A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation

Chemokines play a key role in leukocyte recruitment during inflammation and are implicated in the pathogenesis of a number of autoimmune diseases. As such, inhibiting chemokine signaling has been of keen interest for the development of therapeutic agents. This endeavor, however, has been hampered due to complexities in the chemokine system. Many chemokines have been shown to signal through multiple receptors and, conversely, most chemokine receptors bind to more than one chemokine. One approach to overcoming this complexity is to develop a single therapeutic agent that binds and inactivates multiple chemokines, similar to an immune evasion strategy utilized by a number of viruses. Here, we describe the development and characterization of a novel therapeutic antibody that targets a subset of human CC chemokines, specifically CCL3, CCL4, and CCL5, involved in chronic inflammatory diseases. Using a sequential immunization approach, followed by humanization and phage display affinity maturation, a therapeutic antibody was developed that displays high binding affinity towards the three targeted chemokines. In vitro, this antibody potently inhibits chemotaxis and chemokine-mediated signaling through CCR1 and CCR5, primary chemokine receptors for the targeted chemokines. Furthermore, we have demonstrated in vivo efficacy of the antibody in a SCID-hu mouse model of skin leukocyte migration, thus confirming its potential as a novel therapeutic chemokine antagonist. We anticipate that this antibody will have broad therapeutic utility in the treatment of a number of autoimmune diseases due to its ability to simultaneously neutralize multiple chemokines implicated in disease pathogenesis.     

Neutralization of the chemokine CXCL10 reduces inflammatory cell invasion and demyelination and improves neurological function in a viral model of multiple sclerosis

Intracerebral infection of mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a chronic demyelinating disease with clinical and histological similarities with the human demyelinating disease multiple sclerosis (MS). Following MHV infection, chemokines including CXC chemokine ligand (CXCL)10 (IFN inducible protein 10 kDa), CXCL9 (monokine induced by IFN-gamma), and CC chemokine ligand 5 (RANTES) are expressed during both acute and chronic stages of disease suggesting a role for these molecules in disease exacerbation. Previous studies have shown that during the acute phase of infection, T lymphocytes are recruited into the CNS by the chemokines CXCL10 and CXCL9. In the present study, MHV-infected mice with established demyelination were treated with antisera against these two chemokines, and disease severity was assessed. Treatment with anti-CXCL10 reduced CD4+ T lymphocyte and macrophage invasion, diminished expression of IFN-gamma and CC chemokine ligand 5, inhibited progression of demyelination, and increased remyelination. Anti-CXCL10 treatment also resulted in an impediment of clinical disease progression that was characterized by a dramatic improvement in neurological function. Treatment with antisera against CXCL9 was without effect, demonstrating a critical role for CXCL10 in inflammatory demyelination in this model. These findings document a novel therapeutic strategy using Ab-mediated neutralization of a key chemokine as a possible treatment for chronic human inflammatory demyelinating diseases such as MS.     

CC类趋化因子亚家族与心肌梗死的研究进展

CC类趋化因子亚家族是趋化因子家族中成员最多、研究最广泛的一大类细胞因子,其主要功能参与炎症细胞激活、迁移、粘附等病理生理过程。大量研究表明,CC类趋化因子亚家族成员参与了心肌梗死后病理过程的各个阶段。其中研究最为深入的为单核细胞趋化蛋白-1（monocyte chemoattractant protein-1,MCP-1）及其受体CC趋化因子受体2（CC chemokine receptor 2,CCR2）,在心肌梗死后炎症期、增殖期及疤痕愈合期都发挥了重要作用从而影响梗死后心室重构。近年来,CC类趋化因子亚家族其他成员亦被逐渐揭示参与了心肌梗死的发展。本文结合以往大量文献将对CC类趋化因子亚家族在心肌梗死各个阶段中尤其是梗死后各期对于心室重构的影响进行综述,以期为今后的实验研究提供方向及疾病的预防和治疗提供药物靶点。     

Angiogenesis and chemokines in rheumatoid arthritis and other systemic inflammatory rheumatic diseases

