CXCL17 binds efficaciously to glycosaminoglycans with the potential to modulate chemokine signaling.

Introduction: CXCL17 is a mucosally secreted protein, and the most recently identified human chemokine, an assignment based on protein fold prediction and chemotactic activity for leukocytes. However, these credentials have been the subject of much recent discussion and no experimental evidence has been presented regarding the definitive structure of CXCL17. In this study, we evaluated the structural and chemoattractant credentials of CXCL17 to better characterize this molecule, and gain deeper insights into its functional role as a glycosaminoglycan (GAG) binding protein. Methods: In the absence of structural information, in silico modeling techniques assessed the likelihood of CXCL17 adopting a chemokine fold. Recombinant CXCL17 was synthesized in mammalian and prokaryotic systems. Modified Boyden chamber and real-time chemotaxis assays assessed the ability of CXCL17 to promote chemotaxis of murine splenocytes, human neutrophils, and CXCR1 transfectants. The efficacy of CXCL17 binding to GAGs was quantified with solid-phase assays and bio-layer interferometry techniques. Results: All modeling efforts failed to support classification of CXCL17 as a chemokine based on its predicted conformation. Recombinant CXCL17 was observed to dimerize as a function of concentration, a characteristic of several chemokines. Contrary to a previous report, CXCL17 was not chemotactic for murine splenocytes, although it was a low-potency chemoattractant for human neutrophils at micromolar concentrations, several orders of magnitude higher than those required for CXCL8. As anticipated owing to its highly basic nature, CXCL17 bound to GAGs robustly, with key C-terminal motifs implicated in this process. While inactive via CXCR1, CXCL17 was found to inhibit CXCR1-mediated chemotaxis of transfectants to CXCL8 in a dose-dependent manner. Discussion: In summary, despite finding little evidence for chemokine-like structure and function, CXCL17 readily bound GAGs, and could modulate chemotactic responses to another chemokine in vitro. We postulate that such modulation is a consequence of superior GAG binding, and that C-terminal fragments of CXCL17 may serve as prototypic inhibitors of chemokine function.

Inhibition of pancreatic cancer-cell growth and metastasis in vivo by a pyrazole compound characterized as a cell-migration inhibitor by an in vitro chemotaxis assay.

Pancreatic cancer is recalcitrant to treatment as it is highly metastatic and rapidly progressive. While observing the behavior of human pancreatic BxPC-3 cells using an optical assay device called TAXIScan, we found that several synthetic pyrazole and pyrimidine derivatives inhibited cell migration. One such compound, 14-100, inhibited metastasis of fluorescence-labeled BxPC-3 cells, which were transplanted into the pancreas of nude mice as a subcutaneously grown cancer fragment. Surprisingly, despite its low cytotoxicity, the compound also showed an inhibitory effect on cancer cell proliferation in vivo, suggesting that the compound alters cancer cell characteristics needed to grow in situ. Single-cell RNA-sequencing revealed changes in gene expression associated with metastasis, angiogenesis, inflammation, and epithelial-mesenchymal transition. These data suggest that the compound 14-100 could be a good drug candidate against pancreatic cancer.

A possible way to prevent the progression of bone lesions in multiple myeloma via Src-homology-region-2-domain-containing-phosphatase-1 activation.

