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1.   De novo isolation of antibodies with pH-dependent binding properties 
mAbs  2015;7(2):294-302.
pH-dependent antibodies are engineered to release their target at a slightly acidic pH, a property making them suitable for clinical as well as biotechnological applications. Such antibodies were previously obtained by histidine scanning of pre-existing antibodies, a labor-intensive strategy resulting in antibodies that displayed residual binding to their target at pH 6.0. We report here the de novo isolation of pH-dependent antibodies selected by phage display from libraries enriched in histidines. Strongly pH-dependent clones with various affinity profiles against CXCL10 were isolated by this method. Our best candidate has nanomolar affinity for CXCL10 at pH 7.2, but no residual binding was detected at pH 6.0. We therefore propose that this new process is an efficient strategy to generate pH-dependent antibodies.
PMCID: PMC4623423  PMID: 25608219
antibody recycling; chemokine; histidine; monoclonal antibody; pH-dependency; phage display; phage libraries
2.  Chemokines and Glycosaminoglycans 
PMCID: PMC4443743  PMID: 26074917
chemokines; glycosamininoglycans; RANTES; GAG mutants; oligomerization
3.  Evasin-4, a tick-derived chemokine-binding protein with broad selectivity can be modified for use in preclinical disease models 
The Febs Journal  2013;280(19):4876-4887.
Rhipicephalus sanguineus, the common brown dog tick, produces several chemokine-binding proteins which are secreted into the host in its saliva to modulate the host response during feeding. Two of these demonstrate very restricted selectivity profiles. Here, we describe the characterization of the third, which we named Evasin-4. Evasin-4 was difficult to produce recombinantly using its native signal peptide in HEK cells, but expressed very well using the urokinase-type plasminogen activator signal peptide. Using SPR, Evasin-4 was shown to bind most CC chemokines. Investigation of the neutralization properties by inhibition of chemokine-induced chemotaxis showed that binding and neutralization did not correlate in all cases. Two major anomalies were observed: no binding was observed to CCL2 and CCL13, yet Evasin-4 was able to inhibit chemotaxis induced by these chemokines. Conversely, binding to CCL25 was observed, but Evasin-4 did not inhibit CCL25-induced chemotaxis. Size-exclusion chromatography confirmed that Evasin-4 forms a complex with CCL2 and CCL18. In accordance with the standard properties of unmodified small proteins, Evasin-4 was rapidly cleared following in vivo administration. To enhance the in vivo half-life and optimize its potential as a therapeutic agent, Fc fusions of Evasin-4 were created. Both the N- and C-terminal fusions were shown to retain binding activity, with the C-terminal fusion showing a modest reduction in potency.
PMCID: PMC4240464  PMID: 23910450
anti-inflammatory; binding protein; Fc fusion; hemokine; tick
4.  Chemokine cooperativity is caused by competitive glycosaminoglycan binding 
Chemokines comprise a family of secreted proteins that activate G protein-coupled chemokine receptors and thereby control the migration of leukocytes during inflammation or immune surveillance. The positional information required for such migratory behavior is governed by the binding of chemokines to membrane-tethered glycosaminoglycans (GAGs), which establishes a chemokine concentration gradient. An often-observed but incompletely understood behavior of chemokines is the ability of unrelated chemokines to enhance the potency with which another chemokine subtype can activate its cognate receptor. This phenomenon has been demonstrated to occur between many chemokine combinations and across several model systems, and has been dubbed “chemokine cooperativity”. Here, we have used GAG binding-deficient chemokine mutants and cell-based functional (migration) assays to demonstrate that chemokine cooperativity is caused by competitive binding of chemokines to GAGs. This mechanistic explanation of chemokine cooperativity provides insight into chemokine gradient formation in the context of inflammation, where multiple chemokines are secreted simultaneously.
PMCID: PMC4198333  PMID: 24639348
5.  TSG-6 inhibits neutrophil migration via direct interaction with the chemokine CXCL8 
TNF-stimulated gene/protein-6 (TSG-6) is expressed by many different cell types in response to pro-inflammatory cytokines and plays an important role in the protection of tissues from the damaging consequences of acute inflammation. Recently, TSG-6 was identified as being largely responsible for the beneficial effects of multipotent mesenchymal stem cells, for example in the treatment of animal models of myocardial infarction and corneal injury/allogenic transplant. The protective effect of TSG-6 is due in part to its inhibition of neutrophil migration, but the mechanism(s) underlying this activity remain(s) unknown. Here we have shown that TSG-6 inhibits chemokine-stimulated trans-endothelial migration of neutrophils via a direct interaction (KD ~25 nM) between TSG-6 and the glycosaminoglycan-binding site of CXCL8, which antagonizes the association of CXCL8 with heparin. Furthermore, we found that TSG-6 impairs the binding of CXCL8 to cell surface glycosaminoglycans and the transport of CXCL8 across an endothelial cell monolayer. In vivo this could limit the formation of haptotactic gradients on endothelial heparan sulfate proteoglycans and, hence, integrin-mediated tight adhesion and migration. We further observed that TSG-6 suppresses CXCL8-mediated chemotaxis of neutrophils; this lower potency effect might be important at sites where there is high local expression of TSG-6. Thus, we have identified TSG-6 as a CXCL8-binding protein, making it the first soluble mammalian chemokine-binding protein to be described to date. We have also revealed a potential mechanism whereby TSG-6 mediates its anti-inflammatory and protective effects. This could inform the development of new treatments for inflammation in the context of disease or post transplantation.
