Inhibition of airway inflammation by amino-terminally modified RANTES/CC chemokine ligand 5 analogues is not mediated through CCR3

Chemokines play a key role in the recruitment of activated CD4(+) T cells and eosinophils into the lungs in animal models of airway inflammation. Inhibition of inflammation by N-terminally modified chemokines is well-documented in several models but is often reported with limited dose regimens. We have evaluated the effects of doses ranging from 10 ng to 100 micro g of two CC chemokine receptor antagonists, Met-RANTES/CC chemokine ligand 5 (CCL5) and aminooxypentane-RANTES/CCL5, in preventing inflammation in the OVA-sensitized murine model of human asthma. In the human system, aminooxypentane-RANTES/CCL5 is a full agonist of CCR5, but in the murine system neither variant is able to induce cellular recruitment. Both antagonists showed an inverse bell-shaped inhibition of cellular infiltration into the airways and mucus production in the lungs following allergen provocation. The loss of inhibition at higher doses did not appear to be due to partial agonist activity because neither variant showed activity in recruiting cells into the peritoneal cavity at these doses. Surprisingly, neither was able to bind to the major CCR expressed on eosinophils, CCR3. However, significant inhibition of eosinophil recruitment was observed. Both analogues retained high affinity binding for murine CCR1 and murine CCR5. Their ability to antagonize CCR1 and CCR5 but not CCR3 was confirmed by their ability to prevent RANTES/CCL5 and macrophage inflammatory protein-1beta/CCL4 recruitment in vitro and in vivo, while they had no effect on that induced by eotaxin/CCL11. These results suggest that CCR1 and/or CCR5 may be potential targets for asthma therapy.     

CCR7 ligand-enhanced phagocytosis of various antigens in mature dendritic cells-time course and antigen distribution different from phagocytosis in immature dendritic cells

CC chemokine receptor 7 (CCR7) is selectively expressed on mature dendritic cells (DC). The CCR7 ligands, CC chemokine ligand (CCL) 19 and CCL21, facilitate migration of mature DC from the peripheral tissues to regional lymph nodes. We previously demonstrated that CCR7 ligands induced rapid receptor-mediated endocytosis of dextran in mature DC. In the present study, we further examined the effects of CCR7 ligands on endocytosis of other kinds of antigen, mannosilated bovine serum albumin (Mann-BSA), Escherichia coli(E. coli), or ovalbumin-containing immune complex (OVA-IC), by mature DC. We found that CCR7 ligands enhanced the endocytosis of Mann BSA, E. coli, and OVA-IC in mature DC but not in immature DC. The endocytosis of BSA was not enhanced by CCR7 ligands. Furthermore, the phagocytosis of OVA-IC was significantly inhibited by anti-Fcgamma receptor III/II antibody. These results demonstrate that CCR7 ligands enhance only receptor-mediated endocytosis by mature DC. When rapidly phagocytosed E. coli were traced in CCL19-treated mature DC, most of the phagocytosed E. coli did not colocalize with the lysosomal marker: lysosome-associated membrane protein-1 (Lamp-1), whereas most of E. coli taken up relatively slowly by immature DC colocalized with Lamp-1. These results suggest that phagocytosis of antigens by immature and mature DC plays different functional roles.     

Modulation of dendritic cell trafficking to and from the airways

We investigated the fate of latex (LX) particles that were introduced into mice intranasally. Macrophages acquired the vast majority of particles and outnumbered LX particle-bearing airway dendritic cells (DCs) by at least two orders of magnitude. Yet alveolar macrophages were refractory to migration to the draining lymph node (DLN), and all transport to the DLN could be ascribed to the few LX(+) airway DCs. Upon macrophage depletion, markedly greater numbers of DCs were recruited into the alveolar space. Consequently, the number of DCs that carried particles to the DLN was boosted by 20-fold. Thus, a so far overlooked aspect of macrophage-mediated suppression of airway DC function stems from the modulation of DC recruitment into the airway. This increase in DC recruitment permitted the development of a robust assay to quantify the subsequent migration of DCs to the DLN. Therefore, we determined whether lung DCs use the same molecules that skin DCs use during migration to DLNs. Like skin DCs, lung DCs used CCR7 ligands and CCR8 for emigration to DLN, but the leukotriene C(4) transporter multidrug resistance-related protein 1 did not mediate lung DC migration as it does in skin, indicating that pathways governing DC migration from different tissues partially differ in molecular regulation.     

