Toll-like receptor 4 mutation protects the kidney from Ang-II-induced hypertensive injury.

Cellular autophagy is a protective mechanism where cells degrade damaged organelles to maintain intracellular homeostasis. Apoptosis, on the other hand, is considered as programmed cell death. Interestingly, autophagy inhibits apoptosis by degrading apoptosis regulators. In hypertension, an imbalance of autophagy and apoptosis regulators can lead to renal injury and dysfunction. Previously, we have reported that toll-like receptor 4 (TLR4) mutant mice are protective against renal damage, in part, due to reduced oxidative stress and inflammation. However, the detailed mechanism remained elusive. In this study, we tested the hypothesis of whether TLR4 mutation reduces Ang-II-induced renal injury by inciting autophagy and suppressing apoptosis in the hypertensive kidney. Male mice with normal TLR4 expression (TLR4N, C3H/HeOuJ) and mutant TLR4 (TLR4M, C3H/HeJLps-d) aged 10-12 weeks were infused with Ang-II (1000 ng/kg/d) for 4 weeks to create hypertension. Saline infused appropriate control were used. Blood pressure was increased along with increased TLR4 expression in TLR4N mice receiving Ang-II compared to TLR4N control. Autophagy was downregulated, and apoptosis was upregulated in TLR4N mice treated with Ang-II. Also, kidney injury markers plasma lipocalin-2 (LCN2) and kidney injury molecule 1 (KIM-1) were upregulated in TLR4N mice treated with Ang-II. Besides, increased nuclear translocation and activity of NF-kB were measured in Ang-II-treated TLR4N mice. TLR4M mice remained protected against all these insults in hypertension. Together, these results suggest that Ang-II-induced TLR4 activation suppresses autophagy, induces apoptosis and kidney injury through in part by activating NF-kB signaling, and TLR4 mutation protects the kidney from Ang-II-induced hypertensive injury.

The yin and yang of leukotriene B4 mediated inflammation in cancer.

The high affinity leukotriene B4 receptor, BLT1 mediates chemotaxis of diverse leukocyte subsets to the sites of infection or inflammation. Whereas the pathological functions of LTB4/BLT1 axis in allergy, autoimmunity and cardiovascular disorders are well established; its role in cancer is only beginning to emerge. In this review, we summarize recent findings on LTB4/BLT1 axis enabling distinct outcomes toward tumor progression. In a mouse lung tumor model promoted by silicosis-induced inflammation, genetic deletion of BLT1 attenuated neutrophilic inflammation and tumor promotion. In contrast, in a spontaneous model of intestinal tumorigenesis, absence of BLT1 led to defective mucosal host response, altered microbiota and bacteria dependent colon tumor progression. Furthermore, BLT1 mediated CD8+ T cell recruitment was shown to be essential for initiating anti-tumor immunity in number of xenograft models and is critical for effective PD1 based immunotherapy. BLT2 mediated chemotherapy resistance, tumor promotion and metastasis are also discussed. This new information points to a paradigm shift in our understanding of the LTB4 pathways in cancer.

Temporospatial shifts within commercial laboratory mouse gut microbiota impact experimental reproducibility.

BACKGROUND: Experimental reproducibility in mouse models is impacted by both genetics and environment. The generation of reproducible data is critical for the biomedical enterprise and has become a major concern for the scientific community and funding agencies alike. Among the factors that impact reproducibility in experimental mouse models is the variable composition of the microbiota in mice supplied by different commercial vendors. Less attention has been paid to how the microbiota of mice supplied by a particular vendor might change over time. RESULTS: In the course of conducting a series of experiments in a mouse model of malaria, we observed a profound and lasting change in the severity of malaria in mice infected with Plasmodium yoelii; while for several years mice obtained from a specific production suite of a specific commercial vendor were able to clear the parasites effectively in a relatively short time, mice subsequently shipped from the same unit suffered much more severe disease. Gut microbiota analysis of frozen cecal samples identified a distinct and lasting shift in bacteria populations that coincided with the altered response of the later shipments of mice to infection with malaria parasites. Germ-free mice colonized with cecal microbiota from mice within the same production suite before and after this change followed by Plasmodium infection provided a direct demonstration that the change in gut microbiota profoundly impacted the severity of malaria. Moreover, spatial changes in gut microbiota composition were also shown to alter the acute bacterial burden following Salmonella infection, and tumor burden in a lung tumorigenesis model. CONCLUSION: These changes in gut bacteria may have impacted the experimental reproducibility of diverse research groups and highlight the need for both laboratory animal providers and researchers to collaborate in determining the methods and criteria needed to stabilize the gut microbiota of animal breeding colonies and research cohorts, and to develop a microbiota solution to increase experimental rigor and reproducibility.