On the basis of our recent findings, in which multiple receptor-mediated mast cell functions are regulated via a common signaling cascade, we posit that the formation and functioning of osteoclasts are also controlled by a similar common mechanism. These cells are derived from the same granulocyte/monocyte progenitors and share multiple receptors except those that are cell-specific. In both types of cells, all known receptors reside in lipid rafts, form multiprotein complexes with recruited signaling molecules, and are internalized upon receptor engagement. Signal transduction proceeds in a chain of protein phosphorylations, where adaptor protein LAT (linker-for-activation-of-T-cells) plays a central role. The key kinase that associates LAT phosphorylation and lipid raft internalization is Syk (spleen-tyrosine-kinase) and/or an Src-family-kinase, most probably Lck (lymphocyte-specific-protein-tyrosine-kinase). Dephosphorylation of phosphorylated Syk and Lck by activated SHP-1 (Src-homology-region-2-domain-containing-phosphatase-1) terminates the signal transduction and endocytosis of receptors, resulting in inhibition of osteoclast differentiation and other functions. In malignant plasma cells (MM cells) too, SHP-1 plays a similar indispensable role in controlling signal transduction required for survival and proliferation, though BLNK (B-cell-linker-protein), a functional equivalent of LAT and SLP-76 (SH2-domain-containing-leukocyte-protein-of-76-kDa) in B cells, is used instead of LAT. In both osteoclasts and MM cells, therefore, activated SHP-1 acts negatively in receptor-mediated cellular functions. In osteoblasts, however, activated SHP-1 promotes differentiation, osteocalcin generation, and mineralization by preventing both downregulation of transcription factors, such as Ostrix and Runx2, and degradation of ß-catenin required for activation of the transcription factors. SHP-1 is activated by tyrosine phosphorylation and micromolar doses (M-dose) of CCRI-ligand-induced SHP-1 activation. Small molecular compounds, such as A770041, Sorafenib, Nitedanib, and Dovitinib, relieve the autoinhibitory conformation. Activation of SHP-1 by M-dose CCRI ligands or the compounds described may prevent the progression of bone lesions in MM.

Targeting FROUNT with disulfiram suppresses macrophage accumulation and its tumor-promoting properties.

Tumor-associated macrophages affect tumor progression and resistance to immune checkpoint therapy. Here, we identify the chemokine signal regulator FROUNT as a target to control tumor-associated macrophages. The low level FROUNT expression in patients with cancer correlates with better clinical outcomes. Frount-deficiency markedly reduces tumor progression and decreases macrophage tumor-promoting activity. FROUNT is highly expressed in macrophages, and its myeloid-specific deletion impairs tumor growth. Further, the anti-alcoholism drug disulfiram (DSF) acts as a potent inhibitor of FROUNT. DSF interferes with FROUNT-chemokine receptor interactions via direct binding to a specific site of the chemokine receptor-binding domain of FROUNT, leading to inhibition of macrophage responses. DSF monotherapy reduces tumor progression and decreases macrophage tumor-promoting activity, as seen in the case of Frount-deficiency. Moreover, co-treatment with DSF and an immune checkpoint antibody synergistically inhibits tumor growth. Thus, inhibition of FROUNT by DSF represents a promising strategy for macrophage-targeted cancer therapy.

A common signaling pathway leading to degranulation in mast cells and its regulation by CCR1-ligand.

BACKGROUND: Signal transduction pathways mediated by various receptors expressed on mast cells are thought to be complex, and inhibitory signals that turn off activating signals are not known. METHODS: Upstream signaling cascades mediated by several known receptors in bone marrow-derived mast cells that lead to degranulation and mediator release were studied by immunoblotting and immunoprecipitation. Small interfering RNAs and knockout mice were used to confirm findings. RESULTS: All ligands tested including IgE/Ag, SCF, HSP70, CCL3, and its valiant eMIP induced phosphorylation of linker for activation of T cells (LAT), which triggered their receptor-mediated downstream signaling cascades that controlled degranulation and mediator release. Phosphorylation of lymphocyte-specific protein kinase (Lck) was induced by each ligand, which commonly played an indispensable role in LAT phosphorylation. In contrast, phosphorylation of spleen tyrosine kinase was additionally induced in cells stimulated only with IgE/Ag and SCF, which is also associated with LAT phosphorylation in part. Degranulation and mediator release induced by IgE/Ag, SCF, or HSP70 were enhanced by nanomolar doses of CCR1 ligands CCL3 and eMIP via enhanced LAT phosphorylation. On the other hand, micromolar doses of CCR1 ligand inhibited degranulation and mediator release from mast cells stimulated with IgE/Ag, SCF, or HSP70 by de-phosphorylation of phosphorylated Lck with Src homology region 2 domain-containing phosphatase-1. CONCLUSIONS: Linker for activation of T cells plays a central role in signal transduction pathways in mast cells stimulated with any ligand tested. Dose-dependent alternate costimulation and inhibition of CCR1 ligands in IgE/Ag-, SCF-, or HSP70-stimulated mast cells occur at the level of Lck-LAT phosphorylation.

