cells were treated with nocodazole, or reduced to approximately 20% upon treatment of cells with taxol. Fluorescence recovery of the remaining two-thirds of labeled tubulin occurred with an average half-time (t1/2) of 9-11 min. This pool corresponds to labeled tubulin associated with microtubules, since it was sensitive to treatment of cells with nocodazole and since taxol increased its average t1/2 to greater than 22 min. Movement of photobleached microtubules in the cytoplasm with rates of several micrometers per minute was shown using very small interfringe distances. A significant change in the dynamic properties of microtubules occurred when MDCK cells reached confluency. On a cell average, microtubule half-life was increased about twofold to approximately 16 min. In fact, two populations of cells were detected with respect to their microtubule turnover rates, one with a t1/2 of approximately 9 min and one with a t1/2 of greater than 25 min. Correspondingly, the rate of incorporation of microinjected tubulin into interphase microtubules was reduced about twofold in confluent MDCK cells. In contrast to the MDCK cells, no difference in microtubule dynamics was observed in sparse and confluent populations of Vero fibroblasts, where the average microtubule half- life was approximately 10 min. Thus, microtubules are significantly stabilized in epithelial but not fibroblastic cells grown to confluency.     

Protein kinase R regulates double-stranded RNA induction of TNF-alpha but not IL-1 beta mRNA in human epithelial cells

Epithelial cells represent the initial site of respiratory viral entry and the first line of defense against such infections. This early antiviral response is characterized by an increase in the production of proinflammatory cytokines such as TNF-alpha and IL-1 beta. dsRNA, which is a common factor present during the life cycle of both DNA and RNA viruses, is known to induce TNF-alpha and IL-1 beta in a variety of cells. In this work we provide data showing that dsRNA treatment induces TNF-alpha and IL-1 beta in human lung epithelial cells via two different mechanisms. Our data show that dsRNA activation of dsRNA-activated protein kinase (PKR) is associated with induction of TNF-alpha but not IL-1 beta expression. An inhibitor of PKR activation blocked the dsRNA-induced elevations in TNF-alpha but not IL-1 beta mRNA in epithelial cells. Data obtained from infection of epithelial cells with a vaccinia virus lacking the PKR inhibitory polypeptide, E3L, revealed that PKR activation was essential for TNF-alpha but not for IL-1 beta expression. In this report, we provide experimental support for the differential regulation of proinflammatory cytokine expression by dsRNA and viral infections in human airway epithelial cells.     

The negative-feedback regulation of the IL-13 signal by the IL-13 receptor alpha2 chain in bronchial epithelial cells

Bronchial asthma is a complex disease characterized by airway inflammation involving Th2 cytokines. Among Th2 cytokines, the significance of IL-13 in the pathogenesis of bronchial asthma has recently emerged. Particularly, the direct action of IL-13 on bronchial epithelial cells (BECs) is critical for generation of airway hyperresponsiveness. IL-13 has two binding units; the IL-13 receptor alpha1 chain transduces the IL-13 signal comprising a heterodimer with the IL-4R alpha chain, whereas the IL-13 receptor alpha2 chain (IL-13Ralpha2) is thought to act as a decoy receptor. However, it remains obscure how expression of these molecules is regulated in each cell. In this article, we analyzed the expression of these components in BECs. Either IL-4 or IL-13 induced intracellular expression of IL-13Ralpha2 in BECs, which was STAT6-dependent and required de novo protein synthesis. IL-13Ralpha2 expressed on the cell surface as a monomer inhibited the STAT6-dependent IL-13 signal. Furthermore, expression of IL-13Ralpha2 was induced in lung tissues of ovalbumin-induced asthma model mice. Taken together, our results suggested the possibility that IL-13Ralpha2 induced by its ligand is transferred to the cell surface by an unknown mechanism, and it down-regulates the IL-13 signal in BECs, which functions as a unique negative-feedback system for the cytokine signal.     