Angiogenesis, the formation of new vessels, is important in the pathogenesis of rheumatoid arthritis (RA) and other inflammatory diseases. Chemotactic cytokines termed chemokines mediate the ingress of leukocytes, including neutrophils and monocytes into the inflamed synovium. In this review, authors discuss the role of the most important angiogenic factors and angiogenesis inhibitors, as well as relevant chemokines and chemokine receptors involved in chronic inflammatory rheumatic diseases. RA was chosen as a prototype to discuss these issues, as the majority of studies on the role of angiogenesis and chemokines in inflammatory diseases were carried out in arthritis. However, other systemic inflammatory (autoimmune) diseases including systemic lupus erythematosus (SLE), systemic sclerosis (SSc), Sjögren's syndrome (SS), mixed connective tissue disease (MCTD), polymyositis/dermatomyositis (PM/DM) and systemic vasculites are also discussed in this context. As a number of chemokines may also play a role in neovascularizaton, this issue is also described here. Apart from discussing the pathogenic role of angiogenesis and chemokines, authors also review the regulation of angiogenesis and chemokine production by other inflammatory meditors, as well as the important relevance of neovascularization and chemokines for antirheumatic intervention.     

Chemokine blockade: a new era in the treatment of rheumatoid arthritis?

Blockade of chemokines or chemokine receptors is emerging as a new potential treatment for various immune-mediated conditions. This review focuses on the therapeutic potential in rheumatoid arthritis, based on studies in animal models and patients. Several knockout models as well as in vivo use of chemokine antagonists are discussed. Review of these data suggests that this approach might lead to novel therapeutic strategies in rheumatoid arthritis and other chronic inflammatory disorders.     

Monocyte chemoattractant protein-1--a major contributor to the inflammatory process associated with diabetes

There is evidence that strongly suggests that inflammation plays an important role in diabetes and cardiovascular diseases. The high glucose-induced inflammatory process is characterised by the cooperation of a complex network of inflammatory molecules such as cytokines, adhesion molecules, growth factors, and chemokines. Among the chemokine family, monocyte chemoattractant protein (MCP-1) is a potent chemotactic factor, which is upregulated at sites of inflammation being in control of leukocytes trafficking. Here, we review the current knowledge on MCP-1 and its regulation by high glucose level in vascular cells involved in diabetes-induced accelerated atherosclerosis. The signalling pathways involved in MCP-1 modulation by high glucose, the proximal signalling events that stimulate downstream effects and the role of this chemokine in the pathophysiology of diabetes and its complications, are discussed.     

Evasion and exploitation of chemokines by viruses

Chemokines and chemokine receptors play a critical role in the host defense against viruses by mobilizing leukocytes to sites of infection, injury and inflammation. In order to replicate successfully within their host organisms, viruses have devised novel strategies for exploiting or subverting chemokine networks. This review summarizes various mechanisms that are currently known to be used by viruses for modulating chemokine activities including viral homologs of chemokines and chemokine receptors and soluble viral chemokine binding proteins. Insight into these strategies is providing a wealth of information on viral-host interactions, the function of chemokines in host defense and may help to generate novel anti-chemokine agents for treating against viral diseases or inflammatory disorders.     

Chemokines and chemokine receptors in renal transplantation--from bench to bedside

