Exploiting agonist biased signaling of chemokines to target cancer.

As knowledge of growth-independent functions of cancer cells is expanding, exploration into the role of chemokines in modulating cancer pathogenesis, particularly metastasis, continues to develop. However, more study into the mechanisms whereby chemokines direct the migration of cancer cells is needed before specific therapies can be generated to target metastasis. Herein, we draw attention to the longstanding conundrum in the field of chemokine biology that chemokines stimulate migration in a biphasic manner; and explore this phenomenon's impact on chemokine function in the context of cancer. Typically, low concentrations of chemokines lead to chemotactic migration and higher concentrations halt migration. The signaling mechanisms that govern this phenomenon remain unclear. Over the last decade, we have defined a novel signaling mechanism for regulation of chemokine migration through ligand oligomerization and biased agonist signaling. We provide insight into this new paradigm for chemokine signaling and discuss how it will impact future exploration into chemokine function and biology. In the pursuit of producing more novel cancer therapies, we suggest a framework for pharmaceutical application of the principles of chemokine oligomerization and biased agonist signaling in cancer. © 2016 Wiley Periodicals, Inc.

BCR and chemokine responses upon anti-IgM and anti-IgD stimulation in chronic lymphocytic leukaemia.

Dysregulation of B cell receptor (BCR) signalling is a hallmark of chronic lymphocytic leukaemia (CLL) pathology, and targeting BCR pathway kinases has brought great therapeutic advances. Activation of the BCR in lymphoid organs has been associated with CLL cell proliferation and survival, leading to progressive disease. While these responses are mediated predominantly by IgM, the role of IgD is less clear. Seeking to uncover downstream consequences of individual and combined stimulation of the two BCR isotypes, we found an amplification of IgD expression and IgD-mediated calcium signalling by previous stimulation of IgM in CLL. Furthermore, no heterologous downmodulation of the isotypes, as observed in healthy donors, was present. Only marginal downregulation of the expression of various chemokine receptors by α-IgM and α-IgD stimulation was found as compared to normal B cells. Consistently, calcium responses of CLL cells to different chemokines were only weakly affected by preceding BCR activation. In contrast, migration towards the two homeostatic chemokines CXCL12 and CCL21 was differentially regulated by IgM and IgD. While IgM activation reduced migration of CLL cells towards CXCL12, but not CCL21, IgD activation predominantly impacted on CCL21 but not CXCL12-mediated chemotaxis. This indicates that the preference for one chemokine over the other may depend on the functional presence of the two isotypes in CLL. Inhibitors against the kinases Syk, Lyn, and Btk antagonised both BCR- and chemokine-induced calcium signals.

Pulmonary and hepatic injury after sub-chronic exposure to sublethal doses of microcystin-LR.

We had previously shown that microcystin-LR (MCLR) could induce lung and liver inflammation after acute exposure. The biological outcomes following prolonged exposure to MCLR, although more frequent, are still poorly understood. Thus, we aimed to verify whether repeated doses of MCLR could damage lung and liver and evaluate the dose-dependence of the results. Male Swiss mice received 10 intraperitoneal injections (i.p.) of distilled water (60 µL, CTRL) or different doses of MCLR (5 µg/kg, TOX5), 10 µg/kg (TOX10), 15 µg/kg (TOX15) and 20 µg/kg (TOX20) every other day. On the tenth injection respiratory mechanics (lung resistive and viscoelastic/inhomogeneous pressures, static elastance, and viscoelastic component of elastance) was measured. Lungs and liver were prepared for histology (morphometry and cellularity) and inflammatory mediators (KC and MIP-2) determination. All mechanical parameters and alveolar collapse were significantly higher in TOX5, 10, 15 and 20 than CTRL, but did not differ among them. Lung inflammatory cell content increased dose-dependently in all TOX groups in relation to CTRL, being TOX20 the largest. The production of KC was increased in lung and liver homogenates. MIP-2 increased in the liver of all TOX groups, but in lung homogenates it was significantly higher only in TOX20 group. All TOX mice livers showed steatosis, necrosis, inflammatory foci and a high degree of binucleated hepatocytes. In conclusion, sub-chronic exposure to MCLR damaged lung and liver in all doses, with a more important lung inflammation in TOX20 group.