PMCID: PMC3988464  PMID: 24501198
6.  Treatment with Evasin-3 reduces atherosclerotic vulnerability for ischemic stroke, but not brain injury in mice 
Neutrophilic inflammation might have a pathophysiological role in both carotid plaque rupture and ischemic stroke injury. Here, we investigated the potential benefits of the CXC chemokine-binding protein Evasin-3, which potently inhibits chemokine bioactivity and related neutrophilic inflammation in two mouse models of carotid atherosclerosis and ischemic stroke, respectively. In the first model, the chronic treatment with Evasin-3 as compared with Vehicle (phosphate-buffered saline (PBS)) was investigated in apolipoprotein E-deficient mice implanted of a ‘cast' carotid device. In the second model, acute Evasin-3 treatment (5 minutes after cerebral ischemia onset) was assessed in mice subjected to transient left middle cerebral artery occlusion. Although CXCL1 and CXCL2 were upregulated in both atherosclerotic plaques and infarcted brain, only CXCL1 was detectable in serum. In carotid atherosclerosis, treatment with Evasin-3 was associated with reduction in intraplaque neutrophil and matrix metalloproteinase-9 content and weak increase in collagen as compared with Vehicle. In ischemic stroke, treatment with Evasin-3 was associated with reduction in ischemic brain neutrophil infiltration and protective oxidants. No other effects in clinical and histological outcomes were observed. We concluded that Evasin-3 treatment was associated with reduction in neutrophilic inflammation in both mouse models. However, Evasin-3 administration after cerebral ischemia onset failed to improve poststroke outcomes.
PMCID: PMC3618389  PMID: 23250107
acute stroke; atherosclerosis; brain ischemia; carotid artery; inflammation
7.  CCL18 Exhibits a Regulatory Role through Inhibition of Receptor and Glycosaminoglycan Binding 
PLoS ONE  2013;8(8):e72321.
CCL18 has been reported to be present constitutively at high levels in the circulation, and is further elevated during inflammatory diseases. Since it is a rather poor chemoattractant, we wondered if it may have a regulatory role. CCL18 has been reported to inhibit cellular recruitment mediated by CCR3, and we have shown that whilst it is a competitive functional antagonist as assessed by Schild plot analysis, it only binds to a subset of CCR3 receptor populations. We have extended this inhibitory activity to other receptors and have shown that CCL18 is able to inhibit CCR1, CCR2, CCR4 and CCR5 mediated chemotaxis, but has no effect on CCR7 and CCR9, nor the CXC receptors that we have tested. Whilst CCL18 is able to bind to CCR3, it does not bind to the other receptors that it inhibits. We therefore tested the hypothesis that it may displace glycosaminoglycan (GAG) chemokines bound either in cis- on the leukocyte, or in trans-presentation on the endothelial surface, thereby inhibiting the recruitment of leukocytes into the site of inflammation. We show that CCL18 selectivity displaces heparin bound chemokines, and that chemokines from all four chemokine sub-classes displace cell bound CCL18. We propose that CCL18 has regulatory properties inhibiting chemokine function when GAG-mediated presentation plays a role in receptor activation.