Attraction of mononuclear cells to sites of inflammation requires a close interplay of the inflammatory signal presented via chemokines and specific receptors on effector cells. First studies on acute renal transplant rejection demonstrated the involvement of CC-chemokines, such as RANTES, MIP-1alpha, MIP-1beta and MCP-1, as well as CXC-chemokines such as IL-8 and IP-10, correlating with expression of the corresponding chemokine receptors, CCR1, CCR5 and CCR2 as well as CXCR3. Since then, the pathophysiologic relevance has been extended to chronic allograft nephropathy and transplant glomerulopathy. Chemokine expression can be triggered by different stimuli, e.g. brain death, ischemia, HLA-mismatch and infection. Furthermore, anti-inflammatory chemokines have been identified. Chemokine receptor 7, e.g. enhances homing of lymphocytes to lymphatic tissues and the Duffy antigen receptor, DARC, a non-specific receptor that binds and inactivates different chemokines. While measurement of chemokine expression in clinical transplantation may facilitate the differential diagnosis of allograft dysfunction, knowledge of the chemokine network has also widened the understanding of transplant rejection and opened novel therapeutic approaches. Observations from humans with mutations of the chemokine network as well as transplantation of animals with targeted deletions in this system suggest that manipulations of chemokine signalling may improve the success rates of transplantation. Blocking chemokines unselectively with Met-RANTES or specifically with small molecule inhibitors of various chemokine receptors has lead to improved outcome in animal models. Currently, first human trials are under way to investigate drugs that stimulate lymphocyte homing. Inhibitors of CCR1 and CCR5 are being tested for other human diseases and may eventually be available in transplantation. Nonetheless, chemokine blockade my rather serve as an adjunct in the management of transplant recipients than a new "magic bullet".     

Neutralizing Nanobodies Targeting Diverse Chemokines Effectively Inhibit Chemokine Function

Chemokine receptors and their ligands play a prominent role in immune regulation but many have also been implicated in inflammatory diseases such as multiple sclerosis, rheumatoid arthritis, allograft rejection after transplantation, and also in cancer metastasis. Most approaches to therapeutically target the chemokine system involve targeting of chemokine receptors with low molecular weight antagonists. Here we describe the selection and characterization of an unprecedented large and diverse panel of neutralizing Nanobodies (single domain camelid antibodies fragment) directed against several chemokines. We show that the Nanobodies directed against CCL2 (MCP-1), CCL5 (RANTES), CXCL11 (I-TAC), and CXCL12 (SDF-1α) bind the chemokines with high affinity (at nanomolar concentration), thereby blocking receptor binding, inhibiting chemokine-induced receptor activation as well as chemotaxis. Together, we show that neutralizing Nanobodies can be selected efficiently for effective and specific therapeutic treatment against a wide range of immune and inflammatory diseases.     

Neurochemokines: a menage a trois providing new insights on the functions of chemokines in the central nervous system

Recent observations suggest that besides their role in the immune system, chemokines have important functions in the brain. There is a great line of evidence to suggest that chemokines are a unique class of neurotransmitters/neuromodulators, which regulate many biological aspects as diverse as neurodevelopment, neuroinflammation and synaptic transmission. In physiopathological conditions, many chemokines are synthesized in activated astrocytes and microglial cells, suggesting their involvement in brain defense mechanisms. However, when evoking chemokine functions in the nervous system, it is important to make a distinction between resting conditions and various pathological states including inflammatory diseases, autoimmune or neurodegenerative disorders in which chemokine functions have been extensively studied. We illustrate here the emergent concept of the neuromodulatory/neurotransmitter activities of neurochemokines and their potential role as a regulatory alarm system and as a group of messenger molecules for the crosstalk between neurons and cells from their surrounding microenvironment. In this deliberately challenging review, we provide novel hypotheses on the role of these subtle messenger molecules in brain functions leading to the evidence that previous dogmas concerning chemokines should be reconsidered.     

Chemokine receptor antagonism as an approach to anti-inflammatory therapy: 'just right' or plain wrong?

Inflammation plays a pivotal role in exacerbating a wide array of human diseases. The chemokines are a group of proteins that control the movement and activation of the immune cells involved in all aspects of the inflammatory response. Recently, their cognate receptors have attracted considerable interest as therapeutic targets, in part because they are G-protein-coupled receptors, which have been antagonized successfully before by the pharmaceutical industry. Indeed, several companies have now reported the development of selective small-molecule chemokine receptor antagonists, and some of these compounds have even entered human Phase I clinical trials. Preclinical studies of the responsiveness of murine models of inflammation to either pharmacologic or genetic intervention have suggested that antagonism of some chemokine receptors may well prove to be a safe and efficacious approach to anti-inflammatory therapy.     