Il-4 and IL-13, but not IL-10, protect human synoviocytes from apoptosis

Interleukin-4, which has been contemplated for the treatment of rheumatoid arthritis and/or osteoarthritis because of its anticatabolic properties, has also been shown to modulate apoptosis. Because inadequate apoptosis is thought to contribute to synovial hyperplasia, we have investigated the ability of IL-4 and other Th2 cytokines to protect human synovial cells from apoptosis. Human synoviocytes or synovial explants were pretreated with IL-4, IL-10, and IL-13 before exposure to NO donor sodium-nitro-prusside (SNP). Apoptosis was evaluated by microscopy, annexin V-FITC, 3-(4,5-dimethylthiazol-2-gl)-5-(3-carboxymethoxylphenyl)-2-(4-sulphophenyl-2H: tetrazolium inner salt (MTS) test, pulse field gel electrophoresis, and a method proposed in this study based on (32)P Klenow end labeling of high m.w. DNA. Pretreatment by IL-4 or IL-13, but not IL-10, protected human synoviocytes from apoptosis induced by SNP. Even at doses as high as 2 mM SNP, up to 86% and 56% protection was achieved, after IL-4 and IL-13 treatment, respectively. Cell survival was dependent on IL concentration. IL-4 and IL-13 also had antiapoptotic effects on SNP-treated human synovial explants. Effects of IL-4 and IL-13 varied in the presence of phosphatidylinositol-3 kinase and protein kinase C inhibitors, implying the involvement of these pathways in antiapoptotic signaling. Antiapoptotic effects were dramatically inhibited by LY294002, and partially by the protein kinase C inhibitor G? 6976, while insulin-like growth factor increased synoviocyte survival. The possibility that IL-4 and IL-13 may enhance synovial expansion in vivo by their antiapoptotic effects is discussed.     

Adenosine potentiates stimulatory effects on granulocyte-macrophage hematopoietic progenitor cells in vitro of IL-3 and SCF, but not those of G-CSF, GM-CSF and IL-11

The aim of the studies was to ascertain if adenosine is able to co-operate with selected hematopoietic growth factors and cytokines, namely with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), interleukin-3 (IL-3), and interleukin-11 (IL-11), in inducing the growth of colonies from hematopoietic progenitor cells for granulocytes and macrophages (GM-CFC) from normal bone marrow cells in vitro. Adenosine was found not to produce any colonies when present in the cultures as the only potential stimulator. All the tested cytokines and growth factors were observed to induce the growth of distinct numbers of GM-CFC colonies, with the exception of IL-11. When suboptimal concentrations of the evaluated cytokines and growth factors were tested in the cultures in which various concentrations of adenosine were concomitantly present, mutually potentiating effects were found in the case of IL-3 and SCF. These results confirm the role of adenosine in regulation of granulopoiesis and predict IL-3 and SCF as candidates for further in vivo studies of their combined administration with adenosine.     

IL-10 deficiency augments acute lung but not liver injury in hemorrhagic shock

In hemorrhagic shock and trauma, patients are prone to develop systemic inflammation with remote organ dysfunction, which is thought to be caused by pro-inflammatory mediators. This study investigates the role of the immuno-modulatory cytokine IL-10 in the development of organ dysfunction following hemorrhagic shock. Male C57/BL6 and IL-10 KO mice were subjected to volume controlled hemorrhagic shock for 3 h followed by resuscitation. Animals were either sacrificed 3 or 24 h after resuscitation. To assess systemic inflammation, serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with the Luminex? multiplexing platform; acute lung injury (ALI) was assessed by pulmonary myeloperoxidase (MPO) activity and lung histology and acute liver injury was assessed by hepatic MPO activity, hepatic IL-6 levels, and serum ALT levels. There was a trend towards increased IL-6 and KC serum levels 3 h after resuscitation in IL-10 KO as compared to C57/BL6 mice; however this did not reach statistical significance. Serum MCP-1 levels were significantly increased in IL-10 KO mice 3 and 24 h following resuscitation as compared to C57/BL6 mice. In IL-10 KO mice, pulmonary MPO activity was significantly increased 3 h following resuscitation and after 24 h histological signs of acute lung injury were more apparent than in C57/BL6 mice. In contrast, no significant differences in any liver parameters were detected between IL-10 KO and C57/BL6 mice. Our data indicate that an endogenous IL-10 deficiency augments acute lung but not liver injury following hemorrhagic shock.     