PMCID: PMC3741163  PMID: 23951310
8.  The Activity of CCL18 is Principally Mediated through Interaction with Glycosaminoglycans 
The CC chemokine ligand 18 (CCL18) was first identified as a chemoattractant for naïve T cells. It has been reported to recruit T and B lymphocytes, and we show here, natural killer (NK) cells, but with low efficacy. Investigation of its ability to elicit G-protein-coupled signaling showed that it does not involve extracellular signal-regulated kinase (ERK) phosphorylation, and it is not able to induce receptor internalization, as assessed on CCR3. CCL18 has recently been reported to possess activities unrelated to cellular recruitment, but it had no effect on T lymphocyte proliferation. We postulated that a more potent chemoattractant may be produced under inflammatory conditions but only minor truncations were observed, with the major form being the full-length protein. In view of the lack of potent immunomodulatory properties, we wondered if binding to CCL18 by the tick chemokine binding proteins Evasin-1 and -4 was an artifact of the methods used, but complex formation was confirmed by size exclusion chromatography, and abrogation of its binding to, and antagonism of, CCR3. Its receptor has remained elusive since its cloning in 1997, although it has been reported to induce migration of breast cancer cells by signaling through PITPNM3, but we show that this receptor is not expressed on lymphocytes. We have developed a radiolabeled equilibrium competition binding assay and demonstrated that it bound with high affinity to peripheral blood leukocytes (PBLs), but the binding was displaced similarly by both unlabelled CCL18 as well as heparin. Both heparin binding and binding to PBLs are considerably abrogated by mutation of the BBXB motif in the 40s loop suggesting an essential role of the CCL18-glycosaminoglycan interaction.
PMCID: PMC3711072  PMID: 23874339
chemokines; CCL18; synovial fluid; glycosaminoglycan; BBXB motif; Evasin
9.  HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4 
CXCL12 forms a complex with HMGB1 that binds to the chemokine receptor CXCR4 and increases inflammatory cell migration.
After tissue damage, inflammatory cells infiltrate the tissue and release proinflammatory cytokines. HMGB1 (high mobility group box 1), a nuclear protein released by necrotic and severely stressed cells, promotes cytokine release via its interaction with the TLR4 (Toll-like receptor 4) receptor and cell migration via an unknown mechanism. We show that HMGB1-induced recruitment of inflammatory cells depends on CXCL12. HMGB1 and CXCL12 form a heterocomplex, which we characterized by nuclear magnetic resonance and surface plasmon resonance, that acts exclusively through CXCR4 and not through other HMGB1 receptors. Fluorescence resonance energy transfer data show that the HMGB1–CXCL12 heterocomplex promotes different conformational rearrangements of CXCR4 from that of CXCL12 alone. Mononuclear cell recruitment in vivo into air pouches and injured muscles depends on the heterocomplex and is inhibited by AMD3100 and glycyrrhizin. Thus, inflammatory cell recruitment and activation both depend on HMGB1 via different mechanisms.
PMCID: PMC3302219  PMID: 22370717
10.  Interference with Oligomerization and Glycosaminoglycan Binding of the Chemokine CCL5 Improves Experimental Liver Injury 
PLoS ONE  2012;7(5):e36614.
The chemokine CCL5 is involved in the recruitment of immune cells and a subsequent activation of hepatic stellate cells (HSC) after liver injury. We here investigate whether inhibition of CCL5 oligomerization and glycosaminoglycan binding by a mutated CCL5 protein (44AANA47-CCL5) has the potential to ameliorate liver cell injury and fibrosis in vivo.
Liver injury was induced in C57BL/6 mice by intraperitoneal injection of carbon tetrachloride (CCl4) in an acute and a chronic liver injury model. Simultaneously, mice received either 44AANA47-CCL5 or vehicle. Liver cell necrosis and fibrosis was analyzed by histology, and measurement of serum transaminases and hydroxyproline. Intrahepatic mRNA expression of fibrosis and inflammation related genes were determined by quantitative RT-PCR and infiltration of immune cells was assessed by FACS analysis and immunocytochemistry. In vitro, HSC were stimulated with conditioned media of T-cell enriched splenocytes.
Principal Findings
44AANA47-CCL5 treated mice displayed a significantly reduced degree of acute liver injury (liver cell necrosis, transaminases) and fibrosis (Sirus red positive area and hydroxyproline content) compared to vehicle treated mice. Ameliorated fibrosis by 44AANA47-CCL5 was associated with a decreased expression of fibrosis related genes, decreased α-smoth muscle antigen (αSMA) and a reduction of infiltrating immune cells. In the acute model, 44AANA47-CCL5 treated mice displayed a reduced immune cell infiltration and mRNA levels of TNF, IL-1 and CCL3 compared to vehicle treated mice. In vitro, conditioned medium of T-cell enriched splenocytes of 44AANA47-CCL5 treated mice inhibited the chemotaxis and proliferation of HSC.
The results provide evidence that inhibition of oligomerization and glycosaminoglycan binding of the chemokine CCL5 is a new therapeutic strategy for the treatment of acute and chronic liver injuries and represents an alternative to chemokine receptor antagonism.