Pivotal role of dermal IL-17-producing γδ T cells in skin inflammation.

Interleukin-23 (IL-23) and CD4(+) T helper 17 (Th17) cells are thought to be critical in psoriasis pathogenesis. Here, we report that IL-23 predominantly stimulated dermal γδ T cells to produce IL-17 that led to disease progression. Dermal γδ T cells constitutively expressed the IL-23 receptor (IL-23R) and transcriptional factor RORγt. IL-17 production from dermal γδ T cells was independent of αß T cells. The epidermal hyperplasia and inflammation induced by IL-23 were significantly decreased in T cell receptor δ-deficient (Tcrd(-/-)) and IL-17 receptor-deficient (Il17ra(-/-)) mice but occurred normally in Tcra(-/-) mice. Imiquimod-induced skin pathology was also significantly decreased in Tcrd(-/-) mice. Perhaps further promoting disease progression, IL-23 stimulated dermal γδ T cell expansion. In psoriasis patients, γδ T cells were greatly increased in affected skin and produced large amounts of IL-17. Thus, IL-23-responsive dermal γδ T cells are the major IL-17 producers in the skin and may represent a novel target for the treatment of psoriasis.

Inflammasome-Independent Leukotriene B4 Production Drives Crystalline Silica-Induced Sterile Inflammation.

Silicosis is a lung inflammatory disease caused by chronic exposure to crystalline silica (CS). Leukotriene B4 (LTB4) plays an important role in neutrophilic inflammation, which drives silicosis and promotes lung cancer. In this study, we examined the mechanisms involved in CS-induced inflammatory pathways. Phagocytosis of CS particles is essential for the production of LTB4 and IL-1ß in mouse macrophages, mast cells, and neutrophils. Phagosomes enclosing CS particles trigger the assembly of lipidosome in the cytoplasm, which is likely the primary source of CS-induced LTB4 production. Activation of the JNK pathway is essential for both CS-induced LTB4 and IL-1ß production. Studies with bafilomycin-A1- and NLRP3-deficient mice revealed that LTB4 synthesis in the lipidosome is independent of inflammasome activation. Small interfering RNA knockdown and confocal microscopy studies showed that GTPases Rab5c, Rab40c along with JNK1 are essential for lipidosome formation and LTB4 production. BI-78D3, a JNK inhibitor, abrogated CS-induced neutrophilic inflammation in vivo in an air pouch model. These results highlight an inflammasome-independent and JNK activation-dependent lipidosome pathway as a regulator of LTB4 synthesis and CS-induced sterile inflammation.

Intestinal HIF-1α deletion exacerbates alcoholic liver disease by inducing intestinal dysbiosis and barrier dysfunction.