CCR2 and CCR6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen

Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II(+)CD11c(mod) cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.     

The chemokine RANTES is a crucial mediator of the progression from acute to chronic colitis in the rat

Chemokines have well characterized proinflammatory actions, including the ability to induce extravasation of leukocytes that participate in chronic inflammation. In this study, we evaluated the role of a C-C chemokine, RANTES, in the chronic phase of a rat model of colitis. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various timepoints thereafter (2 h to 14 days), colonic tissue levels of several chemokines were measured. Unlike the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant, the expression of RANTES was significantly elevated during the chronic phase of colitis (> or =7 days after induction). Colonic RANTES mRNA expression was also significantly elevated during the chronic phase of colitis. The numbers of macrophages and monocytes in the colonic mucosa increased substantially during the chronic phase, as did expression of two of the receptors (CCR1 and CCR5) to which RANTES is known to bind. Administration on days 7 through 14 after trinitrobenzene sulfonic acid administration of a CCR1/CCR5 receptor antagonist, Met-RANTES, resulted in a significant reduction of both macroscopic and microscopic colonic damage, as well as reducing the recruitment into the colon of monocytes, mast cells, and neutrophils. In some rats, treatment with Met-RANTES resulted in a near-complete resolution of colonic damage and inflammation. These results suggest a crucial role of RANTES in the progression from acute to chronic inflammation in a rat model of colitis.     

CCR2 mediates conventional dendritic cell recruitment and the formation of bronchovascular mononuclear cell infiltrates in the lungs of mice infected with Cryptococcus neoformans

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. CCR2-deficient mice infected with C. neoformans develop a non-protective T2 immune response and persistent infection. The mechanisms responsible for this aberrant response are unknown. The objective of this study was to define the number, phenotype, and microanatomic location of dendritic cells (DC) residing within the lung of CCR2+/+ or CCR2-/- mice throughout a time course following infection with C. neoformans. Results demonstrate the CCR2-mediated recruitment of conventional DC expressing modest amounts of costimulatory molecules. DC recruitment was preceded by the up-regulation in the lung of the CCR2 ligands CCL2 and CCL7. Colocalization of numerous DC and CD4+ T cells within bronchovascular infiltrates coincided with increased expression of IL-12 and IFN-gamma. By contrast, in the absence of CCR2, DC recruitment was markedly impaired, bronchovascular infiltrates were diminished, and mice developed features of T2 responses, including bronchovascular collagen deposition and IL-4 production. Our results demonstrate that CCR2 is required for the recruitment of large numbers of conventional DC to bronchovascular infiltrates in mice mounting a T1 immune response against a fungal pathogen. These findings shed new insight into the mechanism(s) by which DC recruitment alters T cell polarization in response to an infectious challenge within the lung.     

Signal Relay by CC Chemokine Receptor 2 (CCR2) and Formylpeptide Receptor 2 (Fpr2) in the Recruitment of Monocyte-derived Dendritic Cells in Allergic Airway Inflammation