The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks.

BACKGROUND: The toxicity of multi-walled carbon nanotubes (MWCNT) may be related to the immune system. The objective of this study was to obtain information for immunotoxic mechanisms of MWCNT in situ. METHODS: Using whole-body inhalation, male and female rats were exposed to 0, 0.2, 1 or 5 mg MWCNT/m³ for 13 weeks. Thereafter, spleens were recovered from the rats. Real-time PCR was done to assess expression of TNFα, IL-1ß, IL-6, IL-10, MCP-1 and MIP-1α mRNA in the splenic macrophages; splenic T-lymphocytes were examined for IL-2 and TGF-ß1 mRNA expression. RESULTS: The relative expression of IL-1ß mRNA in the cells from female rats exposed to 5 mg MWCNT/m³ was significantly higher than that in control cells. For IL-6 and IL-10, cells from rats in the 0.2 and 5 mg MWCNT/m³ had significantly higher mRNA expressions than did cells from controls. Expression of IL-1ß, IL-6 and TNFα genes in cells from males in all exposure groups were higher than in control cells. Expression of MIP-1α in the cells from female 5-mg group was significantly higher than that in cells in the control. Only IL-2 was expression reduced, i.e. cells from male and female rats in all MWCNT groups had significantly lower mRNA expressions than control cells. CONCLUSIONS: Systemic inflammation would likely occur in rats (or other hosts) exposed to MWCNT via inhalation due to increases in the expression of inflammatory cytokines in splenic macrophages. Moreover, decreases in IL-2 expression in T-lymphocytes may be critical to the potential reductions in anti-tumor responses in MWCNT-exposed hosts.

Effects of oral exposure to naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory cytokine and chemokine mRNA expression in the mouse.

The foodborne mycotoxin deoxynivalenol (DON) induces a ribotoxic stress response in mononuclear phagocytes that mediate aberrant multi-organ upregulation of TNF-α, interleukins and chemokines in experimental animals. While other DON congeners also exist as food contaminants or pharmacologically-active derivatives, it is not known how these compounds affect expression of these cytokine genes in vivo. To address this gap, we compared in mice the acute effects of oral DON exposure to that of seven relevant congeners on splenic expression of representative cytokine mRNAs after 2 and 6h. Congeners included the 8-ketotrichothecenes 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), nivalenol (NIV), the plant metabolite DON-3-glucoside (D3G) and two synthetic DON derivatives with novel satiety-inducing properties (EN139528 and EN139544). DON markedly induced transient upregulation of TNF-α IL-1ß, IL-6, CXCL-2, CCL-2 and CCL-7 mRNA expressions. The two ADONs also evoked mRNA expression of these genes but to a relatively lesser extent. FX induced more persistent responses than the other DON congeners and, compared to DON, was: 1) more potent in inducing IL-1ß mRNA, 2) approximately equipotent in the induction of TNF-α and CCL-2 mRNAs, and 3) less potent at upregulating IL-6, CXCL-2, and CCL-2 mRNAs. EN139528's effects were similar to NIV, the least potent 8-ketotrichothecene, while D3G and EN139544 were largely incapable of eliciting cytokine or chemokine mRNA responses. Taken together, the results presented herein provide important new insights into the potential of naturally-occurring and synthetic DON congeners to elicit aberrant mRNA upregulation of cytokines associated with acute and chronic trichothecene toxicity.

Diverging mechanisms of activation of chemokine receptors revealed by novel chemokine agonists.

CXCL8/interleukin-8 is a pro-inflammatory chemokine that triggers pleiotropic responses, including inflammation, angiogenesis, wound healing and tumorigenesis. We engineered the first selective CXCR1 agonists on the basis of residue substitutions in the conserved ELR triad and CXC motif of CXCL8. Our data reveal that the molecular mechanisms of activation of CXCR1 and CXCR2 are distinct: the N-loop of CXCL8 is the major determinant for CXCR1 activation, whereas the N-terminus of CXCL8 (ELR and CXC) is essential for CXCR2 activation. We also found that activation of CXCR1 cross-desensitized CXCR2 responses in human neutrophils co-expressing both receptors, indicating that these novel CXCR1 agonists represent a new class of anti-inflammatory agents. Further, these selective CXCR1 agonists will aid at elucidating the functional significance of CXCR1 in vivo under pathophysiological conditions.