PMCID: PMC3344902  PMID: 22574195
11.  Glycosaminoglycan analogs as a novel anti-inflammatory strategy 
Heparin, a glycosaminoglycan (GAG), has both anti-inflammatory and anti-coagulant properties. The clinical use of heparin against inflammation, however, has been limited by concerns about increased bleeding. While the anti-coagulant activity of heparin is well understood, its anti-inflammatory properties are less so. Heparin is known to bind to certain cytokines, including chemokines, small proteins which mediate inflammation through their control of leukocyte migration and activation. Molecules which can interrupt the chemokine-GAG interaction without inhibiting coagulation could therefore, represent a new class of anti-inflammatory agents. In the present study, two approaches were undertaken, both focusing on the heparin-chemokine relationship. In the first, a structure based strategy was used: after an initial screening of potential small molecule binders using protein NMR on a target chemokine, binding molecules were optimized through structure-based design. In the second approach, commercially available short oligosaccharides were polysulfated. In vitro, these molecules prevented chemokine-GAG binding and chemokine receptor activation without disrupting coagulation. However, in vivo, these compounds caused variable results in a murine peritoneal recruitment assay, with a general increase of cell recruitment. In more disease specific models, such as antigen-induced arthritis and delayed-type hypersensitivity, an overall decrease in inflammation was noted, suggesting that the primary anti-inflammatory effect may also involve factors beyond the chemokine system.
PMCID: PMC3472544  PMID: 23087686
chemokine; chemokine antagonist; glycosaminoglycans; anti-inflammatory; NMR
12.  Antagonism of the chemokine Ccl5 ameliorates experimental liver fibrosis in mice 
The Journal of Clinical Investigation  2010;120(11):4129-4140.
Activation of hepatic stellate cells in response to chronic inflammation represents a crucial step in the development of liver fibrosis. However, the molecules involved in the interaction between immune cells and stellate cells remain obscure. Herein, we identify the chemokine CCL5 (also known as RANTES), which is induced in murine and human liver after injury, as a central mediator of this interaction. First, we showed in patients with liver fibrosis that CCL5 haplotypes and intrahepatic CCL5 mRNA expression were associated with severe liver fibrosis. Consistent with this, we detected Ccl5 mRNA and CCL5 protein in 2 mouse models of liver fibrosis, induced by either injection of carbon tetrachloride (CCl4) or feeding on a methionine and choline–deficient (MCD) diet. In these models, Ccl5–/– mice exhibited decreased hepatic fibrosis, with reduced stellate cell activation and immune cell infiltration. Transplantation of Ccl5-deficient bone marrow into WT recipients attenuated liver fibrosis, identifying infiltrating hematopoietic cells as the main source of Ccl5. We then showed that treatment with the CCL5 receptor antagonist Met-CCL5 inhibited cultured stellate cell migration, proliferation, and chemokine and collagen secretion. Importantly, in vivo administration of Met-CCL5 greatly ameliorated liver fibrosis in mice and was able to accelerate fibrosis regression. Our results define a successful therapeutic approach to reduce experimental liver fibrosis by antagonizing Ccl5 receptors.
PMCID: PMC2964968  PMID: 20978355
13.  Viral macrophage inflammatory protein-II improves acute rejection in allogeneic rat kidney transplants 
World Journal of Urology  2010;28(4):537-542.
During rejection, leukocytes are recruited from the peripheral circulation into the graft leading to the damage of endothelial cells, capillary perfusion failure and graft loss. Chemokines play a pivotal role in the recruitment of leukocytes to the endothelium. Viral macrophage inflammatory protein-II (vMIP-II), a human herpes virus-8 DNA-encoded protein, is a broad-spectrum chemokine antagonist. The aim of the study was to prove the beneficial activity of vMIP-II treatment on acute rat kidney allograft damage.
Heterotopic rat kidney transplantation was performed in the Fischer 344 to Lewis transplantation model and animals were treated with vMIP-II (2 × 15 µg or 100 µg/day) for 7 days. Rejection-induced damage was analyzed by histology, and microcirculatory changes within the graft were analyzed by in vivo microscopy.
Viral macrophage inflammatory protein-II significantly improved acute glomerular damage and tubulointerstitial inflammation and lowered the extent of vascular and tubulointerstitial damage of the treated allografts. Functional microcirculation of peritubular capillaries was significantly improved in vivo, and the firm adherence of leukocytes was significantly reduced by vMIP-II treatment.
The administration of the broad-spectrum antagonist vMIP-II improved acute renal allograft damage, mainly by a reduction in leukocyte recruitment with a subsequently improved renal cortical microcirculation in vivo.
PMCID: PMC2908759  PMID: 20401660
Chemokine; Virokine; vMIP-II; Inflammation; Allograft rejection
14.  Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice 
PLoS Pathogens  2010;6(2):e1000755.
Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3−/− mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.