Chemoattractant receptors regulate leukocyte accumulation at sites of inflammation. In allergic airway inflammation, although a chemokine receptor CCR2 was implicated in mediating monocyte-derived dendritic cell (DC) recruitment into the lung, we previously also discovered reduced accumulation of DCs in the inflamed lung in mice deficient in formylpeptide receptor Fpr2 (Fpr2^−/−). We therefore investigated the role of Fpr2 in the trafficking of monocyte-derived DCs in allergic airway inflammation in cooperation with CCR2. We report that in allergic airway inflammation, CCR2 mediated the recruitment of monocyte-derived DCs to the perivascular region, and Fpr2 was required for further migration of the cells into the bronchiolar area. We additionally found that the bronchoalveolar lavage liquid from mice with airway inflammation contained both the CCR2 ligand CCL2 and an Fpr2 agonist CRAMP. Furthermore, similar to Fpr2^−/− mice, in the inflamed airway of CRAMP^−/− mice, DC trafficking into the peribronchiolar areas was diminished. Our study demonstrates that the interaction of CCR2 and Fpr2 with their endogenous ligands sequentially mediates the trafficking of DCs within the inflamed lung.     

The functional antagonist Met-RANTES: A modified agonist that induces differential CCR5 trafficking

CC chemokine receptor 5 (CCR5) is a pro-inflammatory chemokine receptor that is expressed on cells of the immune system, and specializes in cell migration in response to inflammation and tissue damage. Due to its key role in cell communication and migration, this receptor is involved in various inflammatory and autoimmune diseases, in addition to HIV infection. Met-RANTES is a modified CCR5 ligand that has previously been shown to antagonize CCR5 activation and function in response to its natural ligands in vitro. In vivo, Met-RANTES is able to reduce inflammation in models of induced inflammatory and autoimmune diseases. However, due to the fact that Met-RANTES is also capable of partial agonist activity regarding receptor signaling and internalization, it is clear that Met-RANTES does not function as a conventional receptor antagonist. To further elucidate the effect of Met-RANTES on CCR5, receptor trafficking was investigated in a CHO-CCR5-GFP cell line using the Opera confocal plate reader. The internalization response of CCR5 was quantified, and showed that Met-RANTES internalized CCR5 in a slower, less potent manner than the agonists CCL3 and CCL5. Fluorescent organelle labeling and live cell imaging showed CCL3 and CCL5 caused CCR5 to traffic through sorting endosomes, recycling endosomes and the Golgi apparatus. In contrast, Met-RANTES caused CCR5 to traffic through sorting endosomes and the Golgi apparatus in a manner that was independent of recycling endosomes. As receptor trafficking impacts on cell surface expression and the ability of the receptor to respond to more ligand, this information may indicate an alternative regulation of CCR5 by Met-RANTES that allows the modified ligand to reduce inflammation through stimulation of a pro-inflammatory receptor.     

Treatment of chronically Trypanosoma cruzi-infected mice with a CCR1/CCR5 antagonist (Met-RANTES) results in amelioration of cardiac tissue damage

The comprehension of the molecular mechanisms leading to Trypanosoma cruzi-elicited heart dysfunction might contribute to design novel therapeutic strategies aiming to ameliorate chronic Chagas disease cardiomyopathy. In C3H/He mice infected with the low virulence T. cruzi Colombian strain, the persistent cardiac inflammation composed mainly of CCR5⁺ T lymphocytes parallels the expression of CC-chemokines in a pro-inflammatory IFN-γ and TNF-α milieu. The chronic myocarditis is accompanied by increased frequency of peripheral CCR5⁺LFA-1⁺ T lymphocytes. The treatment of chronically T. cruzi-infected mice with Met-RANTES, a selective CCR1/CCR5 antagonist, led to a 20–30% decrease in CD4⁺ cell numbers as well as IL-10, IL-13 and TNF-α expression. Further, Met-RANTES administration impaired the re-compartmentalization of the activated CD4⁺CCR5⁺ lymphocytes. Importantly, Met-RANTES treatment resulted in significant reduction in parasite load and fibronectin deposition in the heart tissue. Moreover, Met-RANTES treatment significantly protected T. cruzi-infected mice against connexin 43 loss in heart tissue and CK-MB level enhancement, markers of heart dysfunction. Thus, our results corroborate that therapeutic strategies based on the modulation of CCR1/CCR5-mediated cell migration and/or effector function may contribute to cardiac tissue damage limitation during chronic Chagas disease.     