BACKGROUND & AIMS: Alcoholic liver disease (ALD) is characterized by gut dysbiosis and increased gut permeability. Hypoxia inducible factor 1α (HIF-1α) has been implicated in transcriptional regulation of intestinal barrier integrity and inflammation. We aimed to test the hypothesis that HIF-1α plays a critical role in gut microbiota homeostasis and the maintenance of intestinal barrier integrity in a mouse model of ALD. METHODS: Wild-type (WT) and intestinal epithelial-specific Hif1a knockout mice (IEhif1α-/-) were pair-fed modified Lieber-DeCarli liquid diet containing 5% (w/v) alcohol or isocaloric maltose dextrin for 24 days. Serum levels of alanine aminotransferase and endotoxin were determined. Fecal microbiota were assessed. Liver steatosis and injury, and intestinal barrier integrity were evaluated. RESULTS: Alcohol feeding increased serum levels of alanine aminotransferase and lipopolysaccharide, hepatic triglyceride concentration, and liver injury in the WT mice. These deleterious effects were exaggerated in IEhif1α-/- mice. Alcohol exposure resulted in greater reduction of the expression of intestinal epithelial tight junction proteins, claudin-1 and occludin, in IEhif1α-/- mice. In addition, cathelicidin-related antimicrobial peptide and intestinal trefoil factor were further decreased by alcohol in IEhif1α-/- mice. Metagenomic analysis showed increased gut dysbiosis and significantly decreased Firmicutes/Bacteroidetes ratio in IEhif1α-/- mice compared to the WT mice exposed to alcohol. An increased abundance of Akkermansia and a decreased level of Lactobacillus in IEhif1α-/- mice were also observed. Non-absorbable antibiotic treatment reversed the liver steatosis in both WT and IEhif1α-/- mice. CONCLUSION: Intestinal HIF-1α is essential for the adaptative response to alcohol-induced changes in intestinal microbiota and barrier function associated with elevated endotoxemia and hepatic steatosis and injury. LAY SUMMARY: Alcohol consumption alters gut microbiota and multiple intestinal barrier protecting factors that are regulated by intestinal hypoxia-inducible factor 1α (HIF-1α). Absence of intestinal HIF-1α exacerbates gut leakiness leading to an increased translocation of bacteria and bacterial products to the liver, consequently causing alcoholic liver disease. Intestinal specific upregulation of HIF-1α could be developed as a novel approach for the treatment of alcoholic liver disease.

Chemoattractant Receptors BLT1 and CXCR3 Regulate Antitumor Immunity by Facilitating CD8+ T Cell Migration into Tumors.

Immunotherapies have shown considerable efficacy for the treatment of various cancers, but a multitude of patients remain unresponsive for various reasons, including poor homing of T cells into tumors. In this study, we investigated the roles of the leukotriene B4 receptor, BLT1, and CXCR3, the receptor for CXCL9, CXCL10, and CXCL11, under endogenous as well as vaccine-induced antitumor immune response in a syngeneic murine model of B16 melanoma. Significant accelerations in tumor growth and reduced survival were observed in both BLT1(-/-) and CXCR3(-/-) mice as compared with wild-type (WT) mice. Analysis of tumor-infiltrating leukocytes revealed significant reduction of CD8(+) T cells in the tumors of BLT1(-/-) and CXCR3(-/-) mice as compared with WT tumors, despite their similar frequencies in the periphery. Adoptive transfer of WT but not BLT1(-/-) or CXCR3(-/-) CTLs significantly reduced tumor growth in Rag2(-/-) mice, a function attributed to reduced infiltration of knockout CTLs into tumors. Cotransfer experiments suggested that WT CTLs do not facilitate the infiltration of knockout CTLs to tumors. Anti-programmed cell death-1 (PD-1) treatment reduced the tumor growth rate in WT mice but not in BLT1(-/-), CXCR3(-/-), or BLT1(-/-)CXCR3(-/-) mice. The loss of efficacy correlated with failure of the knockout CTLs to infiltrate into tumors upon anti-PD-1 treatment, suggesting an obligate requirement for both BLT1 and CXCR3 in mediating anti-PD-1 based antitumor immune response. These results demonstrate a critical role for both BLT1 and CXCR3 in CTL migration to tumors and thus may be targeted to enhance efficacy of CTL-based immunotherapies.

Evidence for a link between gut microbiota and hypertension in the Dahl rat.