Chemokines and their role in airway hyper-reactivity

Airway hyper-reactivity is a characteristic feature of many inflammatory lung diseases and is defined as an exaggerated degree of airway narrowing. Chemokines and their receptors are involved in several pathological processes that are believed to contribute to airway hyper-responsiveness, including recruitment and activation of inflammatory cells, collagen deposition and airway wall remodeling. These proteins are therefore thought to represent important therapeutic targets in the treatment of airway hyper-responsiveness. This review highlights the processes thought to be involved in airway hyper-responsiveness in allergic asthma, and the role of chemokines in these processes. Overall, the application of chemokines to the prevention or treatment of airway hyper-reactivity has tremendous potential.     

Identification of a gammaherpesvirus selective chemokine binding protein that inhibits chemokine action

Chemokines are involved in recruitment and activation of hematopoietic cells at sites of infection and inflammation. The M3 gene of gammaHV68, a gamma-2 herpesvirus that infects and establishes a lifelong latent infection and chronic vasculitis in mice, encodes an abundant secreted protein during productive infection. The M3 gene is located in a region of the genome that is transcribed during latency. We report here that the M3 protein is a high-affinity broad-spectrum chemokine scavenger. The M3 protein bound the CC chemokines human regulated upon activation of normal T-cell expressed and secreted (RANTES), murine macrophage inflammatory protein 1alpha (MIP-1alpha), and murine monocyte chemoattractant protein 1 (MCP-1), as well as the human CXC chemokine interleukin-8, the murine C chemokine lymphotactin, and the murine CX(3)C chemokine fractalkine with high affinity (K(d) = 1. 6 to 18.7 nM). M3 protein chemokine binding was selective, since the protein did not bind seven other CXC chemokines (K(d) > 1 microM). Furthermore, the M3 protein abolished calcium signaling in response to murine MIP-1alpha and murine MCP-1 and not to murine KC or human stromal cell-derived factor 1 (SDF-1), consistent with the binding data. The M3 protein was also capable of blocking the function of human CC and CXC chemokines, indicating the potential for therapeutic applications. Since the M3 protein lacks homology to known chemokines, chemokine receptors, or chemokine binding proteins, these studies suggest a novel herpesvirus mechanism of immune evasion.     

Chemokines in hepatitis C virus infection: pathogenesis, prognosis and therapeutics

Hepatitis C virus infection and its associated liver inflammatory disease is a major global health problem affecting over 170 million people worldwide. Following viral infection, multiple pro-inflammatory mediators contribute to recruitment of immune cells to the liver and to the generation of an anti-viral immune response. However, when this vigorous immune response fails to eliminate the virus, chronic infection is established. This in turn, results in an ongoing process of inflammation, regeneration and fibrosis that in many cases leads to the development of cirrhosis and of hepatocellular carcinoma. Multiple recent publications mark chemokines and their receptors as key players in leukocyte recirculation through the inflamed liver. Furthermore, chemokines may also be involved in liver regeneration, fibrosis, and in malignant transformation, which is induced by the persistence of inflammation. Accumulating data indicates that distinct chemokines and chemokine receptors may be associated with different stages of the chronic hepatitis C virus infection-associated liver disease. Multiple small molecules and peptide antagonizing chemokines and their receptors are in advanced phase 3 and phase 2 clinical trials. In the near future, such drugs are expected to enter clinical use raising the question whether they may be applicable for the treatment of chronic viral infection-associated liver disease. In this review, recent advances in understanding the role of chemokines and their receptors in the pathogenesis of chronic viral infection-associated liver disease are presented. Furthermore, the clinical implications of these novel findings, which mark chemokines as prognostic markers and therapeutic targets for immune-modulation during chronic liver viral infection, are documented.     