Anti-Metastatic Benefits Produced by Hyperthermia and a CCL3 Derivative.

Significant numbers of malignant tumor cells that have spread to surrounding tissues and other distant organs are often too small to be picked up in a diagnostic test, and prevention of even such small metastases should improve patient outcomes. Using a mouse model, we show in this article that intravenous administration of a human CCL3 variant carrying a single amino acid substitution after mild local hyperthermia not only induces tumor growth inhibition at the treated site but also inhibits metastasis. Colon26 adenocarcinoma cells (1 × 105 cells/mouse) were grafted subcutaneously into the right hind leg of syngeneic BALB/c mice and after nine days, when tumor size reached ~11 mm in diameter, the local tumor mass was exposed to high-frequency waves, by which intratumoral temperature was maintained at 42 °C for 30 min. Mice received the CCL3 variant named eMIP (2 µg/mouse/day) intravenously for five consecutive days starting one day after heat treatment. We found that tumor growth in eMIP recipients after hyperthermia was inhibited markedly but no effect was seen in animals treated with either hyperthermia or eMIP alone. Furthermore, the number of lung metastases evaluated after 18 days was dramatically reduced in animals receiving the combination therapy compared with all other controls. These results encourage future clinical application of this combination therapy.

Reduced Number of Lymphocytes by X-ray Irradiation: A Problem in a Combination Therapy Trial that Elicits the Abscopal Effect in Preclinical Studies Using Electron Beam Irradiation.

In preclinical studies with model animals, intravenous administration of a derivative of chemokine CCL3, named eMIP, after local electron-beam irradiation, not only enhanced tumor growth inhibition at a target site but also induced tumor killing beyond the treated site (a phenomenon known as the abscopal effect). eMIP works with alarmins such as high mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70) released from overexpressed tumor cells by irradiation. These alarmins at the irradiated tumor bed trap injected eMIP and, by forming complexes with eMIP, play a key role to recruit and activate tumor inhibitory natural killer (NK) cells and CD4+ and CD8+ T cells. Tumor type-specific secretion of gamma interferon from splenocytes was also demonstrated, which may also activate NK cells. During Phase 1 clinical studies using X-rays, however, no apparent abscopal effect was observed. Instead, we saw frequent reduction in numbers of lymphocytes in the peripheral blood of irradiated patients. The reduced number of lymphocytes recovered poorly once depleted, in contrast to neutrophils, and persisted for months after the treatment. This might have affected outcome after combination treatment of irradiation and eMIP. To enhance host defense mechanisms during and after photon-beam (X-ray) radiotherapy of a deep-seated tumor, it seems essential to keep lymphocytes undamaged by eliminating reactive oxygen species that are formed in the peripheral blood during irradiation.

A Requirement for Neutrophil Glycosaminoglycans in Chemokine:Receptor Interactions Is Revealed by the Streptococcal Protease SpyCEP.