The gut microbiota plays a critical role in maintaining physiological homeostasis. This study was designed to evaluate whether gut microbial composition affects hypertension. 16S rRNA genes obtained from cecal samples of Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats were sequenced. Bacteria of the phylum Bacteroidetes were higher in the S rats compared with the R rats. Furthermore, the family S24-7 of the phylum Bacteroidetes and the family Veillonellaceae of the phylum Firmicutes were higher in the S rats compared with the R rats. Analyses of the various phylogenetic groups of cecal microbiota revealed significant differences between S and R rats. Both strains were maintained on a high-salt diet, administered antibiotics for ablation of microbiota, transplanted with S or R rat cecal contents, and monitored for blood pressure (BP). Systolic BP of the R rats remained unaltered irrespective of S or R rat cecal transplantation. Surprisingly, compared with the S rats given S rat cecal content, systolic BP of the S rats given a single bolus of cecal content from R rats was consistently and significantly elevated during the rest of their life, and they had a shorter lifespan. A lower level of fecal bacteria of the family Veillonellaceae and increased plasma acetate and heptanoate were features associated with the increased BP observed in the S rats given R rat microbiota compared with the S rats given S rat microbiota. These data demonstrate a link between microbial content and BP regulation and, because the S and R rats differ in their genomic composition, provide the necessary basis to further examine the relationship between the host genome and microbiome in the context of BP regulation in the Dahl rats.

Validation of IMP dehydrogenase inhibitors in a mouse model of cryptosporidiosis.

Cryptosporidium parasites are a major cause of diarrhea and malnutrition in the developing world, a frequent cause of waterborne disease in the developed world, and a potential bioterrorism agent. Currently, available treatment is limited, and Cryptosporidium drug discovery remains largely unsuccessful. As a result, the pharmacokinetic properties required for in vivo efficacy have not been established. We have been engaged in a Cryptosporidium drug discovery program targeting IMP dehydrogenase (CpIMPDH). Here, we report the activity of eight potent and selective inhibitors of CpIMPDH in the interleukin-12 (IL-12) knockout mouse model, which mimics acute human cryptosporidiosis. Two compounds displayed significant antiparasitic activity, validating CpIMPDH as a drug target. The best compound, P131 (250 mg/kg of body weight/day), performed equivalently to paromomycin (2,000 mg/kg/day) when administered in a single dose and better than paromomycin when administered in three daily doses. One compound, A110, appeared to promote Cryptosporidium infection. The pharmacokinetic, uptake, and permeability properties of the eight compounds were measured. P131 had the lowest systemic distribution but accumulated to high concentrations within intestinal cells. A110 had the highest systemic distribution. These observations suggest that systemic distribution is not required, and may be a liability, for in vivo antiparasitic activity. Intriguingly, A110 caused specific alterations in fecal microbiota that were not observed with P131 or vehicle alone. Such changes may explain how A110 promotes parasitemia. Collectively, these observations suggest a blueprint for the development of anticryptosporidial therapy.

Deficiency of the leukotriene B4 receptor, BLT-1, protects against systemic insulin resistance in diet-induced obesity.

Chronic inflammation is an underlying factor linking obesity with insulin resistance. Diet-induced obesity promotes an increase in circulating levels of inflammatory monocytes and their infiltration into expanding adipose tissue. Nevertheless, the endogenous pathways that trigger and sustain chronic low-grade inflammation in obesity are incompletely understood. In this study, we report that a high-fat diet selectively increases the circulating levels of CD11b(+) monocytes in wild-type mice that express leukotriene B(4) receptor, BLT-1, and that this increase is abolished in BLT-1-null mice. The accumulation of classically activated (M1) adipose tissue macrophages (ATMs) and the expression of proinflammatory cytokines and chemokines (i.e., IL-6 and Ccl2) was largely blunted in adipose tissue of obese BLT-1(-/-) mice, whereas the ratio of alternatively activated (M2) ATMs to M1 ATMs was increased. Obese BLT-1(-/-) mice were protected from systemic glucose and insulin intolerance and this was associated with a decrease in inflammation in adipose tissue and liver and a decrease in hepatic triglyceride accumulation. Deletion of BLT-1 prevented high fat-induced loss of insulin signaling in liver and skeletal muscle. These observations elucidate a novel role of chemoattractant receptor, BLT-1, in promoting monocyte trafficking to adipose tissue and promoting chronic inflammation in obesity and could lead to the identification of new therapeutic targets for treating insulin resistance in obesity.

Decellularized Tissue-Induced Cellular Recruitment for Tissue Engineering and Regenerative Medicine.