An Important Role of the SDF-1/CXCR4 Axis in Chronic Skin Inflammation

Inflammatory angiogenesis and vascular remodeling play key roles in the chronic inflammatory skin disease psoriasis, but little is known about the molecular mediators of vascular activation. Based on the reported elevated mRNA levels of the angiogenic chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 in psoriasis, we investigated the relevance of the SDF-1/CXCR4 axis in two experimental models of chronic psoriasis-like skin inflammation. The cutaneous expression of both SDF-1 and CXCR4 was upregulated in the inflamed skin of K14-VEGF-A transgenic mice and in imiquimod-induced skin inflammation, with expression of CXCR4 by blood vessels and macrophages. Treatment with the CXCR4 antagonist AMD3100 potently inhibited skin inflammation in both models, associated with reduced inflammatory angiogenesis and inflammatory cell accumulation, including dermal CD4⁺ cells and intraepidermal CD8⁺ T cells. Similar anti-inflammatory effects were seen after treatment with a neutralizing anti-SDF-1 antibody. In vitro, inhibition of CXCR4 blocked SDF-1-induced chemotaxis of CD11b⁺ splenocytes, in agreement with the reduced number of macrophages after in vivo CXCR4 blockade. Our results reveal an important role of the SDF-1/CXCR4 axis in skin inflammation and inflammatory angiogenesis, and they indicate that inhibition of the SDF-1/CXCR4 axis might serve as a novel therapeutic strategy for chronic inflammatory skin diseases.     

Study of gastric fluid induced cytokine and chemokine expression in airway smooth muscle cells and airway remodeling

Asthma is a chronic airway inflammatory disease. Chronic aspiration by gastric fluid in gastroesophageal reflux disease (GERD) is considered a primary inflammatory factor exacerbating or predisposing patients to asthma. Airway smooth muscle cells (SMCs) are considered an important component in airway remodeling. To investigate the role of gastric fluid in airway SMC inflammation and airway remodeling, we examined gastric fluid-induced cytokine and chemokine profiles, airway SMC migration and matrix metalloproteinase expression in rat primary rat airway SMCs. The T helper cell type 2 (Th2) cytokines interleukin 4, interleukin 6 and tumor necrosis factor 2 (TNF-α) and the chemokines, lipopolysaccharide-induced CXC chemokine (LIX/CXCL5), cytokine-induced neutrophil chemoattractant 2 (CINC-2), CINC-3, fractalkine, ciliary neurotrophic factor (CNTF), and vascular endothelial growth factor were induced by gastric fluid in primary cultured rat airway SMCs. Migration of rat airway SMCs was enhanced by gastric fluid and conditioned medium. The migration of rat airway SMCs enhanced by gastric fluid was associated with actin polymerization and activation of focal adhesion kinase. Matrix metalloproteinase 2 expressions in airway SMCs was enhanced by gastric fluid and conditioned medium. The results suggest potential mechanisms by which gastric fluid aspiration might influence SMC-mediated airway remodeling.     

Macrophages heterogeneity in atherosclerosis – implications for therapy

Atherosclerosis is a chronic inflammatory disease occurring within the artery wall and is an underlying cause of cardiovascular complications, including myocardial infarction, stroke and peripheral vascular disease. Its pathogenesis involves many immune cell types with a well accepted role for monocyte/macrophages. Cholesterol-loaded macrophages are a characteristic feature of plaques and are major players in all stages of plaque development. As well as modulating lipid metabolism, macrophages secrete inflammatory cytokines, chemokines and reactive oxygen and nitrogen species that drive pathogenesis. They also produce proteases and tissue factor that contribute to plaque rupture and thrombosis. Macrophages are however heterogeneous cells and when appropriately activated, they phagocytose cytotoxic lipoproteins, clear apoptotic bodies, secrete anti-inflammatory cytokines and synthesize matrix repair proteins that stabilize vulnerable plaques. Pharmacological modulation of macrophage activity therefore represents a potential therapeutic strategy for atherosclerosis. The aim of this review is to provide an overview of the current understanding of the different macrophage subsets and their monocyte precursors, and, the implications of these subsets for atherosclerosis. This will present a foundation for highlighting novel opportunities to exploit the heterogeneity of macrophages as important diagnostic and therapeutic targets for atherosclerosis and its associated diseases.     

