Effects of propofol and its formulation components on macrophages and neutrophils in obese and lean animals.

We hypothesized whether propofol or active propofol component (2,6-diisopropylphenol [DIPPH] and lipid excipient [LIP-EXC]) separately may alter inflammatory mediators expressed by macrophages and neutrophils in lean and obese rats. Male Wistar rats (n = 10) were randomly assigned to receive a standard (lean) or obesity-inducing diet (obese) for 12 weeks. Animals were euthanized, and alveolar macrophages and neutrophils from lean and obese animals were exposed to propofol (50 µM), active propofol component (50 µM, 2,6-DIPPH), and lipid excipient (soybean oil, purified egg phospholipid, and glycerol) for 1 h. The primary outcome was IL-6 expression after propofol and its components exposure by alveolar macrophages extracted from bronchoalveolar lavage fluid. The secondary outcomes were the production of mediators released by macrophages from adipose tissue, and neutrophils from lung and adipose tissues, and neutrophil migration. IL-6 increased after the exposure to both propofol (median [interquartile range] 4.14[1.95-5.20]; p = .04) and its active component (2,6-DIPPH) (4.09[1.67-5.91]; p = .04) in alveolar macrophages from obese animals. However, only 2,6-DIPPH increased IL-10 expression (7.59[6.28-12.95]; p = .001) in adipose tissue-derived macrophages. Additionally, 2,6-DIPPH increased C-X-C chemokine receptor 2 and 4 (CXCR2 and CXCR4, respectively) in lung (10.08[8.23-29.01]; p = .02; 1.55[1.49-3.43]; p = .02) and adipose tissues (8.78[4.15-11.57]; p = .03; 2.86[2.17-3.71]; p = .01), as well as improved lung-derived neutrophil migration (28.00[-3.42 to 45.07]; p = .001). In obesity, the active component of propofol affected both the M1 and M2 markers as well as neutrophils in both alveolar and adipose tissue cells, suggesting that lipid excipient may hinder the effects of active propofol.

Administration of a CXC Chemokine Receptor 2 (CXCR2) Antagonist, SCH527123, Together with Oseltamivir Suppresses NETosis and Protects Mice from Lethal Influenza and Piglets from Swine-Influenza Infection.

Excessive neutrophil influx, their released neutrophil extracellular traps (NETs), and extracellular histones are associated with disease severity in influenza-infected patients. Neutrophil chemokine receptor CXC chemokine receptor 2 (CXCR2) is a critical target for suppressing neutrophilic inflammation. Herein, temporal dynamics of neutrophil activity and NETosis were investigated to determine the optimal timing of treatment with the CXCR2 antagonist, SCH527123 (2-hydroxy-N,N-dimethyl-3-[2-([(R)-1-(5-methyl-furan-2-yl)-propyl]amino)-3,4-dioxo-cyclobut-1-enylamino]-benzamide), and its efficacy together with antiviral agent, oseltamivir, was tested in murine and piglet influenza-pneumonia models. SCH527123 plus oseltamivir markedly improved survival of mice infected with lethal influenza, and diminished lung pathology in swine-influenza-infected piglets. Mechanistically, addition of SCH527123 in the combination treatment attenuated neutrophil influx, NETosis, in both mice and piglets. Furthermore, neutrophils isolated from influenza-infected mice showed greater susceptibility to NETotic death when stimulated with a CXCR2 ligand, IL-8. In addition, CXCR2 stimulation induced nuclear translocation of neutrophil elastase, and enhanced citrullination of histones that triggers chromatin decondensation during NET formation. Studies on temporal dynamics of neutrophils and NETs during influenza thus provide important insights into the optimal timing of CXCR2 antagonist treatment for attenuating neutrophil-mediated lung pathology. These findings reveal that pharmacologic treatment with CXCR2 antagonist together with an antiviral agent could significantly ameliorate morbidity and mortality in virulent and sublethal influenza infections.

Paeoniflorin inhibited nod-like receptor protein-3 inflammasome and NF-κB-mediated inflammatory reactions in diabetic foot ulcer by inhibiting the chemokine receptor CXCR2.