Biomaterials that recapitulate the native in vivo microenvironment are promising to facilitate tissue repair and regeneration when used in combination with relevant growth factors (GFs), chemokines, cytokines, and other small molecules and cell sources. However, limitations with the use of exogenous factors and ex vivo cell expansion has prompted cell-/GF-free tissue engineering strategies. Additionally, conventional chemotaxis assays for studying cell migration behavior provide limited information, lack long-term stability, and fail to recapitulate physiologically relevant conditions. In this study, articular cartilage tissue-based biomaterials were developed via a rapid tissue decellularization protocol. The decellularized tissue was further processed into a hydrogel through solubilization and self-assembly. Chemotactic activity of the tissue-derived gel was investigated using sophisticated cellular migration assays. These tissue-derived extracellular matrix (ECM) biomaterials retain biochemical cues of native tissue and stimulate the chemotactic migration of hBMSCs in 2D and 3D cell migration models using a real-time chemotaxis assay. This strategy, in a way, developed a new paradigm in tissue engineering where cartilage tissue repair and regeneration can be approached with decellularized cartilage tissue in the place of an engineered matrix. This strategy can be further expanded for other tissue-based ECMs to develop cell-/GF-free tissue engineering and regenerative medicine strategies for recruiting endogenous cell populations to facilitate tissue repair and regeneration.

Synergistic cytotoxic action of cisplatin and withaferin A on ovarian cancer cell lines.

Cisplatin derivatives are used as the mainline treatment of ovarian cancer, despite their severe side effects and development of resistance. We developed a novel combination therapy by combining cisplatin with withaferin A. Treatment of ovarian cancer cell lines with combination therapy acted synergistically to induce cell death, thus required a lower dose of cisplatin to achieve the same therapeutic effect. WFA and cisplatin combination induced cell death through the generation of reactive oxygen species (ROS) for WFA, while DNA damage for cisplatin, suggesting that cisplatin binds directly to DNA to form adducts while WFA indirectly damages DNA through ROS generation. Our results for the first time suggest that combining low dose of cisplatin with suboptimal dose of WFA can serve as a potential combination therapy for the treatment of ovarian cancer with the potential to minimize/eliminate the side effects associated with high doses of cisplatin.

Effects of biaxial oscillatory shear stress on endothelial cell proliferation and morphology.

Wall shear stress (WSS) on anchored cells affects their responses, including cell proliferation and morphology. In this study, the effects of the directionality of pulsatile WSS on endothelial cell proliferation and morphology were investigated for cells grown in a Petri dish orbiting on a shaker platform. Time and location dependent WSS was determined by computational fluid dynamics (CFD). At low orbital speed (50 rpm), WSS was shown to be uniform (0-1 dyne/cm(2)) across the bottom of the dish, while at higher orbital speed (100 and 150 rpm), WSS remained fairly uniform near the center and fluctuated significantly (0-9 dyne/cm(2)) near the side walls of the dish. Since WSS on the bottom of the dish is two-dimensional, a new directional oscillatory shear index (DOSI) was developed to quantify the directionality of oscillating shear. DOSI approached zero for biaxial oscillatory shear of equal magnitudes near the center and approached one for uniaxial pulsatile shear near the wall, where large tangential WSS dominated a much smaller radial component. Near the center (low DOSI), more, smaller and less elongated cells grew, whereas larger cells with greater elongation were observed in the more uniaxial oscillatory shear (high DOSI) near the periphery of the dish. Further, cells aligned with the direction of the largest component of shear but were randomly oriented in low magnitude biaxial shear. Statistical analyses of the individual and interacting effects of multiple factors (DOSI, shear magnitudes and orbital speeds) showed that DOSI significantly affected all the responses, indicating that directionality is an important determinant of cellular responses.

Nonredundant roles for leukotriene B4 receptors BLT1 and BLT2 in inflammatory arthritis.