Differential activation of CC chemokine receptors by AOP-RANTES

RANTES (regulated on activation normal T cell expressed) has been found at elevated levels in biological fluids from patients with a wide range of allergic and autoimmune diseases and is able to attract several subtypes of leukocytes including eosinophils and monocytes into inflamed tissue. Amino-terminal modifications of RANTES produce receptor antagonists which are candidates for blocking this cellular recruitment. Met-RANTES has been shown to modulate inflammation in vivo, while AOP-RANTES is a potent inhibitor of R5 human immunodeficiency virus type 1 (HIV-1) strains and has been shown to down-modulate CCR5 and prevent recycling of the receptor. We have studied the effect of AOP-RANTES in eosinophil activation and have found that it is able to efficiently elicit eosinophil effector functions through CCR3, as measured by the release of reactive oxygen species and calcium mobilization, whereas Met-RANTES is inactive in these assays. AOP-RANTES is found to inhibit CCR3-mediated HIV-1 infection with moderate potency, in contrast to its potent inhibition of CCR5-mediated HIV-1 infection. Furthermore, we have investigated the abilities of these modified proteins to down-modulate CCR1 and CCR3 from the surface of monocytes and eosinophils. We show here that AOP-RANTES is much less effective than RANTES in down-modulation of CCR1. Surprisingly, recycling of CCR1 was minimal after incubation with RANTES while there was complete recycling with AOP-RANTES. In the case of CCR3, no significant difference was found between RANTES and AOP-RANTES in down-modulation and recycling. It therefore appears that trafficking of RANTES receptors follows different patterns, which opens up potential new targets for therapeutic intervention.     

Angiotensin II-induced mononuclear leukocyte interactions with arteriolar and venular endothelium are mediated by the release of different CC chemokines

Angiotensin II (Ang-II) is associated with atherogenesis and arterial subendothelial mononuclear leukocyte infiltration. We have demonstrated that Ang-II causes the initial attachment of mononuclear cells to the arteriolar endothelium. We now report on the contribution of CC chemokines to this response. Intraperitoneal administration of 1 nM Ang-II induced MCP-1, RANTES, and MIP-1alpha generation, maximal at 4 h, followed by mononuclear leukocyte recruitment at 8 and 24 h. Using intravital microscopy within the rat mesenteric microcirculation 4 h after exposure to 1 nM Ang-II, arteriolar mononuclear cell adhesion was 80-90% inhibited by pretreatment with Met-RANTES, a CCR1 and CCR5 antagonist, or an anti-MCP-1 antiserum, without affecting the increased endothelial expression of P-selectin and VCAM-1. Conversely, leukocyte interactions with the venular endothelium, although inhibited by Met-RANTES, were little affected by the anti-MCP-1. Using rat whole blood in vitro, Ang-II (100 nM) induced the expression of monocyte CD11b that was inhibited by Met-RANTES but not by anti-MCP-1. Stimulation of human endothelial cells (human umbilical arterial endothelial cells and HUVECs) with 1-1000 nM Ang-II, predominantly acting at its AT(1) receptor, induced the release of MCP-1 within 1 h, RANTES within 4 h, and MCP-3 within 24 h. Eotaxin-3, a natural CCR2 antagonist, was released within 1 h and may delay mononuclear cell responses to MCP-1. Therefore, Ang-II-induced mononuclear leukocyte recruitment at arterioles and venules is mediated by the production of different CC chemokines. Thus, Ang-II may be a key molecule in the initial attachment of mononuclear cells to the arterial endothelium in cardiovascular disease states where this event is a characteristic feature.     

CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephritis despite reduction of glomerular leukocyte infiltration

The chemokine CC chemokine ligand (CCL)5/RANTES as well as its respective receptor CCR5 mediate leukocyte infiltration during inflammation and are up-regulated early during the course of glomerulonephritis (GN). We tested the effects of the two CCL5/RANTES blocking analogs, Met-RANTES and amino-oxypentane-RANTES, on the course of horse apoferritin (HAF)-induced GN. HAF-injected control mice had proliferative GN with mesangial immune complex deposits of IgG and HAF. Daily i.p. injections of Met-RANTES or amino-oxypentane-RANTES markedly reduced glomerular cell proliferation and glomerular macrophage infiltration, which is usually associated with less glomerular injury and proteinuria in HAF-GN. Surprisingly, however, HAF-GN mice treated with both analogs showed worse disease with mesangiolysis, capillary obstruction, and nephrotic range albuminuria. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state, characterized by a distinct morphology and increased inducible NO synthetase expression in vitro and in vivo, but a reduced uptake of apoptotic cells in vivo. The humoral response and the Th1/Th2 balance in HAF-GN and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs. We conclude that, despite blocking local leukocyte recruitment, chemokine analogs can aggravate some specific disease models, most likely due to interactions with systemic immune reactions, including the removal of apoptotic cells and inducible NO synthetase expression.     

Amino-terminally modified RANTES analogues demonstrate differential effects on RANTES receptors.

Modification of the amino terminus of regulated on activated normal T-cell expressed (RANTES) has been shown to have a significant effect on biological activity and produces proteins with antagonist properties. Two amino-terminally modified RANTES proteins, Met-RANTES and aminooxypentane-RANTES (AOP-RANTES), exhibit differential inhibitory properties on both monocyte and eosinophil chemotaxis. We have investigated their binding properties as well as their ability to activate the RANTES receptors CCR1, CCR3, and CCR5 in cell lines overexpressing these receptors. We show that Met-RANTES has weak activity in eliciting a calcium response in Chinese hamster ovary cells expressing CCR1, CCR3, and CCR5, whereas AOP-RANTES has full agonist activity on CCR5 but is less effective on CCR3 and CCR1. Their ability to induce chemotaxis of the murine pre-B lymphoma cell line, L1.2, transfected with the same receptors, consolidates these results. Monocytes have detectable mRNA for CCR1, CCR2, CCR3, CCR4, and CCR5, and they respond to the ligands for these receptors in chemotaxis but not always in calcium mobilization. AOP-RANTES does not induce calcium mobilization in circulating monocytes but is able to do so as these cells acquire the macrophage phenotype, which coincides with a concomitant up-regulation of CCR5. We have also tested the ability of both modified proteins to induce chemotaxis of freshly isolated monocytes and eosinophils. Cells from most donors do not respond, but occasionally cells from a particular donor do respond, particularly to AOP-RANTES. We therefore hypothesize that the occasional activity of AOP-RANTES to induce leukocyte chemotaxis is due to donor to donor variation of receptor expression.     

Expression and regulation of CCR1 by airway smooth muscle cells in asthma

C-C chemokines such as CCL11, CCL5, and CCL3 are central mediators in the pathogenesis of asthma. They are mainly associated with the recruitment and the activation of specific inflammatory cells, such as eosinophils, lymphocytes, and neutrophils. It has recently been shown that they can also activate structural cells, such as airway smooth muscle and epithelial cells. The aims of this study were to examine the expression of the CCL3 receptor, CCR1, on human airway smooth muscle cells (ASMC) and to document the regulation of this receptor by cytokines involved in asthma pathogenesis. We first demonstrated that CCR1 mRNA is increased in the airways of asthmatic vs control subjects and showed for the first time that ASMC express CCR1 mRNA and protein, both in vitro and in vivo. Calcium mobilization by CCR1 ligands confirmed its functionality on ASMC. Stimulation of ASMC with TNF-alpha and, to a lesser extent, IFN-gamma resulted in an up-regulation of CCR1 expression, which was totally suppressed by both dexamethasone or mithramycin. Taken together, our data suggest that CCR1 might be involved in the pathogenesis of asthma, through the activation of ASMC by its ligands.     