Diabetic foot ulcer (DFU) is an invariably common complication of diabetes, characterized by delayed wound healing process and increased inflammation. Evidence has indicated that paeoniflorin exerts an anti-inflammatory effect in diabetic retinopathy. The current work was aimed to investigate the effect of paeoniflorin on inflammation and wound healing in DFU. DFU rat models by streptozotocin and skin biopsy punch, as well as high glucose-treated human immortalized keratinocytes (HaCaT) were established. Levels of blood glucose, wound contraction and proinflammatory cytokine were detected after paeoniflorin administration. Several essential targets associated with the NF-κB and Nod-like receptor protein-3 (NLRP3) signaling pathways were examined. Results showed markedly down-regulation of interleukin (IL)-1ß, IL-18 and tumor necrosis factor-alpha in paeoniflorin-treated DFU rats. Paeoniflorin decreased the expression levels of chemokine receptor CXCR2, nuclear NF-κB and p-IκB (Ser36), as well as increased IκB level. Histological analysis and immunostaining showed lower inflammatory cells with decreased NLRP3 and cleaved caspase-1 levels following paeoniflorin treatment. Further in vitro evidence confirmed that paeoniflorin efficiently inhibited NLRP3 and NF-κB-mediated inflammation in DFU by inhibiting CXCR2. These findings are suggestive of greatly attenuated wound inflammation and better wound healing in paeoniflorin-treated DFU rats. Our study demonstrates that paeoniflorin is a potential therapeutic agent for DFU.

The Chemokine Receptor CXCR2 Supports Nociceptive Sensitization after Traumatic Brain Injury.

Abstract: Chronic pain after traumatic brain injury (TBI) is very common, but the mechanisms linking TBI to pain and the pain-related interactions of TBI with peripheral injuries are poorly understood. Chemokine receptors play an important role in both pain and brain injury. In the current work, we pursued the hypothesis that the epigenetically regulated CXC chemokine receptor 2 (CXCR2) is a crucial modulator of nociceptive sensitization induced by TBI. For these studies, we used the rat lateral fluid percussion model of TBI. Histone actyltransferase activity was blocked using anacardic acid beginning immediately following injury, or delayed for seven days prior to administration. The selective CXCR2 antagonist SCH527123 administered systemically or intrathecally was used to probe the role of chemokine signaling on mechanical hindpaw sensitization after TBI. The expression of the CXCR2 receptor was accomplished using real-time PCR, immunohistochemistry, and Western blotting, while epigenetic regulation was assessed using chromatin immunoprecipitation assay. The spinal levels of several pain-related mediators including CXCL1, an endogenous ligand for CXCR2, as well as brain-derived neurotrophic factor and prodynorphin were measured by enzyme-linked immunosorbent assay. We observed that anacardic acid potently blocked and reversed mechanical hindpaw sensitization after TBI. The same drug was able to prevent the upregulation of CXCR2 after TBI, but did not affect the spinal expression of other pain mediators. On the other hand, both systemically and intrathecally administered SCH527123 reversed hindpaw allodynia after TBI. Most of the spinal CXCR2 appeared to be expressed by spinal cord neurons. Chromatin immunoprecipitation experiments demonstrated TBI-enhanced association of the CXCR2 promoter with acetylated-H3K9 histone protein that was also reversible using anacardic acid. Taken together, our findings suggested that TBI causes the upregulation of spinal CXCR2 through an epigenetic mechanism ultimately supporting nociceptive sensitization. The use of CXCR2 antagonists may, therefore, be useful in pain resulting from TBI.

Transcriptome profiling of gene expression during immunisation trial against Fasciola hepatica: identification of genes and pathways involved in conferring immunoprotection in a murine model.