Lipid mediators derived from arachidonic acid through the cyclooxygenase and lipoxygenase pathways are known to be important mediators of inflammation. Studies in mouse models demonstrated an important role for the high-affinity leukotriene B(4) receptor BLT1 in arthritis, atherosclerosis, and asthma. BLT2, a low-affinity leukotriene B(4) receptor, was also shown to be a high-affinity receptor for cyclooxygenase-1 derived 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. However, its biochemical activities and physiological roles remain unknown. In this study, we developed mice deficient in BLT2 by targeted disruption. The BLT2(-/-) mice developed normally, and analysis of immune cells showed that disruption of BLT2 did not alter BLT1 expression or function. Mast cells from the C57BL/6 mice but not from the BLT2(-/-) mice showed intracellular calcium mobilization in response to 12(S)-hydroxyheptadeca-5Z, 8E, 10E-trienoic acid. In an autoantibody-induced inflammatory arthritis model, the BLT2(-/-) mice showed reduced incidence and severity of disease, including protection from bone and cartilage loss. Reciprocal bone marrow transplant experiments identified that loss of BLT2 expression on a bone marrow-derived cell lineage offers protection against severe disease. Thus, BLT2, a unique receptor for 5-lipoxygenase- and cyclooxygenase-1-derived lipid mediators, represents a novel target for therapies directed at treating inflammation associated with arthritis.

Modulation of Gut Barrier Functions in Ulcerative Colitis by Hyaluronic Acid System.

The active stages of intestinal inflammation and the pathogenesis of ulcerative colitis are associated with superficial mucosal damage and intermittent wounding that leads to epithelial barrier defects and increased permeability. The standard therapeutic interventions for colitis have focused mainly on maintaining the remission levels of the disease. Nonetheless, such treatment strategies (using anti-inflammatory, immunomodulatory agents) do not address colitis' root cause, especially the mucosal damage and dysregulated intestinal barrier functions. Restoration of barrier functionality by mucosal healing or physical barrier protecting strategies shall be considered as an initial event in the disease suppression and progression. Herein, a biphasic hyaluronan (HA) enema suspension, naïve-HA systems that protect the dysregulated gut epithelium by decreasing the inflammation, permeability, and helping in maintaining the epithelial barrier integrity in the dextran sodium sulfate-induced colitis mice model is reported. Furthermore, HA-based system modulates intestinal epithelial junctional proteins and regulatory signaling pathways, resulting in attenuation of inflammation and mucosal protection. The results suggest that HA-based system can be delivered as an enema to act as a barrier protecting system for managing distal colonic inflammatory diseases, including colitis.

Inflammation-specific targeted carriers for local drug delivery to inflammatory bowel disease.

Delivering drugs directly to the inflamed intestinal sites to treat inflammatory bowel disease (IBD), particularly Crohn's and ulcerative colitis, is highly challenging. Recent advances in colitis therapy medications are expanding opportunities for improving local on-site drug availability by minimising the associated systemic side-effects. Drug delivery with targeted carrier systems has shown the potential to increase site-specificity, stability, and therapeutic efficacy. Herein, we report the development of a strong anionic charged inflammation targeted nanocarriers (IT-NCs) loaded with an immunosuppressant model drug. This system showed preferential adhesion on a charge-modified surface in vitro, and in both dextran sulfate sodium (DSS) and TNBS colitis mice in vivo models. IT-NCs showed improved colitis phenotype therapeutic efficacy in both animal models compared to free drug. Furthermore, ex vivo study of colon tissue biopsies from patients with colitis revealed that IT-NCs adhered preferentially to inflamed biopsies compared to normal. Together, our results suggest that IT-NCs have promising therapeutic potential as delivery carriers' in colitis management.

Activin A induces dendritic cell migration through the polarized release of CXC chemokine ligands 12 and 14.

Activin A is a dimeric protein, member of the transforming growth factor (TGF)-beta family that plays a crucial role in wound repair and in fetal tolerance. Emerging evidence also proposes activin A as a key mediator in inflammation. This study reports that activin A induces the directional migration of immature myeloid dendritic cells (iDCs) through the activation of ALK4 and ActRIIA receptor chains. Conversely, activin A was not active on plasmacytoid dendritic cells (DCs) or mature myeloid DCs. iDC migration to activin A was phosphatidylinositol 3-kinase gamma-dependent, Bordetella pertussis toxin- and cycloheximide-sensitive, and was inhibited by M3, a viral-encoded chemokine-binding protein. In a real-time video microscopy-based migration assay, activin A induced polarization of iDCs, but not migration. These characteristics clearly differentiated the chemotactic activities of activin A from TGF-beta and classic chemokines. By the use of combined pharmacologic and low-density microarray analysis, it was possible to define that activin-A-induced migration depends on the selective and polarized release of 2 chemokines, namely CXC chemokine ligands 12 and 14. This study extends the proinflammatory role of activin A to DC recruitment and provides a cautionary message about the reliability of the in vitro chemotaxis assays in discriminating direct versus indirect chemotactic agonists.