To evade the immune system, the lethal human pathogen Streptococcus pyogenes produces SpyCEP, an enzyme that cleaves the C-terminal α-helix of CXCL8, resulting in markedly impaired recruitment of neutrophils to sites of invasive infection. The basis for chemokine inactivation by SpyCEP is, however, poorly understood, as the core domain of CXCL8 known to interact with CXCL8 receptors is unaffected by enzymatic cleavage. We examined the in vitro migration of human neutrophils and observed that their ability to efficiently navigate a CXCL8 gradient was compromised following CXCL8 cleavage by SpyCEP. SpyCEP-mediated cleavage of CXCL8 also impaired CXCL8-induced migration of transfectants expressing the human chemokine receptors CXCR1 or CXCR2. Despite possessing an intact N terminus and preserved disulfide bonds, SpyCEP-cleaved CXCL8 had impaired binding to both CXCR1 and CXCR2, pointing to a requirement for the C-terminal α-helix. SpyCEP-cleaved CXCL8 had similarly impaired binding to the glycosaminoglycan heparin. Enzymatic removal of neutrophil glycosaminoglycans was observed to ablate neutrophil navigation of a CXCL8 gradient, whereas navigation of an fMLF gradient remained largely intact. We conclude, therefore, that SpyCEP cleavage of CXCL8 results in chemokine inactivation because of a requirement for glycosaminoglycan binding in productive chemokine:receptor interactions. This may inform strategies to inhibit the activity of SpyCEP, but may also influence future approaches to inhibit unwanted chemokine-induced inflammation.

CXCL4/Platelet Factor 4 is an agonist of CCR1 and drives human monocyte migration.

Activated platelets release micromolar concentrations of the chemokine CXCL4/Platelet Factor-4. Deposition of CXCL4 onto the vascular endothelium is involved in atherosclerosis, facilitating monocyte arrest and recruitment by an as yet, unidentified receptor. Here, we demonstrate that CXCL4 drives chemotaxis of the monocytic cell line THP-1. Migration and intracellular calcium responses induced by CXCL4 were pertussis toxin-sensitive, implicating a GPCR in signal transduction. Cell treatment with chondroitinase ABC ablated migration, suggesting that cis presentation of CXCL4 by cell surface glycosaminoglycans to a GPCR is required. Although CXCR3 has been previously described as a CXCL4 receptor, THP-1 cells were unresponsive to CXCR3 ligands and CXCL4-induced migration was insensitive to a CXCR3 antagonist, suggesting that an alternative receptor is involved. Interrogating CC-class chemokine receptor transfectants, we unexpectedly found that CXCL4 could induce the migration of CCR1-expressing cells and also induce CCR1 endocytosis. Extending our findings to primary human monocytes, we observed that CXCL4 induced CCR1 endocytosis and could induce monocyte chemotaxis in a CCR1 antagonist-sensitive manner. Collectively, our data identify CCR1 as a previously elusive monocyte CXCL4 receptor and suggest that CCR1 may play a role in inflammation where the release of CXCL4 is implicated.

Identification of the active portion of the CCL3 derivative reported to induce antitumor abscopal effect.

BACKGROUND AND PURPOSE: Intravenous administration of a single amino acid-substituted chemokine CCL3 derivative named eMIP elicits the abscopal effect (an effect distal to the target), after local irradiation at a tumor-bearing site. To distinguish the active portion of eMIP, we tested the antitumor activity of chemically synthesized partial peptides of eMIP. Synthetic peptide has various advantages in its clinical application. MATERIAL AND METHODS: Colon26 adenocarcinoma cells were implanted subcutaneously in the right and left flanks of mice. eMIP, CCL3 or any of synthesized peptides was administered intravenously, either after irradiating the right flank. The effect was evaluated by tumor-growth inhibition. RESULTS: Q/C peptide, a synthetic peptide of amino acids 22-51 of eMIP has no chemotaxis-inducing ability but yet enhanced tumor growth inhibition at the non-irradiated sites, recapitulating the effect of eMIP with local irradiation. Co-administration of this peptide and HSP70 also inhibited tumor growth. CONCLUSIONS: Q/C peptide maps to the eMIP ß-sheet: 3 adjacent anti-parallel strands connected by the ß-hairpins, is the active portion of eMIP necessary for an immunomodulatory antitumor effect. This experimental reduction furthers our understanding of the underlying mechanism of the abscopal effect. The data will open the way for therapeutic application of like peptides.

Macrophage inflammatory protein derivative ECI301 enhances the alarmin-associated abscopal benefits of tumor radiotherapy.