BACKGROUND: Fasciolosis remains a significant food-borne trematode disease causing high morbidity around the world and affecting grazing animals and humans. A deeper understanding concerning the molecular mechanisms by which Fasciola hepatica infection occurs, as well as the molecular basis involved in acquiring protection is extremely important when designing and selecting new vaccine candidates. The present study provides a first report of microarray-based technology for describing changes in the splenic gene expression profile for mice immunised with a highly effective, protection-inducing, multi-epitope, subunit-based, chemically-synthesised vaccine candidate against F. hepatica. METHODS: The mice were immunised with synthetic peptides containing B- and T-cell epitopes, which are derived from F. hepatica cathepsin B and amoebapore proteins, as novel vaccine candidates against F. hepatica formulated in an adjuvant adaptation vaccination system; they were experimentally challenged with F. hepatica metacercariae. Spleen RNA from mice immunised with the highest protection-inducing synthetic peptides was isolated, amplified and labelled using Affymetrix standardised protocols. Data was then background corrected, normalised and the expression signal was calculated. The Ingenuity Pathway Analysis tool was then used for analysing differentially expressed gene identifiers for annotating bio-functions and constructing and visualising molecular interaction networks. RESULTS: Mice immunised with a combination of three peptides containing T-cell epitopes induced high protection against experimental challenge according to survival rates and hepatic damage scores. It also induced differential expression of 820 genes, 168 genes being up-regulated and 652 genes being down-regulated, p value <0.05, fold change ranging from -2.944 to 7.632. A functional study of these genes revealed changes in the pathways related to nitric oxide and reactive oxygen species production, Interleukin-12 signalling and production in macrophages and Interleukin-8 signalling with up-regulation of S100 calcium-binding protein A8, Matrix metallopeptidase 9 and CXC chemokine receptor 2 genes. CONCLUSION: The data obtained in the present study provided us with a more comprehensive overview concerning the possible molecular pathways implied in inducing protection against F. hepatica in a murine model, which could be useful for evaluating future vaccine candidates.

Therapeutic implications of 'neutrophilic asthma'.

PURPOSE OF REVIEW: This review examines the association between airway neutrophilia and severe asthma, potential mechanisms, and the effect on asthma control of therapies directed at reducing airway neutrophil numbers or activity. RECENT FINDINGS: The majority of studies that observe an association between airway neutrophilia and severe asthma are cross-sectional in nature, and the intensity of neutrophilia is low and may be a reflection of the age of the patients, effect of tobacco smoke exposure, or the high doses of corticosteroids used to treat their asthma. There may be a small proportion of patients who may have abnormal innate immune responses that may lead to airway neutrophilia. However, these neutrophils may not be any more activated than in patients with milder asthma. Novel strategies using small molecule antagonists against the interleukin-8 receptor, CXCR2, are able to reduce airway neutrophilia, and their clinical efficacies are being investigated. SUMMARY: Although cross-sectional studies suggest that airway neutrophilia may be observed in some patients with severe asthma, it is not clearly established if this is a consequence of treatment with corticosteroids or if it contributes directly to asthma pathobiology and severity. New therapies such as anti-CXCR2 provide an opportunity to investigate the contribution of neutrophils to asthma severity.

Volatile anaesthetics reduce neutrophil inflammatory response by interfering with CXC receptor-2 signalling.

BACKGROUND: Growing evidence suggests a protective effect of volatile anaesthetics in ischaemia-reperfusion (I/R)-injury, and the accumulation of neutrophils is a crucial event. Pro-inflammatory cytokines carrying the C-X-C-motif including interleukin-8 (IL-8) and CXC-ligand 1 (CXCL1) activate CXC receptor-1 (CXCR1; stimulated by IL-8), CXC receptor-2 (CXCR2; stimulated by IL-8 and CXCL1), or both to induce CD11b-dependent neutrophil transmigration. Inhibition of CXCR1, CXCR2, or both reduces I/R-injury by preventing neutrophil accumulation. We hypothesized that interference with CXCR1/CXCR2 signalling contributes to the well-established beneficial effect of volatile anaesthetics in I/R-injury. METHODS: Isolated human neutrophils were stimulated with IL-8 or CXCL1 and exposed to volatile anaesthetics (sevoflurane/desflurane). Neutrophil migration was assessed using an adapted Boyden chamber. Expression of CD11b, CXCR1, and CXCR2 was measured by flow cytometry. Blocking antibodies against CXCR1/CXCR2/CD11b and phorbol myristate acetate were used to investigate specific pathways. RESULTS: Volatile anaesthetics reduced CD11b-dependent neutrophil transmigration induced by IL-8 by >30% and CD11b expression by 18 and 27% with sevoflurane/desflurane, respectively. This effect was independent of CXCR1/CXCR2 expression and CXCR1/CXCR2 endocytosis. Inhibition of CXCR1 signalling did not affect downregulation of CD11b with volatile anaesthetics. Blocking of CXCR2-signalling neutralized effects by volatile anaesthetics on CD11b expression. Specific stimulation of CXCR2 with CXCL1 was sufficient to induce upregulation of CD11b, which was impaired with volatile anaesthetics. No effect of volatile anaesthetics was observed with direct stimulation of protein kinase C located downstream of CXCR1/CXCR2. CONCLUSION: Volatile anaesthetics attenuate neutrophil inflammatory responses elicited by CXC cytokines through interference with CXCR2 signalling. This might contribute to the beneficial effect of volatile anaesthetics in I/R-injury.