Inducing P-selectin ligand formation in CD8 T cells: IL-2 and IL-12 are active in vitro but not required in vivo

In vitro studies have demonstrated that IL-2 and IL-12 can support formation of P-selectin ligands (P-SelL) in activated T cells, ligands that are variably required for efficient lymphocyte recruitment to sites of inflammation. To ascertain whether these cytokines were required for P-SelL formation in vivo, TCR transgenic CD8 T cells specific for male Ag (HY) were transferred into male mice under conditions in which either IL-2 and/or IL-15 or IL-12Rp40 were absent. P-SelL formation at day 2 was unperturbed in HY-TCR IL-2(null) CD8 T cells responding in doubly deficient IL-2(null)IL-12(null) or IL-2(null)IL-15(null) male recipients. HY-specific CD8 T cell proliferative responses detected in both spleen and peritoneum occurred vigorously, but only splenic CD8 T cells up-regulated P-SelL, demonstrating that in vivo induction of P-SelL is an active, nonprogrammed event following T cell activation and that despite the efficacy of IL-2 and IL-12 in supporting P-SelL formation in vitro, these cytokines appear to be dispensable for this purpose in vivo.     

Effects of Th2 cytokines on chemokine expression in the lung: IL-13 potently induces eotaxin expression by airway epithelial cells

Airway inflammation associated with asthma is characterized by massive infiltration of eosinophils, mediated in part by specific chemoattractant factors produced in the lung. Allergen-specific Th2 cells appear to play a central role in asthma; for example, adoptively transferred Th2 cells induced lung eosinophilia associated with induction of specific chemokines. Interestingly, Th2 supernatant alone administered intranasally to naive mice induced eotaxin, RANTES, monocyte-chemotactic protein-1, and KC expression along with lung eosinophilia. We tested the major cytokines individually and found that IL-4 and IL-5 induced higher levels of macrophage-inflammatory protein-1alpha and KC; IL-4 also increased the production of monocyte-chemotactic protein-1; IL-13 and IL-4 induced eotaxin. IL-13 was by far the most potent inducer of eotaxin; indeed, a neutralizing anti-IL-13 Ab removed most of the eotaxin-inducing activity from Th2 supernatants, although it did not entirely block the recruitment of eosinophils. While TNF-alpha did not stimulate eotaxin production by itself, it markedly augmented eotaxin induction by IL-13. IL-13 was able to induce eotaxin in the lung of JAK3-deficient mice, suggesting that JAK3 is not required for IL-13 signaling in airway epithelial cells; however, eosinophilia was not induced in this situation, suggesting that JAK3 transduces other IL-13-mediated mechanisms critical for eosinophil recruitment. Our study suggests that IL-13 is an important mediator in the pathogenesis of asthma and therefore a potential target for asthma therapy.     

A dystroglycan/plectin scaffold mediates mechanical pathway bifurcation in lung epithelial cells

In alveolar epithelial cells (AECs), the membrane-anchored proteoglycan dystroglycan (DG) is a mechanoreceptor that transmits mechanical stretch forces to activate independently the ERK1/2 and the adenosine 5'-monophosphate-activated protein kinase (AMPK) signaling cascades in a process called pathway bifurcation. We tested the hypothesis that the cytoskeleton cross-linker plectin, known to bind both DG and AMPK in muscle cells, acts as a scaffold to regulate DG-mediated mechanical stimulation and pathway bifurcation. We demonstrate that plectin and DG form a complex in AECs and that this complex interacts with ERK1/2 and AMPK. Plectin knockdown reduces DG interaction with AMPK but not with ERK1/2. Despite this, mechanoactivation of both signaling pathways is significantly attenuated in AECs deficient in plectin. Thus, DG has the dual role of mechanical receptor and scaffold for ERK1/2, whereas plectin acts as a scaffold for AMPK signaling but is also required for DG-mediated ERK1/2 activation. We conclude that the DG-plectin complex plays a central role in transmitting mechanical stress from the extracellular matrix to the cytoplasm.