Radiotherapy can produce antitumor benefits beyond the local site of irradiation, an immune-based phenomenon known as the abscopal effect, but the mechanisms underlying these benefits are poorly understood. Preclinical studies of ECI301, a mutant derivative of macrophage inhibitory protein-1α, have shown that its administration can improve the antitumor effects of radiotherapy in a manner associated with a tumor-independent abscopal effect. In this article, we report that i.v. administration of ECI301 after intratumoral injection of tumor cell lysates can inhibit tumor growth, not only at the site of injection but also at nontreated sites. Effects of the tumor lysate were further recapitulated by intratumoral injection [corrected] of the alarmins HSP70 or HMGB1, but not HSP60, and i.v. administration [corrected] of ECI301 + HSP70 were sufficient to inhibit tumor growth. Although i.v. administration [corrected] of ECI301 + HMGB1 did not inhibit tumor growth, we found that administration of a neutralizing HMGB1 antibody neutralized the cooperative effects of ECI301 on tumor irradiation. Moreover, mice genetically deficient in TLR4, an immune pattern receptor that binds alarmins, including HMGB1 and HSP70, did not exhibit antitumor responses to irradiation with ECI301 administration. Although ECI301 was cleared rapidly from peripheral blood, it was found to bind avidly to HSP70 and HMGB1 in vitro. Our results suggest a model in which sequential release of the alarmins HSP70 and HMGB1 from a tumor by irradiation may trap circulating ECI301, thereby licensing or restoring tumor immunosurveillance capabilities of natural killer cells or CD4(+) and CD8(+) T cells against tumor cells that may evade irradiation. Cancer Res; 74(18); 5070-8. ©2014 AACR.

Aurantio-obtusin stimulates chemotactic migration and differentiation of MC3T3-E1 osteoblast cells.

Osteoporosis is one of the major metabolic bone diseases and is among the most challenging noncommunicable diseases to treat. Although there is an increasing interest in identifying bioactive molecules for the prevention and management of osteoporosis, such studies principally focus only on differentiation and mineralization of osteoblasts or inhibition of osteoclast activity. Stimulation of osteoblast migration must be a promising osteoanabolic strategy for improved metabolic bone disease therapy. In this study, we show that an anthraquinone derivative, aurantio-obtusin, stimulated chemotactic migration of MC3T3-E1 osteoblast cells in a concentration-dependent manner. The use of a real-time chemotaxis analyzing system, TAXIScan, facilitated the evaluation of both velocity and directionality of osteoblast migration in response to the compound. Besides migration, the compound stimulated osteoblast differentiation and mineralization. Taken together, the data presented in this paper demonstrate that aurantio-obtusin is a promising osteoanabolic compound of natural origin with potential therapeutic applications in the prevention of osteoporosis and other metabolic bone diseases.

Identification of a binding element for the cytoplasmic regulator FROUNT in the membrane-proximal C-terminal region of chemokine receptors CCR2 and CCR5.

Chemokine receptors mediate the migration of leucocytes during inflammation. The cytoplasmic protein FROUNT binds to chemokine receptors CCR2 [chemokine (C-C motif) receptor 2] and CCR5, and amplifies chemotactic signals in leucocytes. Although the interaction between FROUNT and chemokine receptors is important for accurate chemotaxis, the interaction mechanism has not been elucidated. In the present study we identified a 16-amino-acid sequence responsible for high-affinity binding of FROUNT at the membrane-proximal C-terminal intracellular region of CCR2 (CCR2 Pro-C) by yeast two-hybrid analysis. Synthesized peptides corresponding to the CCR2 Pro-C sequence directly interacted with FROUNT in vitro. CCR2 Pro-C was predicted to form an amphipathic helix structure. Residues on the hydrophobic side are completely conserved among FROUNT-binding receptors, suggesting that the hydrophobic side is the responsible element for FROUNT binding. The L316T mutation to the hydrophobic side of the predicted helix decreased the affinity for FROUNT. Co-immunoprecipitation assays revealed that the CCR2 L316T mutation diminished the interaction between FROUNT and full-length CCR2 in cells. Furthermore, this mutation impaired the ability of the receptor to mediate chemotaxis. These findings provide the first description of the functional binding element in helix 8 of CCR2 for the cytosolic regulator FROUNT that mediates chemotactic signalling.