Author Summary
When infectious agents enter our body, neutrophils are the first cells recruited to the scene. In addition to their capacity to kill microbes, neutrophils secrete molecules that attract other cells also involved in immune defense, such as dendritic cells (DCs). Here, we investigate the secretion of DC-attracting chemokines by neutrophils after inoculation of mice with Leishmania major, a protozoan parasite that can cause long-lasting, skin ulcers in man. Following parasite inoculation, most inbred strains of mice (e.g.C57BL/6) develop self-resolving lesions, while in a few strains (e.g. BALB/c) lesions fail to heal. We report here that in “healer” C57BL/6 mice, higher numbers of DCs were attracted at the site of infection than in “non-healer” BALB/c mice. DC recruitment correlated with secretion by neutrophils of the chemokine CCL3, as indeed a markedly decreased DC recruitment was observed in C57BL/6 mice depleted of neutrophils or deprived of the capacity to produce CCL3. DC recruitment was restored upon transfer of normal neutrophils to CCL3 deficient mice. Our results reveal a crucial role for neutrophil-secreted CCL3 in early recruitment of DCs in L. major-infected “healer” mice, and suggest that the type of chemokine secreted by neutrophils will have consequences in the launching of pathogen-specific immune response.
PMCID: PMC2816696  PMID: 20140197
15.  Structural Basis of Chemokine Sequestration by a Tick Chemokine Binding Protein: The Crystal Structure of the Complex between Evasin-1 and CCL3 
PLoS ONE  2009;4(12):e8514.
Chemokines are a subset of cytokines responsible for controlling the cellular migration of inflammatory cells through interaction with seven transmembrane G protein-coupled receptors. The blocking of a chemokine-receptor interaction results in a reduced inflammatory response, and represents a possible anti-inflammatory strategy, a strategy that is already employed by some virus and parasites. Anti-chemokine activity has been described in the extracts of tick salivary glands, and we have recently described the cloning and characterization of such chemokine binding proteins from the salivary glands, which we have named Evasins.
Methodology/Principal Findings
We have solved the structure of Evasin-1, a very small and highly selective chemokine-binding protein, by x-ray crystallography and report that the structure is novel, with no obvious similarity to the previously described structures of viral chemokine binding proteins. Moreover it does not possess a known fold. We have also solved the structure of the complex of Evasin-1 and its high affinity ligand, CCL3. The complex is a 1∶1 heterodimer in which the N-terminal region of CCL3 forms numerous contacts with Evasin-1, including prominent π-π interactions between residues Trp89 and Phe14 of the binding protein and Phe29 and Phe13 of the chemokine.
However, these interactions do not appear to be crucial for the selectivity of the binding protein, since these residues are found in CCL5, which is not a ligand for Evasin-1. The selectivity of the interaction would appear to lie in the N-terminal residues of the chemokine, which form the “address” whereas the hydrophobic interactions in the rest of the complex would serve primarily to stabilize the complex. A thorough understanding of the binding mode of this small protein, and its other family members, could be very informative in the design of potent neutralizing molecules of pro-inflammatory mediators of the immune system, such as chemokines.
PMCID: PMC2796168  PMID: 20041127
16.  Ticks produce highly selective chemokine binding proteins with antiinflammatory activity 
The Journal of Experimental Medicine  2008;205(9):2019-2031.
Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126–1136), and may be therapeutically useful as novel antiinflammatory agents in the future.
PMCID: PMC2526197  PMID: 18678732
17.  Amelioration of Rat Adjuvant-Induced Arthritis by Met-RANTES 
Arthritis and rheumatism  2005;52(6):1907-1919.
CC chemokines and their receptors play a fundamental role in trafficking and activation of leukocytes at sites of inflammation, contributing to joint damage in rheumatoid arthritis. Met-RANTES, an amino-terminal–modified methionylated form of RANTES (CCL5), antagonizes the binding of the chemokines RANTES and macrophage inflammatory protein 1α (MIP-1α; CCL3) to their receptors CCR1 and CCR5, respectively. The aim of this study was to investigate whether Met-RANTES could ameliorate adjuvant-induced arthritis (AIA) in the rat.
Using immunohistochemistry, enzyme-linked immunosorbent assay, real-time reverse transcription–polymerase chain reaction, Western blot analysis, adoptive transfer, and chemotaxis, we defined joint inflammation, bony destruction, neutrophil and macrophage migration, Met-RANTES binding affinity to rat receptors, proinflammatory cytokine and bone marker levels, CCR1 and CCR5 expression and activation, and macrophage homing into joints with AIA.