Differential activation and regulation of CXCR1 and CXCR2 by CXCL8 monomer and dimer.

CXCL8 (also known as IL-8) activates CXCR1 and CXCR2 to mediate neutrophil recruitment and trigger cytotoxic effect at sites of infection. Under physiological conditions, CXCL8 could exist as monomers, dimers, or a mixture of monomers and dimers. Therefore, both forms of CXCL8 could interact with CXCR1 and CXCR2 with different affinities and potencies to mediate different cellular responses. In the present study, we have used a "trapped" nonassociating monomer (L25NMe) and a nondissociating dimer (R26C) to investigate their activities for human neutrophils that express both receptors and for RBL-2H3 cells stably expressing either CXCR1(RBL-CXCR1) or CXCR2 (RBL-CXCR2). The monomer was more active than the dimer for activities such as intracellular Ca(2+) mobilization, phosphoinositide hydrolysis, chemotaxis. and exocytosis. Receptor regulation, however, is distinct for each receptor. The rate of monomer-mediated regulation of CXCR1 is greater for activities such as phosphorylation, desensitization, beta-arrestin translocation, and internalization. In contrast, for CXCR2, both monomeric and dimeric CXCL8 mediate these activities to a similar extent. Interestingly, receptor-mediated signal-regulated kinase (ERK) phosphorylation in response to all three CXCL8 variants was more sustained for CXCR2 relative to CXCR1. Taken together, the results indicate that the CXCL8 monomer and dimer differentially activate and regulate CXCR1 and CXCR2 receptors. These distinct properties of the ligand and the receptors play a critical role in orchestrating neutrophil recruitment and eliciting cytotoxic activity during an inflammatory response.

Regulation of D6 chemokine scavenging activity by ligand- and Rab11-dependent surface up-regulation.

The decoy receptor D6 plays a nonredundant role in the control of inflammatory processes through scavenging of inflammatory chemokines. However it remains unclear how it is regulated. Here we show that D6 scavenging activity relies on unique trafficking properties. Under resting conditions, D6 constitutively recycled through both a rapid wortmannin (WM)-sensitive and a slower brefeldin A (BFA)-sensitive pathway, maintaining low levels of surface expression that required both Rab4 and Rab11 activities. In contrast to "conventional" chemokine receptors that are down-regulated by cognate ligands, chemokine engagement induced a dose-dependent BFA-sensitive Rab11-dependent D6 re-distribution to the cell membrane and a corresponding increase in chemokine degradation rate. Thus, the energy-expensive constitutive D6 cycling through Rab11 vesicles allows a rapid, ligand concentration-dependent increase of chemokine scavenging activity by receptor redistribution to the plasma membrane. D6 is not regulated at a transcriptional level in a variety of cellular contexts, thus ligand-dependent optimization of its scavenger performance represents a rapid and unique mechanism allowing D6 to control inflammation.

Ligand-induced nuclear translocation of S1P(1) receptors mediates Cyr61 and CTGF transcription in endothelial cells.

Sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P(1)), a G-protein coupled receptor (GPCR), regulates many biological activities of endothelial cells (ECs). In this report, we show that S1P(1) receptors are present in the nuclei of ECs by using various biochemical and microscopic techniques such as cellular fractionation, immunogold labeling, and confocal microscopic analysis. Live cell imaging showed that plasma membrane S1P(1) receptors are rapidly internalized and subsequently translocated to nuclear compartment upon S1P stimulation. Utilizing membrane biotinylation technique further supports the notion that nuclear S1P(1) receptors were internalized from plasma membrane S1P(1) after ligand treatment. Moreover, nuclear S1P(1) is able to regulate the transcription of Cyr61 and CTGF, two growth factors functionally important in the regulation of vasculature. Collectively, these data suggest a novel S1P-S1P(1) signaling axis present in the nuclear compartment of endothelial cells, which may regulate biological responses of endothelium.