Alarmins released during local antitumor treatments play an essential role in enhancing tumor growth inhibition at treated and non-treated sites via a derivative of CCL3.

ECI301 (eMIP), a single amino-acid substituted CCL3 (MIP-1α), enhanced tumor growth inhibition and the abscopal effect (an effect distal to the target) following local antitumor therapy such as radiation, radiofrequency ablation (RFA), or hyperthermia treatment. The recent elucidation of the underlying mechanism may lead to a better antitumor therapy.

Systematic single cell analysis of migration and morphological changes of human neutrophils over stimulus concentration gradients.

To compare the responses of individual neutrophils to chemoattractants, migration pathway data were obtained using TAXIScan, an optically accessible/horizontal apparatus in which a concentration gradient is established reproducibly for a given stimulus. The observed linear-mode trajectory pattern of neutrophils toward N-formyl-methionyl-leucyl-phenylalanine (fMLP) or Interleukin (IL)-8/CXCL8 was distinguished from random migration patterns toward leukotriene (LT) B4 or platelet activating factor (PAF). The median values of velocity and directionality calculated for individual cells toward fMLP and IL-8 were both relatively similar and high, whereas the values toward LTB4 and PAF were widely dispersed over a lower range of directionality and from low to high ranges of velocity. The different patterns between the groups may be explained by unique morphology with single polarity toward fMLP and IL-8, and unstable morphology with multiple polarities toward LTB4 and PAF. Unique morphologies toward fMLP and IL-8 were not affected by coexisting LTB4 or PAF. On the other hand, the addition of suboptimum concentrations of fMLP or IL-8 to LTB4 or PAF induced a nearly maximum chemotactic response in most cells. These data suggest that exogenous formyl peptides and endogenous chemokines augment neutrophil accumulation at inflammation sites, whereas lipid mediators may play a role in supporting activation of the inflammatory cells for recruitment.

Inhibition of lung metastasis by chemokine CCL17-mediated in vivo silencing of genes in CCR4+ Tregs.

Despite significant attractiveness of antisense oligonucleotide/RNAi technology, its clinical application has been precluded by a lack of methods for targeted delivery and transduction of primary immune cells in vivo. Here, we devised a chemokine CCL17-based strategy (TARC-arp) that transiently silences expression of genes in immune cells by delivering inhibitory oligonucleotides through their chemokine receptors. In modeling studies using mice with established 4T1.2 breast cancer, we show that IL10 produced by CCR4 cells, in particular FoxP3 regulatory T cells (Tregs), plays an important role in lung metastasis. As such, TARC-arp-mediated silencing of IL10 or FoxP3 in CCR4 Tregs is sufficient to block lung metastasis. Thus, we provide a simple solution that circumvents the problems of RNAi use in vivo, indicating that a disease outcome can be successfully controlled by delivering inhibitory oligonucleotides with chemokines to inactivate a selective subset of immune cells.

FROUNT is a common regulator of CCR2 and CCR5 signaling to control directional migration.

FROUNT is a known CCR2-binding protein that facilitates monocyte/macrophage infiltration. Here we report that FROUNT also binds to the C-terminal region of CCR5 and enhances CCR5-mediated cellular chemotaxis. We show that FROUNT overexpression enhances the directionality of chemotaxis, while FROUNT suppression results in impaired responsiveness. Furthermore, we found an increase in consolidated pseudopodium formation in FROUNT-overexpressing cells (FNT cells) on uniform stimulation with CCL4 (MIP1-beta), a specific ligand of CCR5. In most FNT cells, one to two pseudopodia directed toward higher chemokine concentration were found, whereas most FNT-suppressed cells had multiple pseudopodia. The data indicate that FROUNT is involved in sensing and amplifying a shallow extracellular chemokine gradient that leads to a limited number of accurate pseudopodia directed toward the chemokine concentration. In addition to its separate roles in CCR2- and CCR5-mediated chemotaxis, FROUNT, as a common regulator of these receptors, possibly plays a crucial role in the recruitment of immune cells expressing these receptors.