Interference with heparin binding and oligomerization creates a novel anti-inflammatory strategy targeting the chemokine system

A hallmark of autoimmunity and other chronic diseases is the overexpression of chemokines resulting in a detrimental local accumulation of proinflammatory immune cells. Chemokines play a pivotal role in cellular recruitment through interactions with both cell surface receptors and glycosaminoglycans (GAGs). Anti-inflammatory strategies aimed at neutralizing the chemokine system have to-date targeted inhibition of the receptor-ligand interaction with receptor antagonists. In this study, we describe a novel strategy to modulate the inflammatory process in vivo through mutation of the essential heparin-binding site of a proinflammatory chemokine, which abrogates the ability of the protein to form higher-order oligomers, but retains receptor activation. Using well-established protocols to induce inflammatory cell recruitment into the peritoneal cavity, bronchoalveolar air spaces, and CNS in mice, this non-GAG binding variant of RANTES/CCL5 designated [44AANA47]-RANTES demonstrated potent inhibitory capacity. Through a combination of techniques in vitro and in vivo, [44AANA47]-RANTES appears to act as a dominant-negative inhibitor for endogenous RANTES, thereby impairing cellular recruitment, not through a mechanism of desensitization. [44AANA47]-RANTES is unable to form higher-order oligomers (necessary for the biological activity of RANTES in vivo) and importantly forms nonfunctional heterodimers with the parent chemokine, RANTES. Therefore, although retaining receptor-binding capacity, altering the GAG-associated interactive site of a proinflammatory chemokine renders it a dominant-negative inhibitor, suggesting a powerful novel approach to generate disease-modifying anti-inflammatory reagents.     

Targeting chemokine receptors in allergic disease

The directed migration of cells in response to chemical cues is known as chemoattraction, and plays a key role in the temporal and spatial positioning of cells in lower- and higher-order life forms. Key molecules in this process are the chemotactic cytokines, or chemokines, which, in humans, constitute a family of approx. 40 molecules. Chemokines exert their effects by binding to specific GPCRs (G-protein-coupled receptors) which are present on a wide variety of mature cells and their progenitors, notably leucocytes. The inappropriate or excessive generation of chemokines is a key component of the inflammatory response observed in several clinically important diseases, notably allergic diseases such as asthma. Consequently, much time and effort has been directed towards understanding which chemokine receptors and ligands are important in the allergic response with a view to therapeutic intervention. Such strategies can take several forms, although, as the superfamily of GPCRs has historically proved amenable to blockade by small molecules, the development of specific antagonists has been has been a major focus of several groups. In the present review, I detail the roles of chemokines and their receptors in allergic disease and also highlight current progress in the development of relevant chemokine receptor antagonists.     

The Multiple Chemokine–Binding Bovine Herpesvirus 1 Glycoprotein G (BHV1gG) Inhibits Polymorphonuclear Cell but Not Monocyte Migration into Inflammatory Sites

Chemokines facilitate the recruitment of inflammatory cells into tissues, contributing to target organ injury in a wide range of inflammatory and autoimmune diseases. Targeting either single chemokines or chemokine receptors alters the progression of disease in animal models of rheumatoid arthritis and lupus with varying degrees of efficacy, but clinical trials in humans have been less successful. Given the redundancy of chemokine–chemokine receptor interactions, targeting of more than one chemokine may be required to inhibit active inflammatory disease. To test the effects of multiple chemokine blockade in inflammation, we generated an adenovirus expressing bovine herpesvirus 1 glycoprotein G (BHV1gG), a viral chemokine antagonist that binds to a wide spectrum of murine and human chemokines, fused to the fragment crystallizable (Fc) portion of murine immunoglobulin (IgG)2a. Administration of the adenovirus significantly inhibited thioglycollate-induced migration of polymorphonuclear leukocytes into the peritoneal cavity of BALB/c mice and reduced both clinical severity and articular damage in K/BxN serum transfer-induced arthritis. However, treatment with BHV1gG-Ig fusion protein did not prevent monocyte infiltration into the peritoneum in the thioglycollate model and did not prevent renal monocyte infiltration or nephritis in lupus-prone NZB/W mice. These observations suggest that the simultaneous inhibition of multiple chemokines by BHV1gG has the potential to interfere with acute inflammatory responses mediated by polymorphonuclear leukocytes, but is less effective in chronic inflammatory disease mediated by macrophages.