Deletion of CCR1 attenuates pathophysiologic responses during respiratory syncytial virus infection

The role of chemokines in chronic inflammatory responses are central to the recruitment of particular subsets of leukocytes. In the present studies, we have examined the role of CCR1 in the developing pathogenesis of respiratory syncytial virus (RSV) in the lungs of infected BALB/c mice. Although we did not observe significant differences in clearance of RSV, we were able to identify decreased pathophysiologic responses in CCR1(-/-) mice. CCR1(-/-) mice displayed a significant reduction in both airway hyperresponsiveness and mucus production that corresponded to significant increases in IFN-gamma and CXCL10. The goblet cell hyper/metaplasia and the expression of mucus-associated gene, gob5, were correspondingly reduced in the CCR1(-/-) mice. In addition, the Western blot analysis of gob5 protein indicated that CCR1(-/-) mice have virtually no up-regulation of the protein at day 6 of infection compared with wild-type-infected mice. Results from bone marrow chimeric mice indicated that partial reconstitution of the response could be achieved in the CCR1(-/-) mice with wild-type bone marrow cells, suggesting that these cells have a role in the response. However, transplanting of CCR1(-/-) bone marrow into wild-type mice did demonstrate an incomplete deficit in RSV-induced responses, indicating that CCR1(+) parenchymal cells may also play a significant role in the process. Thus, the presence of CCR1 appears to have a significant role in the development of detrimental airway physiologic responses during RSV infection. These data suggest that CCR1 may be a potential target during detrimental pulmonary responses during infection.     

Double-stranded RNA stimulation or CD40 ligation of monocyte-derived dendritic cells as models to study their activation and maturation process

Monocyte-derived dendritic cells (DCs) were used as an in vitro model of myeloid DCs in order to determine a minimum marker pattern with which to characterize and distinguish different stages of DC activation and maturation. Phenotypic changes induced on immature DCs by two prototypic stimuli, poly I:C and CD40 ligation, were first examined. Both elicited HLA-DR, CD40, CD86 and CXCR4 upregulation, and CCR5 downregulation, but only CD40 ligand-stimulated DCs became CD83(+)\CCR7(+), whereas poly I:C-stimulated DCs expressed lower CD83 levels and were mostly CCR7(--). CD40 ligation and poly I:C elicited increased production of inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha, of IL-10 and the CCL5 chemokine, but profiles differed as to higher IL-10, IL-12 and CCL22 (a CCR4 ligand important for T cell recruitment) levels for the former, and of CCL4 and CCL5 for the latter. Thus, a limited set of phenotypic markers, cytokine and chemokine production assays, may be used to distinguish the three stages in the life of DCs: immaturity, activation and full maturation. The ability of purified protein derivative-loaded DCs to stimulate autologous T cells to produce IL-2, IL-4 and interferon-gamma indeed depended on their activation stage and endocytic activity, which decreased upon maturation. We then examined whether ligation of CD4, CCR5 and\or CXCR4, the receptor and coreceptors of human immunodeficiency virus envelope gp120, respectively, affected DC activation or maturation, neither a monoclonal antibody to the gp120-binding site on CD4 nor CCL5 nor CXCL12, the natural ligands of CCR5 and CXCR4, respectively, nor gp120 altered the DC activation and maturation processes.     

Role of CCL5 (RANTES) in viral lung disease

CCL5/RANTES is a key proinflammatory chemokine produced by virus-infected epithelial cells and present in respiratory secretions of asthmatics. To examine the role of CCL5 in viral lung disease, we measured its production during primary respiratory syncytial virus (RSV) infection and during secondary infection after sensitizing vaccination that induces Th2-mediated eosinophilia. A first peak of CCL5 mRNA and protein production was seen at 18 to 24 h of RSV infection, before significant lymphocyte recruitment occurred. Treatment in vivo with Met-RANTES (a competitive chemokine receptor blocker) throughout primary infection decreased CD4+ and CD8+ cell recruitment and increased viral replication. In RSV-infected, sensitized mice with eosinophilic disease, CCL5 production was further augmented; Met-RANTES treatment again reduced inflammatory cell recruitment and local cytokine production. A second wave of CCL5 production occurred on day 7, attributable to newly recruited T cells. Paradoxically, mice treated with Met-RANTES during primary infection demonstrated increased cellular infiltration during reinfection. We therefore show that RSV induces CCL5 production in the lung and this causes the recruitment of RSV-specific cells, including those making additional CCL5. If this action is blocked with Met-RANTES, inflammation decreases and viral clearance is delayed. However, the exact effects of chemokine modulation depend critically on time of administration, a factor that may potentially complicate the use of chemokine blockers in inflammatory diseases.     