Administration of Met-RANTES as a preventative reduced the severity of joint inflammation. Administration of Met-RANTES to ankles with AIA showed decreases in inflammation, radiographic soft tissue swelling, and bone erosion. Met-RANTES significantly reduced the number of neutrophils and macrophages at the peak of arthritis compared with saline-injected controls. Competitive chemotaxis in peripheral blood mononuclear cells demonstrated that Met-RANTES inhibited MIP-1α and MIP-1β at 50% inhibition concentrations of 5 nM and 2 nM, respectively. Furthermore, levels of tumor necrosis factor α, interleukin-1β, macrophage colony-stimulating factor, and RANKL were decreased in joints with AIA in the Met-RANTES group compared with the control group. Interestingly, the expression and activation of CCR1 and CCR5 in the joint were down-regulated in the Met-RANTES group compared with the control group. Functionally, Met-RANTES administration decreased adoptively transferred peritoneal macrophage homing into the joint.
The data suggest that the targeting of Th1-associated chemokine receptors reduce joint inflammation, bone destruction, and cell recruitment into joints with AIA.
PMCID: PMC1282452  PMID: 15934086
18.  CC Chemokine Receptor (CCR)4 and the CCR10 Ligand Cutaneous T Cell–attracting Chemokine (CTACK) in Lymphocyte Trafficking to Inflamed Skin 
The Journal of Experimental Medicine  2001;194(10):1541-1547.
The chemokine thymus and activation-regulated chemokine (TARC; CCL17) is displayed by cutaneous (but not intestinal) venules, and is thought to trigger vascular arrest of circulating skin homing memory T cells, which uniformly express the TARC receptor CC chemokine receptor (CCR)4. Cutaneous T cell–attracting chemokine (CTACK; CCL27), expressed by skin keratinocytes, also attracts cutaneous memory T cells, and is hypothesized to assist in lymphocyte recruitment to skin as well. Here we show that chronic cutaneous inflammation induces CD4 T cells expressing E-selectin binding activity (a marker of skin homing memory cells) in draining lymph node, and that these E-selectin ligand+ T cells migrate efficiently to TARC and to CTACK. In 24 h in vivo homing assays, stimulated lymph node T cells from wild-type mice or, surprisingly, from CCR4-deficient donors migrate efficiently to inflamed skin; and an inhibitory anti-CTACK antibody has no effect on wild-type lymphocyte recruitment. However, inhibition with anti-CTACK monoclonal antibody abrogates skin recruitment of CCR4-deficient T cells. We conclude that CTACK and CCR4 can both support homing of T cells to skin, and that either one or the other is required for lymphocyte recruitment in cutaneous delayed type hypersensitivity.
PMCID: PMC2193675  PMID: 11714760
lymphocyte homing; chemokines; inflammation; delayed type hypersensitivity; selectins
19.  Interaction of the CC-Chemokine RANTES with Glycosaminoglycans Activates a p44/p42 Mitogen-Activated Protein Kinase-Dependent Signaling Pathway and Enhances Human Immunodeficiency Virus Type 1 Infectivity 
Journal of Virology  2002;76(5):2245-2254.
The interaction of the CC-chemokine RANTES with its cell surface receptors transduces multiple intracellular signals: low concentrations of RANTES (1 to 10 nM) stimulate G-protein-coupled receptor (GPCR) activity, and higher concentrations (1 μM) activate a phosphotyrosine kinase (PTK)-dependent pathway. Here, we show that the higher RANTES concentrations induce rapid tyrosine phosphorylation of multiple proteins. Several src-family kinases (Fyn, Hck, Src) are activated, as is the focal adhesion kinase p125 FAK and, eventually, members of the p44/p42 mitogen-activated protein kinase (MAPK) family. This PTK signaling pathway can be activated independently of known seven-transmembrane GPCRs for RANTES because it occurs in cells that lack any such RANTES receptors. Instead, activation of the PTK signaling pathway is dependent on the expression of glycosaminoglycans (GAGs) on the cell surface, in that it could not be activated by RANTES in GAG-deficient cells. We have previously demonstrated that RANTES can both enhance and inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). Here we show that activation of both PTK and MAPK is involved in the enhancement of HIV-1 infectivity caused by RANTES in cells that lack GPCRs for RANTES but which express GAGs.
PMCID: PMC135942  PMID: 11836402
20.  Endocytosis and Recycling of the HIV Coreceptor Ccr5 
The Journal of Cell Biology  2000;151(6):1281-1294.
The chemokine receptor CCR5 is a cofactor for the entry of R5 tropic strains of human immunodeficiency viruses (HIV)-1 and -2 and simian immunodeficiency virus. Cells susceptible to infection by these viruses can be protected by treatment with the CCR5 ligands regulated on activation, normal T cell expressed and secreted (RANTES), MIP-1α, and MIP-1β. A major component of the mechanism through which chemokines protect cells from HIV infection is by inducing endocytosis of the chemokine receptor. Aminooxypentane (AOP)-RANTES, an NH2-terminal modified form of RANTES, is a potent inhibitor of infection by R5 HIV strains. AOP-RANTES efficiently downmodulates the cell surface expression of CCR5 and, in contrast with RANTES, appears to prevent recycling of CCR5 to the cell surface. Here, we investigate the cellular basis of this effect.