Activation of cellular chemotactic responses to chemokines coupled with oxidation of plasma membrane proteins by lysyl oxidase.

Lysyl oxidase (LOX) is a potent chemokine inducing the migration of varied cell types. Here we demonstrate that inhibition of cellular LOX activity by preincubation of vascular smooth muscle cells (VSMC) with ß-aminopropionitrile (BAPN), the irreversible inhibitor of LOX activity, resulted in the marked suppression of the chemotactic response and sensitivity of these cells toward LOX and toward PDGF-BB. Plasma membranes purified from VSMC not previously exposed to BAPN contained a group of oxidized plasma membrane proteins, including the PDGF receptor, PDGFR-ß. The oxidation of this receptor and other membrane proteins was largely prevented in cells preincubated with BAPN. Addition of purified LOX to BAPN-free cells, which had been previously exposed to BAPN, restored the profile of oxidized proteins towards that of control cells. The high affinity and capacity for the binding of PDGF-BB by cells was significantly diminished when compared with cells in which oxidation by LOX was prevented by BAPN. The chemotactic responses of LOX knock-out mouse embryonic fibroblasts mirrored those obtained with VSMC treated with BAPN. These novel findings suggest that LOX activity is essential to generate optimal chemotactic sensitivity of cells to chemoattractants by oxidizing specific cell surface proteins, such as PDGFR-ß.

Chemokine decoy receptors: new players in reproductive immunology.

Chemokines are multifunctional molecules with roles in leukocyte trafficking and developmental processes. Both fetal and maternal components of the placenta produce chemokines, which control leukocyte trafficking observed in the placenta. Thus, chemokines play roles in the balance between protection of the developing embryo/fetus and tolerance of its hemiallogeneic tissues. Recently, a group of chemokine receptors, which include D6, DARC, and CCX-CKR, have been described as "silent" receptors by virtue of their inability to activate signal transduction events leading to cell chemoattraction. Here we review in vitro and in vivo evidence indicating that chemokine "silent" receptors regulate innate and adaptive immunity behaving as decoy receptors that support internalization and degradation of chemotactic factors, and discuss available information on their potential role in reproductive immunology.

Antagonism of the chemokine receptors CXCR3 and CXCR4 reduces the pathology of experimental autoimmune encephalomyelitis.

Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11((4-79)), was obtained. CXCL11((4-79)) strongly inhibited the migration of activated mouse T cells in response to all three high-affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12((P2G2)), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4(+) T cells in the CNS. Further investigation demonstrated that CXCL12((P2G2)) inhibited the sensitization phase, whereas CXCL11((4-79)) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.

Targeting cellular adhesion molecules, chemokines and chemokine receptors in rheumatoid arthritis.

The development of specific targeted therapies, such as anti-TNF-alpha treatment, for chronic inflammatory disorders such as rheumatoid arthritis, has significantly improved treatment, although not all patients respond. Targeting cellular adhesion molecules and chemokines/chemokine receptors as regulators of the extravasation and migration of leukocytes may provide a novel approach for the treatment of these diseases. Moreover, the possibility of developing small-molecule antagonists offers an excellent method for the oral delivery of compounds with a short half-life.

Effect of oral CCK-1 agonist GI181771X on fasting and postprandial gastric functions in healthy volunteers.