Embryonic stem cell transplantation correlates with endogenous neurogenin 3 expression and pancreas regeneration in streptozotocin-injured mice.

Pancreatic beta cell regeneration remains poorly understood, yet stimulation of adult beta cell neogenesis could lead to therapies for type 1 and type 2 diabetes. We studied the effect of embryonic stem (ES) cell transplantation on pancreas regeneration following beta cell injury. Female Balb/c nude mice were treated with streptozotocin to induce hyperglycemia and received an ES cell transplant 24 hr later beneath the renal capsule. Transplantation of ES cells prevented hyperglycemia in a subset of mice, maintaining euglycemia and mild glucose tolerance up to 5 weeks. Pancreata of euglycemic mice showed histological evidence of beta cell regeneration and expression of pancreas and duodenum transcription factor-1 (PDX-1) and neurogenin 3 (Ngn3) in ductal epithelium. Cell tracing analysis indicated that significant beta cell neogenesis from progenitor cells occurred between 2 to 3 weeks following injury in ES cell-transplanted mice but not in sham-transplanted animals. Significantly, whereas pancreas-localized ES cells or their derivatives were adjacent to sites of regeneration, neogenic pancreatic epithelia, including Ngn3+ cells, were endogenous. In conclusion, transplanted ES cells can migrate to the injured pancreas. Transplantation is associated with enhanced endogenous regeneration characterized by expression of Ngn3 and increased beta cell differentiation from endogenous progenitor cells.

Image analysis of mast cell degranulation in a concentration gradient of stimuli formed in the channel between a glass plate and a silicon substrate.

Measurement of released granule components, popularly used to quantify mast cell exocytosis, does not deliver real-time information about degranulation at the single-cell level nor the ratio of responding/non-responding cells. Rather it provides, only end-point, bulk-population data. Here we studied degranulation of rat peritoneal mast cells dispersed in a narrow horizontal channel between a silicon substrate and a glass plate. Upon exposure to a concentration gradient of a soluble stimulus, degranulation started from those cells facing towards the highest concentration of stimulus. We captured images of exocytosing cells without the need for phase-contrast or differential interference-contrast microscopy. This was achieved using the reflection caused by the silicon substrate. The time-lapse images of cells in the channel were segmented into multiple concentration belts to identify the proportion of degranulated cells in each belt region. Maximum ratios of degranulated cells in the belt regions determined by time-course curve fitting calculations were then plotted against the distance from the stimulus injection site, resulting in a sigmoidal response curve. This method provides a powerful means for real-time analysis of concentration- and stimulus-dependent degranulation of mast cells and allows comparison of cell responses under different conditions. To show its effectiveness, we evaluated the effect of a protein kinase C (PKC) inhibitor, Gö6976, on degranulation induced by various stimuli. In contrast to stimulation with concanavalin A+lysophosphatidylserine (lysoPS) or nerve growth factor+lysoPS (completely inhibited by Gö6976 over the whole range of stimulus concentrations used) or compound 48/80 and mastoparan (no inhibition by Gö6976), stimulation with ionomycin, a known Ca(2+) ionophore, showed a concentration-dependent inhibition by Gö6976, with a major inhibition at low stimulus concentrations and a diminished one at higher ionomycin concentrations. The results indicate that ionomycin-induced degranulation is mainly induced via a PKC-independent signal cascade at high stimulus concentrations, whereas below a certain concentration, degranulation is completely dependent on PKC.

Differential regulatory function of resting and preactivated allergen-specific CD4+ CD25+ regulatory T cells in Th2-type airway inflammation.

Although CD4(+)CD25(+) regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.