Secretory leukocyte protease inhibitor plays an important role in the regulation of allergic asthma in mice

Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.     

Unexpected role of plasminogen activator inhibitor 1 in cell adhesion and detachment

Plasminogen activator inhibitor 1 (PAI-1) is the primary physiological inhibitor of plasminogen activation in vivo, and thus it is one of the main regulators of the fibrinolytic system. In this regard, individuals with elevated PAI-1 seem to have an increased risk for thrombotic disease, whereas those lacking the inhibitor develop a lifelong bleeding diathesis. Unexpectedly, recent observations demonstrate that cancer patients with high PAI-1 levels have a poor prognosis for survival. This correlation with metastatic disease may be related to the observation that high PAI-1 levels decrease the adhesive strength of cells for their substratum, and that this de-adhesive activity of PAI-1 is not related to its role as a protease inhibitor. Initial insights into potential mechanisms by which PAI-1 regulates the attachment, detachment, and migration of cells are addressed in this review.     

Synthesis and regulation of C1 inhibitor in human skin fibroblasts

Proteins of the C1 complex, C1q, C1r, and C1s, of the classical pathway of complement activation are known to be synthesized in human skin fibroblasts. Using metabolic labeling with [35S]methionine, immunoprecipitation, and SDS-PAGE, we demonstrate that human skin fibroblasts synthesize and secrete C1 inhibitor with an apparent molecular mass of 78 kDa in the cell lysate and 102 kDa in the extracellular medium. This C1 inhibitor had the capacity to bind activated C1s. Fibroblasts synthesized 30- to 50-fold more C1 inhibitor than was synthesized in monocytes. As previously reported, fibroblasts also synthesized C1r and C1s. IFN-gamma, IFN-beta 1, and TNF had significant, but distinct, effects on synthesis of C1 inhibitor, C1r, and C1s. Incubation of the cells with IFN-gamma, 1000 U/ml, for 24 h induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 4.2-, 1.9- and 1.6-fold, respectively. IFN-beta 1 had effects similar to IFN-gamma, although smaller in magnitude. TNF, 12.5 ng/ml, induced increases in the synthesis of C1 inhibitor, C1r, and C1s by 1.5-, 1.4- and 2.6-fold. IL-1, IFN-beta 2 (IL-6), and LPS did not affect synthesis of C1 inhibitor, C1r, or C1s. Fibroblasts are present in large amounts in most tissues. Synthesis of C1 inhibitor, C1r, and C1s by these cells could provide a source of these important proteins in body tissues. In addition, fibroblasts should be a good model for the in vitro study of genetic diseases involving the synthesis of these proteins.     

C1 inhibitor prevents endotoxin shock via a direct interaction with lipopolysaccharide

C1 inhibitor (C1INH) is beneficial in animal models of endotoxemia and sepsis. However, the mechanism(s) of C1INH protection remain(s) ill-defined. In this study, we demonstrated that both active C1INH and reactive center-cleaved, inactive C1INH protected mice from lethal Gram-negative endotoxemia. Both forms of C1INH blocked the LPS-binding protein-dependent binding of Salmonella typhimurium LPS to the murine macrophage cell line, RAW 264.7, and suppressed LPS-induced TNF-alpha mRNA expression. Inhibition of LPS binding to RAW 264.7 cells was reversed with anti-C1INH Ab and was more efficient when C1INH was incubated first with LPS rather than with the cells. C1INH also suppressed LPS-induced up-regulation of TNF-alpha mRNA in whole human blood. The interaction of C1INH with LPS was directly demonstrated both by ELISA and by nondenaturing PAGE, but deletion of the amino-terminal 97-aa residues abrogated this binding. Therefore, C1INH, in addition to its function as a serine protease inhibitor, has a novel anti-inflammatory function mediated via its heavily glycosylated amino-terminal non-serpin domain.     

Functional role of gap junctions in cytokine-induced leukocyte adhesion to endothelium in vivo

To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch and also in the cremaster muscle of wild-type (WT) and endothelium-specific connexin 43 (Cx43) null mice (Cx43e(-/-)). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-alpha; 150 ng/ml, 15 min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18-alpha-glycyrrhetinic acid (AGA; 75 microM) or 18-beta-glycyrrhetinic acid (50 microM) abolished the TNF-alpha-induced increase in LAV and LPV; carbenoxolone (75 microM) or oleamide (100 microM) reduced LAV by 50 and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75 microM), a non-GJ blocker analog of AGA, was devoid of effect. Interestingly, when AGA was removed 90 min after TNF-alpha stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90 min after TNF-alpha reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF-alpha (0.5 microg/0.3 ml) increased LAV (fourfold) and LPV (threefold) compared with saline-injected controls. In contrast to the observations in WT animals, TNF-alpha stimulation did not increase LAV or LPV in Cx43e(-/-) mice. These results demonstrate an important role for GJ communication in leukocyte adhesion and transmigration during acute inflammation in vivo and further suggest that endothelial Cx43 is key in these processes.     

Mechanisms of leukocyte adhesion

The interaction between granulocytes and endothelial walls may be influenced by the blood flow. This possibility was investigated by studying the influence of fluid flow on the adhesion and detachment of 51Cr-labeled rat granulocytes interacting with protein-coated glass surfaces. It is concluded that: i) Adhesion is markedly decreased when the wall shear rate becomes higher than about 20 s-1. ii) Pretreating glass with concanavalin A or polylysine significantly decreased adhesion, whereas fibronectin had little effect on binding. iii) Very high flow rates (about one thousandfold higher than those compatible with bond formation) were required to provoke substantial detachment of substrate-bound cells. iv) Coating glass with laminin or polylysine decreased binding strength whereas fibronectin or concanavalin A did not substantially influence this parameter. v) Exposing granulocytes to phorbol myristate acetate might increase the cell ability to form strong adhesions, whereas labile adhesion was unaffected or even decreased by this treatment.     