The chemokine receptors CXCR1 and CXCR2 regulate oral cancer cell behaviour.

BACKGROUND: Chemokines regulate physiological and pathological leucocyte trafficking, and chemokine receptors play a role in tumorigenesis. Expression of interleukin-8 (IL-8) receptors CXCR1 and CXCR2 has been shown in oral squamous cell carcinoma (OSCC) but remains poorly characterised. This aim of this study was to investigate CXCR1 and CXCR2 expression on normal oral keratinocytes (NOKs) and oral cancer cell lines (OCCL) and their relative response when exposed to IL-8 and growth-related oncogene-α (which selectively binds CXCR2). METHODS: mRNA and protein expression was studied using RT-PCR, immunocytochemistry and flow cytometry. ELISAs were used to investigate ERK1/2 phosphorylation and MMP production, whereas a MTS-based assay was employed to study proliferation. Migration assays were carried out using modified Boyden chambers with a matrigel coating used for invasion assays. RESULTS: mRNA expression of CXCR1 and CXCR2 was seen in both NOKs and OCCL with significantly higher protein expression in OCCL. Exposure to IL-8 and GROα increased intracellular ERK phosphorylation, proliferation, migration and invasion with OCCL showing a greater response than NOKs. These effects were mediated through CXCR1 and CXCR2 (for IL-8) and CXCR2 (for GROα) as receptor-blocking antibodies significantly inhibited the responses. IL-8 and GROα also increased MMP-9 release from NOKs and OCCL with significantly higher amounts released by OCCL. However, an increase in MMP-7 production was only seen in OCCL. CONCLUSIONS: Functional CXCR1 and CXCR2 exist on normal and cancerous oral epithelial cells, and our data suggests a role for these receptors in oral cancer biology.

Blockage of CXCR2 suppresses tumor growth of intrahepatic cholangiocellular carcinoma.

BACKGROUND/AIMS: Complete operative resection is the only approach to cure for intrahepatic cholangiocellular carcinoma (ICC), but the disease's prognosis is notably poor. A novel therapeutic approach is urgently required. CXC chemokine receptor 2 (CXCR2) has been associated with tumorigenesis and metastasis in human cancers. In this study, we investigated the suppressive effect of ICC growth by blocking CXCR2. MATERIAL AND METHODS: The role of CXCR2 was estimated using the human ICC cell lines, RBE and SSP25. CXCR2 small interfering RNA (siRNA) and an antagonist (SB225002) were used to block CXCR2. Proliferation assays, migration assays, and invasion assays were performed to confirm the suppressive effect of blocking CXCR2. Subcutaneous SSP25 tumors were established in athymic nude mice, and the mice were given SB225002. The expression of CXCR2 in ICC was determined by immunohistochemical staining of 34 ICC specimens. We investigated the relationship between CXCR2 expression and prognosis in ICC. RESULTS: The prognosis of patients who had higher CXCR2 expression in ICC was significantly poor (P = .004). CXCR2 siRNA treatment significantly suppressed CXCR2 expression in both RBE and SSP25. Cell proliferation, migration, and invasion were significantly suppressed by both CXCR2 siRNA and SB225002 compared with the control group. SB225002 also suppressed the growth of transplanted subcutaneous tumors (P = .02) CONCLUSION: Our results demonstrated that blocking CXCR2 clearly suppressed the development of ICC. Blocking CXCR2 may be a promising therapeutic approach for ICC.

CXCR2 modulators: a patent review (2009 - 2013).