Regulatory T cells control dendritic cell/NK cell cross-talk in lymph nodes at the steady state by inhibiting CD4+ self-reactive T cells

The CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) play an important role in the control of peripheral tolerance by directly inhibiting conventional T cell proliferative and effector functions. However, the mechanisms by which Treg regulate the homeostasis of lymph nodes remain unclear. In this study, we show in a mouse model that Treg control two major checkpoints dictated by the interaction between self-reactive CD4(+) T cells and resident dendritic cell (DC) in secondary lymphoid organs. First, Treg inhibit the production of CCR5 ligands, limiting the CCR5-dependent recruitment of DC in the lymph nodes. Second, Treg prevent the DC exposure of IL-15Ralpha, markedly interfering in the DC-mediated NK cell proliferation in vivo. Therefore, the DC/T cell autoreactivity leading to NK cell triggering could potentially be controlled by the coinhibition of both IL-15Ralpha and CCR5 in autoimmune disorders in which NK cells play a deleterious role.     

Met-RANTES reduces vascular and tubular damage during acute renal transplant rejection: blocking monocyte arrest and recruitment.

Chemokines are thought to contribute to the cellular infiltrate characteristic of renal transplant rejection. We show that Met-RANTES, a chemokine receptor antagonist, suppresses recruitment of inflammatory cells into renal allografts. In a renal transplant model (Fisher RT1(lvl) rat kidney into Lewis RT1(l) rat) where no additional immune suppressant was used, Met-RANTES-treated animals showed a significant reduction in vascular injury score (16.10 +/- 5.20 vs. 62.67 +/- 18.64) and tubular damage score (15.70 +/- 5.22 vs. 33.00 +/- 6.44) relative to untreated animals. In a more severe rejection model (Brown-Norway RT1(n) rat kidney into Lewis RT1(1) rat), Met-RANTES significantly augmented low-dose cyclosporin A treatment to reduce all aspects of renal injury including interstitial inflammation (score 71.00 +/- 6.10 vs. 157.30 +/- 21.30). The majority of infiltrating cells in these models (60-70%) consisted of monocytes. Potential mechanisms of action of Met-RANTES were tested using monocyte attachment assays on microvascular endothelium under physiological flow conditions. Preexposure of microvascular endothelium to RANTES resulted in RANTES immobilization and RANTES-induced firm adhesion of monocytes only after prestimulation of the endothelium with IL-1beta. Met-RANTES completely inhibited this RANTES-mediated arrest. Thus, Met-RANTES may counter acute rejection by blocking leukocyte firm adhesion to inflamed endothelium.     

Chemokine receptor CCR2 but not CCR5 or CCR6 mediates the increase in pulmonary dendritic cells during allergic airway inflammation

Increased numbers of pulmonary dendritic cells (DCs) are recruited to the lungs during allergic airway inflammation and contribute to the maintenance of the inflammatory immune response. The chemokine receptors that directly control DC accumulation into the lungs are largely unknown. To explore this issue, we generated mixed bone marrow chimeric mice containing both wild-type and knockout cells for a given chemokine receptor. After induction of allergic airway inflammation, we specifically tracked and compared chemokine receptor knockout vs wild-type DC populations through various lung compartments. Using this approach, we show that CCR2, but not CCR5 or CCR6, directly controls the accumulation of DCs into allergic lungs. Furthermore, the size of inflammatory monocyte populations in peripheral blood was strikingly CCR2 dependent, suggesting that CCR2 primarily mediates the release of monocytic DC precursors into the bloodstream.