A role for calcium and protein kinase C in agonist-stimulated adhesion of human neutrophils.

Stimulated adherence of human neutrophils to plastic and changes in cytosolic free Ca2+ concn. [( Ca2+]i) were measured in the same cell preparations. [Ca2+]i-activation curves were constructed to compare the relation between [Ca2+]i and adhesion in response to ionomycin and formylmethionyl-leucyl-phenylalanine (FMLP). This showed that FMLP-stimulated adhesion required less increase in [Ca2+]i than did ionomycin's effect, a result suggesting that an additional stimulatory component might be involved in the response to FMLP. Protein kinase C activation was a possibility, and activation of protein kinase C with a phorbol ester (PMA) was found to stimulate adhesion with no change in [Ca2+]i. A low concentration of PMA was found to synergize with ionomycin to stimulate a greater adhesion response than with each alone, and the [Ca2+]i-activation curve for ionomycin in the presence of PMA was shifted towards that for FMLP. Thus, synergy between [Ca2+]i and protein kinase C (each of which is sufficient alone) probably explains the stimulatory effects of FMLP on adhesion of neutrophils.     

Endogenous regulatory T cells adhere in inflamed dermal vessels via ICAM-1: association with regulation of effector leukocyte adhesion

Regulatory T cells (Tregs) must express appropriate skin-homing adhesion molecules to exert suppressive effects on dermal inflammation. However, the mechanisms whereby they control local inflammation remain unclear. In this study we used confocal intravital microscopy in wild-type and Foxp3-GFP mice to examine adhesion of effector T cells and Tregs in dermal venules. These experiments examined a two-challenge model of contact sensitivity (CS) in which Treg abundance in the skin progressively increases during the course of the response. Adhesion of CD4(+) T cells increased during CS, peaking 8-24 h after an initial hapten challenge, and within 4 h of a second challenge. At these time points, 40% of adherent CD4(+) T cells were Foxp3(+) Tregs. CD4(+) T cell adhesion was highly dependent on ICAM-1, and consistent with this finding, anti-ICAM-1 prevented Treg adhesion. Skin TGF-β levels were elevated in skin during both challenges, in parallel with Treg adhesion. In the two-challenge CS model, inhibition of ICAM-1 eliminated Treg adhesion, an effect associated with a significant increase in neutrophil adhesion. Similarly, total CD4(+) T cell depletion caused an increase in adhesion of CD8(+) T cells. Because Treg adhesion was restricted by both of these treatments, these experiments suggest that adherent Tregs can control adhesion of proinflammatory leukocytes in vivo. Moreover, the critical role of ICAM-1 in Treg adhesion provides a potential explanation for the exacerbation of inflammation reported in some studies of ICAM-1-deficient mice.     

Complement regulatory protein C1 inhibitor binds to selectins and interferes with endothelial-leukocyte adhesion

C1 inhibitor (C1INH), a member of the serine proteinase inhibitor (serpin) family, is an inhibitor of proteases in the complement system, the contact system of kinin generation, and the intrinsic coagulation pathway. It is the most heavily glycosylated plasma protein, containing 13 definitively identified glycosylation sites as well as an additional 7 potential glycosylation sites. C1INH consists of two distinct domains: a serpin domain and an amino-terminal domain. The serpin domain retains all the protease-inhibitory function, while the amino-terminal domain bears most of the glycosylation sites. The present studies test the hypothesis that plasma C1INH bears sialyl Lewis(x)-related moieties and therefore binds to selectin adhesion molecules. We demonstrated that plasma C1INH does express sialyl Lewis(x)-related moieties on its N-glycan as detected using mAb HECA-452 and CSLEX1. The data also show that plasma C1INH can bind to P- and E-selectins by FACS and immunoprecipitation experiments. In a tissue culture model of endothelial-leukocyte adhesion, C1INH showed inhibition in a dose-dependent manner. Significant inhibition (>50%) was achieved at a concentration of 250 micro g/ml or higher. This discovery may suggest that C1INH plays a role in the endothelial-leukocyte interaction during inflammation. It may also provide another example of the multifaceted anti-inflammatory effects of C1INH in various animal models and human diseases.     

Regulation of brain tumor dispersal by NKCC1 through a novel role in focal adhesion regulation

Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na(+)-K(+)-Cl(-) cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms.     

A direct role for Met endocytosis in tumorigenesis

Compartmentalization of signals generated by receptor tyrosine kinase (RTK) endocytosis has emerged as a major determinant of various cell functions. Here, using tumour-associated Met-activating mutations, we demonstrate a direct link between endocytosis and tumorigenicity. Met mutants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes, activation of the GTPase Rac1, loss of actin stress fibres and increased levels of cell migration. Blocking endocytosis inhibited mutants' anchorage-independent growth, in vivo tumorigenesis and metastasis while maintaining their activation. One mutant resistant to inhibition by a Met-specific tyrosine kinase inhibitor was sensitive to endocytosis inhibition. Thus, oncogenicity of Met mutants results not only from activation but also from their altered endocytic trafficking, indicating that endosomal signalling may be a crucial mechanism regulating RTK-dependent tumorigenesis.