INTRODUCTION: Small-molecule antagonists of CXC chemokine receptor 2 (CXCR2) have attracted a considerable amount of attention due to the key central role that this receptor plays in inflammatory conditions. Recently, several CXCR2 receptor antagonists have demonstrated promising proof of activity in early pulmonary clinical trials, which has stimulated additional efforts to identify new CXCR2 receptor antagonists. AREAS COVERED: During the period 2009 - 2013, there were numerous patent publications from various companies claiming the discovery of novel CXCR2 receptor antagonists. Herein, an interpretation of these new patent publications combined with emerging disclosures from the peer-reviewed literature during this time frame is given. This review highlights the preferred or representative compounds from the patent applications along with relevant biological characterization. EXPERT OPINION: Many of the new CXCR2 receptor antagonists described in this review represent closely related analogs to previously disclosed clinical candidates. With the recent discontinuation of several CXCR2 receptor antagonists in the clinic, additional clinical trial information for CXCR2 receptor antagonists, both past and present, will determine the long-term therapeutic potential of these compounds for the treatment of a variety of inflammatory disorders.

Inhibition of LPS-induced airway neutrophilic inflammation in healthy volunteers with an oral CXCR2 antagonist.

BACKGROUND: Inhaled lipopolysaccharide (LPS) induces a dose-dependent, acute neutrophilic response in the airways of healthy volunteers that can be quantified in induced sputum. Chemokines, such as CXCL1 and CXCL8, play an important role in neutrophilic inflammation in the lung through the activation of CXCR2 and small molecule antagonists of these receptors have now been developed. We investigated the effect of AZD8309, a CXCR2 antagonist, compared with placebo on LPS-induced inflammation measured in sputum of healthy volunteers. METHODS: Twenty healthy subjects were randomized in a double-blind placebo-controlled, cross-over study. AZD8309 (300 mg) or placebo was dosed twice daily orally for 3 days prior to challenge with inhaled LPS and induced sputum was collected 6 h later. RESULTS: Treatment with AZD8309 showed a mean 77% reduction in total sputum cells (p < 0.001) and 79% reduction in sputum neutrophils (p < 0.05) compared with placebo after LPS challenge. There was also a reduction in neutrophil elastase activity (p < 0.05) and CXCL1 (p < 0.05) and trends for reductions in sputum macrophages (47%), leukotriene B4 (39%) and CXCL8 (52%). CONCLUSIONS: AZD8309 inhibited LPS-induced inflammation measured in induced sputum of normal volunteers, indicating that this treatment may be useful in the treatment of neutrophilic diseases of the airways, such as COPD, severe asthma and cystic fibrosis. TRIAL REGISTRATION: NCT00860821.

Volatile anesthetics reduce invasion of colorectal cancer cells through down-regulation of matrix metalloproteinase-9.

BACKGROUND: Invasion of extracellular matrix is a hallmark of malignant tumors. Clamping maneuvers during cancer surgery reduce blood loss, but trigger reperfusion injury (RI). RI increases cancer recurrence in the reperfused organ through up-regulation of matrix metalloproteinase-9 (MMP-9). Interleukin-8 is an important cytokine in RI promoting accumulation of neutrophils, a major source of MMP-9. Volatile anesthetics were demonstrated to reduce RI. We hypothesized that these anesthetics might attenuate MMP-9 up-regulation and consequently tumor cell invasion in RI. METHODS: Isolated human neutrophils (n = 6) were preconditioned with sevoflurane or desflurane, followed by stimulation with interleukin-8, phorbol myristate acetate, or chemokine CXC-ligand 1 (CXCL1) to differentiate intracellular pathways. MMP-9 release and activity were quantified by enzyme-linked immunosorbent assay and zymography, respectively. CXC-receptor-2 (CXCR2) expression and phosphorylation of extracellular signal-regulated kinases 1/2 were assessed by flow cytometry. The impact of MMP-9 on the invasion of neutrophils and MC-38 colon cancer cells was assessed using Matrigel-coated filters (n = 6). RESULTS: Preconditioning reduced interleukin-8-induced MMP-9-release by 41% (±13, 5%, sevoflurane) and 40% (±13%, desflurane). This was also evident following stimulation of CXCR2 with CXCL1. No impact on phosphorylation of extracellular signal-regulated kinases 1/2 and MMP-9 release was observed with receptor-independent stimulation of protein kinase C with phorbol myristate acetate. Preconditioning reduced transmigration of neutrophils and MC-38 tumor cells to baseline levels. DISCUSSION: Volatile anesthetics impair neutrophil MMP-9 release and interfere with pathways downstream of CXCR2, but upstream of protein kinase C. Through down-regulation of MMP-9, volatile anesthetics decrease Matrigel breakdown and reduce subsequent migration of cancer cells in vitro.