CCK influences satiation and gastric and gallbladder emptying. GI181771X is a novel oral CCK-1 agonist; its effects on gastric emptying of solids, accommodation, and postprandial symptoms are unclear. Effects of four dose levels of the oral CCK-1 agonist GI181771X and placebo on gastric functions and postprandial symptoms were compared in 61 healthy men and women in a randomized, gender-stratified, double-blind, double-dummy placebo-controlled, parallel group study. Effects of 0.1, 0.5, and 1.5 mg of oral solution and a 5.0-mg tablet of GI181771X on gastric emptying of solids by scintigraphy, gastric volume by (99m)Tc-single photon emission computed tomographic imaging, maximum tolerated volume of Ensure, and postprandial nausea, bloating, fullness, and pain were studied. On each of 3 study days, participants received their randomly assigned treatment. Adverse effects and safety were monitored. There were overall group effects of GI181771X on gastric emptying (P < 0.01) and fasting and postprandial volumes (P = 0.036 and 0.015, respectively). The 1.5-mg oral solution of GI181771X significantly delayed gastric emptying of solids (P < 0.01) and increased fasting (P = 0.035) gastric volumes without altering postprandial (P = 0.056) gastric volumes or postprandial symptoms relative to placebo. The effect of the 5.0-mg tablet on gastric emptying of solids did not reach significance (P = 0.052). Pharmacokinetic profiles showed the highest area under the curve over 4 h for the 1.5-mg solution and a similar area under the curve for the 0.5-mg solution and 5-mg tablet. Adverse effects were predominantly gastrointestinal and occurred in a minority of participants. GI181771X delays gastric emptying of solids and exhibits an acceptable safety profile in healthy participants. CCK-1 receptors can be modulated to increase fasting gastric volume.

Analysis of strain difference in behavior to Cholecystokinin (CCK) receptor mediated drugs in PVG hooded and Sprague-Dawley rats using elevated plus-maze test apparatus.

This study investigated the behavioral mechanisms underlying the anxiogenic, or anxiolytic mediated effects of CCK(2) receptor mediated agonist (CCK-4) and antagonist drugs (LY225910, LY288513, CR2945) in PVG hooded and Sprague-Dawley (SD) rats using the elevated plus maze test apparatus. In addition, the effects of a CCK(1) antagonist (CR1409) were investigated for its possible mediation in anxiety behavior between PVG hooded and SD rats. PVG hooded rats treated with CCK-4, decreased the time spent in the open arm and increased the time spent in the closed arm and correspondingly showed increase in the number of entries in the open arms while the number of entries in closed arm was insignificant, whereas SD rats decreased the time spent in the closed arm, while other parameters remained insignificant. PVG hooded rats administered with various CCK(2) antagonists (LY225910, LY288513, and CR2945) significantly increased the time spent in the open arm and correspondingly decreased the time spent in the closed arm, while the number of entries in the open or closed arm was insignificant, in contrast, SD rats failed to show any reliable significance. PVG hooded rats administered with the CCK(1) antagonist (CR1409), failed to show any reliable significance, in contrast, SD rats significantly increased the time spent in the open arm. The strain differences observed in this study suggests that CCK plays mainly as a neuromodulator, in which the various CCK(2) antagonists may not affect baseline anxiety state, but instead they modulate heightened states of anxiety through differential effects of CCK(1)/CCK(2) receptors.

CCK1R agonists: a promising target for the pharmacological treatment of obesity.

Almost 30 years have passed since Gibbs, Young, and Smith demonstrated the ability of exogenously administered cholecystokinin (CCK) to inhibit food intake in rats. This observation was the beginning of very extensive studies into the role CCK plays in the regulation of food intake in mammals. CCK is a brain-gut peptide, which exists in multiple forms. CCK peptides exert their action on two distinct receptor subtypes: CCK-A (Alimentary) now called the CCK1R, mostly expressed peripherally; and CCK-B (Brain), renamed the CCK2R, which is primarily present in the brain. Through the use of subtype-selective agonists and antagonists for the CCK receptor, it was determined that the effect of CCK on feeding was dependent on agonist induced activation of peripheral CCK1 receptors. This discovery was followed by intense research with the goal of identifying small molecule agonists on the CCK1 receptor as potentially useful agents for the treatment of obesity. This review will attempt to summarize the results of this research.

Inflammatory agonists induce cyclooxygenase type 2 expression by human neutrophils.