Using CHO cells expressing human CCR5, we show that both RANTES and AOP-RANTES induce rapid internalization of CCR5. In the absence of ligand, CCR5 shows constitutive turnover with a half-time of 6–9 h. Addition of RANTES or AOP-RANTES has little effect on the rate of CCR5 turnover. Immunofluorescence and immunoelectron microscopy show that most of the CCR5 internalized after RANTES or AOP-RANTES treatment accumulates in small membrane-bound vesicles and tubules clustered in the perinuclear region of the cell. Colocalization with transferrin receptors in the same clusters of vesicles indicates that CCR5 accumulates in recycling endosomes. After the removal of RANTES, internalized CCR5 recycles to the cell surface and is sensitive to further rounds of RANTES-induced endocytosis. In contrast, after the removal of AOP-RANTES, most CCR5 remains intracellular. We show that these CCR5 molecules do recycle to the cell surface, with kinetics equivalent to those of receptors in RANTES-treated cells. However, these recycled CCR5 molecules are rapidly reinternalized. Our results indicate that AOP-RANTES–induced changes in CCR5 alter the steady-state distribution of the receptor and provide the first evidence for G protein–coupled receptor trafficking through the recycling endosome compartment.
PMCID: PMC2190598  PMID: 11121442
chemokine receptor; endocytosis; CCR5; recycling endosome; HIV
21.  Lymph-Borne Chemokines and Other Low Molecular Weight Molecules Reach High Endothelial Venules via Specialized Conduits While a Functional Barrier Limits Access to the Lymphocyte Microenvironments in Lymph Node Cortex 
The Journal of Experimental Medicine  2000;192(10):1425-1440.
Lymph-borne, soluble factors (e.g., chemokines and others) influence lymphocyte recirculation and endothelial phenotype at high endothelial venules (HEVs) in lymph node cortex. Yet the route lymph-borne soluble molecules travel from the subcapsular sinus to the HEVs is unclear. Therefore, we injected subcutaneously into mice and rats a wide variety of fluorophore-labeled, soluble molecules and examined their distribution in the draining lymph nodes. Rather than percolating throughout the draining lymph node, all molecules, including microbial lipopolysaccharide, were very visible in the subcapsular and medullary sinuses but were largely excluded from the cortical lymphocyte microenvironments. Exclusion prevailed even during the acute lymph node enlargement accompanying viral infection. However, low molecular mass (MW) molecules, including chemokines, did gain entry into the cortex, but in a very defined manner. Low MW, fluorophore-labeled molecules highlighted the subcapsular sinus, the reticular fibers, and the abluminal and luminal surfaces of the associated HEVs. These low MW molecules were in the fibers of the reticular network, a meshwork of collagen fibers ensheathed by fibroblastic reticular cells that connects the subcapsular sinus floor and the HEVs by intertwining with their basement membranes. Thus, low MW, lymph-borne molecules, including chemokines, traveled rapidly from the subcapsular sinus to the HEVs using the reticular network as a conduit.
PMCID: PMC2193184  PMID: 11085745
reticular network; mouse; rat; lymphocyte recirculation; antigen
22.  A Key Role for Cc Chemokine Receptor 4 in Lipopolysaccharide-Induced Endotoxic Shock 
The Journal of Experimental Medicine  2000;191(10):1755-1764.
CC chemokine receptor (CCR)4, a high affinity receptor for the CC chemokines thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC), is expressed in the thymus and spleen, and also by peripheral blood T cells, macrophages, platelets, and basophils. Recent studies have shown that CCR4 is the major chemokine receptor expressed by T helper type 2 (Th2) polarized cells. To study the in vivo role of CCR4, we have generated CCR4-deficient (CCR4−/−) mice by gene targeting. CCR4−/− mice developed normally. Splenocytes and thymocytes isolated from the CCR4−/− mice failed to respond to the CCR4 ligands TARC and MDC, as expected, but also surprisingly did not undergo chemotaxis in vitro in response to macrophage inflammatory protein (MIP)-1α. The CCR4 deletion had no effect on Th2 differentiation in vitro or in a Th2-dependent model of allergic airway inflammation. However, CCR4−/− mice exhibited significantly decreased mortality on administration of high or low dose bacterial lipopolysaccharide (LPS) compared with CCR4+/+ mice. After high dose LPS treatment, serum levels of tumor necrosis factor α, interleukin 1β, and MIP-1α were reduced in CCR4−/− mice, and decreased expression of MDC and MIP-2 mRNA was detected in peritoneal exudate cells. Analysis of peritoneal lavage cells from CCR4−/− mice by flow cytometry also revealed a significant decrease in the F4/80+ cell population. This may reflect a defect in the ability of the CCR4−/− macrophages to be retained in the peritoneal cavity. Taken together, our data reveal an unexpected role for CCR4 in the inflammatory response leading to LPS-induced lethality.