Maxadilan, the Lutzomyia longipalpis vasodilator, drives plasma leakage via PAC1-CXCR1/2-pathway.

Experiments were designed to determine if the vasodilatory peptides maxadilan and pituitary adenylate cyclase-activating peptide (PACAP-38) may cause plasma leakage through activation of leukocytes and to what extent these effects could be due to PAC1 and CXCR1/2 receptor stimulation. Intravital microscopy of hamster cheek pouches utilizing FITC-dextran and rhodamine, respectively, as plasma and leukocyte markers was used to measure arteriolar diameter, plasma leakage and leukocyte accumulation in a selected area (5mm(2)) representative of the hamster cheek pouch microcirculation. Our studies showed that the sand fly vasodilator maxadilan and PACAP-38 induced arteriolar dilation, leukocyte accumulation and plasma leakage in postcapillary venules. The recombinant mutant of maxadilan M65 and an antagonist of CXCR1/2 receptors, reparixin, and an inhibitor of CD11b/CD18 up-regulation, ropivacaine, inhibited all these effects as induced by maxadilan. Dextran sulfate, a complement inhibitor with heparin-like anti-inflammatory effects, inhibited plasma leakage and leukocyte accumulation but not arteriolar dilation as induced by maxadilan and PACAP-38. In vitro studies with isolated human neutrophils showed that maxadilan is a potent stimulator of neutrophil migration comparable with fMLP and leukotriene B(4) and that M65 and reparixin inhibited such migration. The data suggest that leukocyte accumulation and plasma leakage induced by maxadilan involves a mechanism related to PAC1- and CXCR1/2-receptors on leukocytes and endothelial cells.

Effect of formoterol and budesonide on chemokine release, chemokine receptor expression and chemotaxis in human neutrophils.

Severe persistent asthma and chronic obstructive pulmonary disease (COPD) are associated with neutrophil influx into the airways. It is not clear whether neutrophil chemotaxis is influenced by beta(2)-agonists and glucocorticoids, drugs commonly used in treatment of asthma and COPD. The effect of a long-acting beta(2)-agonist (formoterol), and a glucocorticosteroid (budesonide) on chemokine/cytokine release (CXCL8, CXCL1, IL-6), regulation of chemokine receptors (CXCR1, CXCR2), and migration were assessed in neutrophils from 10 non-allergic, healthy donors. Formoterol enhanced and budesonide inhibited IL-6, CXCL8 and CXCL1 release from LPS-stimulated neutrophils. Formoterol up-regulated both CXCR1 and CXCR2 expression, whereas budesonide up-regulated the expression of CXCR2 only. Despite the effects on chemokine release and drug-induced up-regulation of CXCR1 and CXCR2, no influence on neutrophil chemotaxis could be demonstrated. We conclude that a beta(2)-agonist and a glucocorticoid, commonly used in the treatment of obstructive lung diseases, influence chemokine release and receptor sensitivity but the functional consequences of these findings remain unclear.

Identification of a putative intracellular allosteric antagonist binding-site in the CXC chemokine receptors 1 and 2.