The synthesis of prostanoids is regulated by cyclooxygenases (prostaglandin H synthases), which catalyze the conversion of arachidonic acid to PGH2. Cyclooxygenases are the target of aspirin and other nonsteroidal anti-inflammatory agents. In this study, we found that human polymorphonuclear leukocytes (PMNs) express the inducible isoform of cyclooxygenase, COX-2, when stimulated by LPS whereas the protein was not detectable in freshly isolated human PMNs. We also found by immunohistochemical analysis that COX-2 is expressed in PMNs in inflamed human tissues. COX-2 was induced in a time- and concentration-dependent fashion when isolated human PMNs were exposed to LPS; COX-2 was also induced, or its expression was increased, by TNF-alpha, IL-1, and IL-8. Expression of COX-2 in stimulated PMNs was paralleled by secretion of PGE2. The release of PGE2 was blocked by a selective nonsteroidal inhibitor of COX-2, indicating that the enzyme is responsible for the prostanoids produced, and was inhibited by dexamethasone. The time course of LPS-induced COX-2 expression and other features were different in freshly isolated PMNs, monocytes, and macrophages, indicating that COX-2 expression is differentially regulated in myeloid cells of different lineages and degrees of maturation. Consistent with this, IL-4 and IL-10, which suppressed LPS-induced COX-2 expression in monocytes, had little effect on this response by PMNs. These experiments demonstrate that PMNs express COX-2 when appropriately stimulated. Thus, they may actively influence the eicosanoid composition of the acute inflammatory milieu.

IL-12 exacerbates helminth-mediated corneal pathology by augmenting inflammatory cell recruitment and chemokine expression.

Corneal inflammation (keratitis) is a major cause of visual impairment in Onchocerca volvulus infection. Previous studies showed that onchocercal keratitis can be induced in mice following s.c. immunization and intracorneal injection with soluble O. volvulus Ags (OvAg), and that the inflammatory response is dependent on T cells and IL-4. Since recombinant IL-12 impairs IL-4-dependent, Th2-mediated responses in other parasitic infections and in models of allergic asthma, the present study was undertaken to determine the effect of IL-12 on onchocercal keratitis. Mice were injected i.p. with IL-12 or saline at the time of initial sensitization to OvAg. Surprisingly, IL-12 treatment caused significant exacerbation of corneal pathology, which was associated with increased eosinophil and mononuclear cell infiltration into the corneal stroma. Consistent with the well-documented effect of IL-12 on Th1 cell development, corneas of IL-12-treated animals had elevated expression of the Th1 cytokine IFN-gamma and diminished expression of the Th2 cytokines IL-4, IL-5, IL-10, and IL-13. However, corneas from these animals also had marked elevation of alpha- and beta-chemokines known to be active on eosinophils and mononuclear cells, including IFN-gamma-inducible protein (IP)-10, macrophage inflammatory protein-1alpha, macrophage inflammatory protein-1beta, JE/monocyte chemotactic protein-1, RANTES (regulated upon activation, normal T expressed and secreted), and eotaxin. Together, these data indicate that IL-12 exacerbates OvAg-mediated corneal pathology by enhancing chemokine expression and recruitment of inflammatory cells.

Solution structure and basis for functional activity of stromal cell-derived factor-1; dissociation of CXCR4 activation from binding and inhibition of HIV-1.

The three-dimensional structure of stromal cell-derived factor-1 (SDF-1) was determined by NMR spectroscopy. SDF-1 is a monomer with a disordered N-terminal region (residues 1-8), and differs from other chemokines in the packing of the hydrophobic core and surface charge distribution. Results with analogs showed that the N-terminal eight residues formed an important receptor binding site; however, only Lys-1 and Pro-2 were directly involved in receptor activation. Modification to Lys-1 and/or Pro-2 resulted in loss of activity, but generated potent SDF-1 antagonists. Residues 12-17 of the loop region, which we term the RFFESH motif, unlike the N-terminal region, were well defined in the SDF-1 structure. The RFFESH formed a receptor binding site, which we propose to be an important initial docking site of SDF-1 with its receptor. The ability of the SDF-1 analogs to block HIV-1 entry via CXCR4, which is a HIV-1 coreceptor for the virus in addition to being the receptor for SDF-1, correlated with their affinity for CXCR4. Activation of the receptor is not required for HIV-1 inhibition.