PMCID: PMC2193157  PMID: 10811868
CC chemokine receptor 4; lipopolysaccharide; endotoxic shock; F4/80 antigen; T helper type 2 cells
23.  Similarities and Differences in RANTES- and (AOP)-RANTES–triggered Signals: Implications for Chemotaxis  
The Journal of Cell Biology  1999;144(4):755-765.
Chemokines are a family of proinflammatory cytokines that attract and activate specific types of leukocytes. Chemokines mediate their effects via interaction with seven transmembrane G protein–coupled receptors (GPCR). Using CCR5-transfected HEK-293 cells, we show that both the CCR5 ligand, RANTES, as well as its derivative, aminooxypentane (AOP)- RANTES, trigger immediate responses such as Ca2+ influx, receptor dimerization, tyrosine phosphorylation, and Gαi as well as JAK/STAT association to the receptor. In contrast to RANTES, (AOP)-RANTES is unable to trigger late responses, as measured by the association of focal adhesion kinase (FAK) to the chemokine receptor complex, impaired cell polarization required for migration, or chemotaxis. The results are discussed in the context of the dissociation of the late signals, provoked by the chemokines required for cell migration, from early signals.
PMCID: PMC2132943  PMID: 10037796
chemokines; receptor dimerization; inflammation; HIV-1
24.  The CC-Chemokine RANTES Increases the Attachment of Human Immunodeficiency Virus Type 1 to Target Cells via Glycosaminoglycans and Also Activates a Signal Transduction Pathway That Enhances Viral Infectivity 
Journal of Virology  1999;73(8):6370-6379.
We have studied the mechanisms by which the CC-chemokine RANTES can enhance the infectivities of human immunodeficiency virus type 1 (HIV-1) and other enveloped viruses, when present at concentrations in excess of 500 ng/ml in vitro. Understanding the underlying mechanisms might throw light on fundamental processes of viral infection, in particular for HIV-1. Our principal findings are twofold: firstly, that oligomers of RANTES can cross-link enveloped viruses, including HIV-1, to cells via glycosaminoglycans (GAGs) present on the membranes of both virions and cells; secondly, that oligomers of RANTES interact with cell-surface GAGs to transduce a herbimycin A-sensitive signal which, over a period of several hours, renders the cells more permissive to infection by several viruses, including HIV-1. The enhancement mechanisms require that RANTES oligomerize either in solution or following binding to GAGs, since no viral infectivity enhancement is observed with a mutant form of the RANTES molecule that contains a single-amino-acid change (glutamic acid to serine at position 66) which abrogates oligomerization.
PMCID: PMC112716  PMID: 10400729
25.  Enhancement of Human Immunodeficiency Virus Type 1 Infection by the CC-Chemokine RANTES Is Independent of the Mechanism of Virus-Cell Fusion 
Journal of Virology  1999;73(1):684-694.
We have studied the effects of CC-chemokines on human immunodeficiency virus type 1 (HIV-1) infection, focusing on the infectivity enhancement caused by RANTES. High RANTES concentrations increase the infectivity of HIV-1 isolates that use CXC-chemokine receptor 4 for entry. However, RANTES can have a similar enhancing effect on macrophagetropic viruses that enter via CC-chemokine receptor 5 (CCR5), despite binding to the same receptor as the virus. Furthermore, RANTES enhances the infectivity of HIV-1 pseudotyped with the envelope glycoprotein of murine leukemia virus or vesicular stomatitis virus, showing that the mechanism of enhancement is independent of the route of virus-cell fusion. The enhancing effects of RANTES are not mediated via CCR5 or other known chemokine receptors and are not mimicked by MIP-1α or MIP-1β. The N-terminally modified derivative aminooxypentane RANTES (AOP-RANTES) efficiently inhibits HIV-1 infection via CCR5 but otherwise mimics RANTES by enhancing viral infectivity. There are two mechanisms of enhancement: one apparent when target cells are pretreated with RANTES (or AOP-RANTES) for several hours, and the other apparent when RANTES (or AOP-RANTES) is added during virus-cell absorption. We believe that the first mechanism is related to cellular activation by RANTES, whereas the second is an increase in virion attachment to target cells.
PMCID: PMC103875  PMID: 9847374

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