The chemokine receptors CXCR1 and CXCR2 are G-protein-coupled receptors (GPCRs) implicated in mediating cellular functions associated with the inflammatory response. Potent CXCR2 receptor antagonists have been discovered, some of which have recently entered clinical development. The aim of this study was to identify key amino acid residue differences between CXCR1 and CXCR2 that influence the relative antagonism by two compounds that have markedly different chemical structures. By investigating the effects of domain switching and point mutations, we found that the second extracellular loop, which contained significant amino acid sequence diversity, was not important for compound antagonism. We were surprised to find that switching the intracellular C-terminal 60 amino acid domains of CXCR1 and CXCR2 caused an apparent reversal of antagonism at these two receptors. Further investigation showed that a single amino acid residue, lysine 320 in CXCR2 and asparagine 311 in CXCR1, plays a predominant role in describing the relative antagonism of the two compounds. Homology modeling studies based on the structure of bovine rhodopsin indicated a potential intracellular antagonist binding pocket involving lysine 320. We conclude that residue 320 in CXCR2 forms part of a potential allosteric binding pocket on the intracellular side of the receptor, a site that is distal to the orthosteric site commonly assumed to be the location of antagonist binding to GPCRs. The existence of a common intracellular allosteric binding site at GPCRs related to CXCR2 may be of value in the design of novel antagonists for therapeutic intervention.

Influences of aflatoxin B1 on reactive oxygen species generation and chemotaxis of human polymorphonuclear leukocytes.

The effects of aflatoxin (AF), a hepatotoxic agent and the strongest carcinogen in nature, on polymorphonuclear leukocyte (PMN) chemotaxis and chemiluminescence (CL) were studied. Luminol-dependent CL activity, which reflects the production of reactive oxygen species (ROS) from PMNs, was up-regulated to approximately 150% when PMNs were treated with 0.05 ng/ml of AFB1 upon stimulation with N-formyl-methionine-leucine-phenylalanine (fMLP) or zymosan. On the other hand, PMN CL activity was down-regulated to approximately 25% of the control when PMNs were treated with 50 ng/ml of AFB1 upon stimulation with zymosan. The effect of AFB1 on PMN chemotaxis was also investigated using the Boyden chamber method. The chemotactic ability of PMNs when interleukin (IL)-8 was used as a chemoattractant was inhibited in a dose-dependent manner at AFB1 concentrations of >10 ng/ml. In reverse transcriptase (RT)-PCR analysis, gene expression levels of CXC chemokine receptor (CXCR)1/2, whose ligands are IL-8, granulocyte chemotactic protein (GCP)-2, neutrophil attractant-activation protein (NAP)-2, and epithelial neutrophil-activating protein (ENA)-78, which regulate PMN chemotaxis, were also down-regulated in a dose-dependent manner by 50 ng/ml AFB1. mRNA expression levels of CXCR1/2 were down-regulated to approximately 85% of that in the controls when PMNs were treated with 100 ng/ml AFB1. These results suggest that a high concentration of AFB1 reduces the chemotactic ability of PMNs via the CXCR1/2 cascade indirectly.

Chemokine-mobilized adult stem cells; defining a better hematopoietic graft.

Stem cell research is currently focused on totipotent stem cells and their therapeutic potential, however adult stem cells, while restricted to differentiation within their tissue or origin, also have therapeutic utility. Transplantation with bone marrow hematopoietic stem cells (HSC) has been used for curative therapy for decades. More recently, alternative sources of HSC, particularly those induced to exit marrow or mobilize to peripheral blood by G-CSF, have become the most widely used hematopoietic graft and show significant superiority to marrow HSC. The chemokine/chemokine receptor axis also mobilizes HSC that occurs more rapidly than with G-CSF. In mice, the HSC and progenitor cells (HPC) mobilized by the CXCR2 receptor agonist GRObeta can be harvested within minutes of administration and show significantly lower levels of apoptosis, enhanced homing to marrow, expression of more activated integrin receptors and superior repopulation kinetics and more competitive engraftment than the equivalent cells mobilized by G-CSF. These characteristics suggest that chemokine axis-mobilized HSC represent a population of adult stem cells distinct from those mobilized by G-CSF, with superior therapeutic potential. It remains to be determined if the chemokine mobilization axis can be harnessed to mobilize other populations of unique adult stem cells with clinical utility.

Reparixin, a specific interleukin-8 inhibitor, has no effects on inflammation during endotoxemia.

Reparixin antagonizes interleukin-8 (IL-8) on the level of signal transduction in vitro. We hypothesized that IL-8 mediates some of the reactions occurring during acute inflammation and specifically that IL-8 may be a mediator of endotoxin induced neutrophilia. We therefore tested the effects of reparixin on humoral and cellular parameters in LPS-induced acute systemic inflammation. The study is a randomized (3:2 active:placebo), double-blind, placebo-controlled parallel group trial. Twenty healthy male volunteers randomly received either reparixin (12) or placebo (8) intravenously. One hour after the start of reparixin/placebo infusion a bolus of 2 ng/kg endotoxin was infused over 1-2 min. Blood samples were obtained over 24 h. Reparixin, being metabolized to ibuprofen, suppressed serum thromboxane B2 levels by 78 percent compared to baseline and control at 8 h. LPS-induced neutrophilia was not significantly affected by reparixin in human volunteers. Consistently, reparixin did not alter the lymphocyte or monocyte counts and had no effect on LPS-induced systemic inflammation as measured by tumor necrosis factor alpha (TNF-alpha) or interleukin-6 (IL-6) release. Regulation of IL-8 receptors CXCR1 and 2 and the degranulation marker CD11b showed the expected kinetics. Reparixin had no effect on thrombin formation as measured by prothrombin fragment (F1+2). In conclusion, our study showed that reparixin was safe but had no impact on endotoxin induced inflammation. In contrast to previous studies with its metabolite ibuprofen, reparixin does not enhance inflammation in this model.

Studies on MIP-2 and CXCR2 expression in a mouse model of extrahepatic colorectal metastasis.

AIMS: The CXC chemokine macrophage inflammatory protein (MIP)-2 has been shown to promote outgrowth of colorectal liver metastasis by enhancing angiogenesis and tumor cell migration. However, the effect of MIP-2 on extrahepatic metastasis is not known yet. With a use of a murine model, we therefore studied cell proliferation and microvascularization of extrahepatic CT26.WT-GFP colorectal tumors after exposure to MIP-2. METHODS: Green fluorescent protein (GFP)-transfected CT26.WT colorectal cancer cells were implanted in dorsal skinfold chambers of syngeneic BALB/c mice. After 5 days, the tumors were locally exposed to 100 nM MIP-2. Cell proliferation as well as tumor microvascularization and growth were studied during a further 9-day period using intravital fluorescence microscopy, histology and immunohistochemistry. Tumors exposed to PBS served as controls. RESULTS: MIP-2 induced a marked CXCR2 expression and promoted a distinct tumor cell proliferation. This was associated with a significant increase of tumor size compared to PBS-treated controls. Of interest, MIP-2 did not affect dilation and permeability of the tumor microvessels, which would be indicators for an enhanced VEGF action. Accordingly, the angiogenic response, e.g. the outgrowth of new microvessels, was not affected, and the density of the established tumor microvascular network was even found decreased after MIP-2 exposure when compared to PBS controls. CONCLUSION: With the use of a murine tumor model, we demonstrate that MIP-2 accelerates growth of experimentally established extrahepatic colorectal metastases by inducing tumor cell proliferation rather than promoting vascularization.

Morphine impairs host innate immune response and increases susceptibility to Streptococcus pneumoniae lung infection.

Chronic morphine use impairs host innate immune response and increases susceptibility to bacteria and virus. In this study a novel mouse model of chronic morphine treatment, followed by intranasal inoculation with Streptococcus pneumoniae, was used to investigate microbial events and host innate immune response. Our results show that chronic morphine treatment markedly delayed neutrophil recruitment and increased bacterial burden in the lung, spleen, and blood with a subsequent increase in mortality. In morphine-treated animals, before neutrophil recruitment, a significant decrease in TNF-alpha, IL-1, IL-6, MIP-2, and KC was observed both in bronchoalveolar lavage fluids and in lung tissue. In the early phase of infection, we found that accumulation of galectin-3 in the alveolar space of streptococcus-infected lungs was decreased after morphine treatment. The transcription factor NF-kappaB in lung resident cells was also inhibited after morphine treatment. Taken together, these results suggest that chronic morphine treatment in an S. pneumoniae infection model suppresses NF-kappaB gene transcription in lung resident cells, which, in turn, modulates the transcriptional regulation of MIP-2 and inflammatory cytokines. The decreased synthesis of MIP-2 and inflammatory cytokines coupled with the decreased release of galectin-3 result in reduced migration of neutrophils to the site of infection, thereby increasing susceptibility to S. pneumoniae infection after morphine treatment.