Jörn Elsner

Chemokine receptor antagonists: a novel therapeutic approach in allergic diseases.

The aim of this review is to give an overview of the role of chemokines, particularly ligands of the CC chemokine receptor CCR3, in allergic diseases and to show the new concept in the treatment of allergies using chemokine receptor antagonists. Allergic diseases such as allergic asthma, allergic rhinitis and atopic dermatitis are characterized by a complex interaction of different cell types and mediators. Among this, Th2 cells, mast cells, basophils and eosinophils are found in the inflamed tissue due to the attraction of chemokines. Of all the known chemokine receptors, the chemokine receptor CCR3 seems to play the major role in allergic diseases which is supported by the detection of this receptor on the cell types mentioned above. Therefore, academic and industrial research focus on compounds to block this receptor. To date, certain chemokine receptor antagonists derived from peptides and small molecules exist to block the chemokine receptor CCR3. However, the in vivo data about these compounds and the mechanisms of receptor interaction are poorly understood, as yet. For the development of additional chemokine receptor antagonists, more details about the interaction between the ligands and their receptors are required. Therefore, additional studies will lead to the identification of novel CCR3 chemokine receptor antagonists, which can be therapeutically used in allergic asthma, allergic rhinitis, and atopic dermatitis.

The CC chemokine receptor antagonist Met-RANTES inhibits eosinophil effector functions

Eosinophils play an important role in allergic diseases such as allergic asthma, rhinoconjunctivitis and atopic dermatitis. Recruitement of eosinophils to the side of inflammation, the release of reactive oxygen species, leading to tissue damage, and the propagation of the inflammatory response are mediated by chemokines. Thus, the applicability of agents able to inhibit or antagonize chemokine–induced eosinophil activation seems to be of interest in the treatment of allergic diseases. Therefore, the effect of the CC chemokine antagonist, Met–RANTES, on its effect on human eosinophil effector functions in response to RANTES, MCP–3 and eotaxin was investigated. Met–RANTES had no intrinsic activity on [Ca2+]i transients in eosinophils and was able to dose–dependently inhibit [Ca2+]i transients in eosinophils following stimulation with RANTES, MCP–3 and eotaxin. Besides its effect on [Ca2+]i transients, Met–RANTES dose–dependently inhibited actin polymerization in eosinophils and the release of reactive oxygen species following stimulation with RANTES, MCP–3 and eotaxin. The results of this study lead to the conclusion that Met–RANTES is an effective and powerful compound to antagonize effector functions of human eosinophils following stimulation with RANTES, MCP–3 and eotaxin and is therefore a promising therapeutic approach to prevent the invasion and destructive power of eosinophils in allergic diseases.

n-Nonanoyl-CC Chemokine Ligand 14, a Potent CC Chemokine Ligand 14 Analogue That Prevents the Recruitment of Eosinophils in Allergic Airway Inflammation

CCR3 is responsible for tissue infiltration of eosinophils, basophils, mast cells, and Th2 cells, particularly in allergic diseases. In this context, CCR3 has emerged as a target for the treatment of allergic asthma. It is well known that the N-terminal domain of chemokines is crucial for receptor binding and, in particular, its activation. Based on this background, we investigated a number of N-terminally truncated or modified peptides derived from the chemokine CCL14/hemofiltrate CC chemokine-1 for their ability to modulate the activity of CCR3. Among 10 derivatives tested, n-nonanoyl (NNY)-CCL14[10–74] (NNY-CCL14) was the most potent at evoking the release of reactive oxygen species and inducing chemotaxis of human eosinophils. In contrast, NNY-CCL14 has inactivating properties on human eosinophils, because it is able to induce internalization of CCR3 and to desensitize CCR3-mediated intracellular calcium release and chemotaxis. In contrast to naturally occurring CCL11, NNY-CCL14 is resistant to degradation by CD26/dipeptidyl peptidase IV. Because inhibition of chemokine receptors through internalization is a reasonable therapeutic strategy being pursued for HIV infection, we tested a potential inhibitory effect of NNY-CCL14 in two murine models of allergic airway inflammation. In both OVA- and Aspergillus fumigatus-sensitized mice, i.v. treatment with NNY-CCL14 resulted in a significant reduction of eosinophils in the airways. Moreover, airway hyper-responsiveness was shown to be reduced by NNY-CCL14 in the OVA model. It therefore appears that an i.v. administered agonist internalizing and thereby inhibiting CCR3, such as NNY-CCL14, has the potential to alleviate CCR3-mediated diseases.

Synthesis and Surface Expression of ICAM-1 in Polymorphonuclear Neutrophilic Leukocytes in Normal Subiects and during Inflammatory Disease

During bacterial peritonitis of patients on continuous ambulatory peritoneal dialysis (CAPD) leukocytes, particularly polymorphonuclear neutrophilic granulocytes (PMNs), migrate into the peritoneal cavity. However, at the site of inflammation PMNs are not sufficiently able to protect the host against micro-organisms. Adhesion molecules, such as ICAM-1 (CD54), are involved in the interaction between endothelial cells and PMNs leading to the accumulation of PMNs at the site of inflammation. As PMNs are the predominant cell type in the peritoneal cavity in peritonitis, the aim of this study was to find out whether PMNs from CAPD peritonitis patients were able to express ICAM-1. Flow cytometric analyses with the anti-CD54 monoclonal antibody demonstrated that normal PMNs constitutively express slight amounts of ICAM-1. In contrast to normal PMNs, peritoneal PMNs from patients with CAPD peritonitis expressed high amounts of ICAM-1 (p = 0.003). Furthermore, ICAM-1 expression on peripheral blood PMNs of these patients significantly differed from PMNs from healthy donor (p = 0.01). Furthermore, Northern blot analysis revealed a weak signal of ICAM-1 mRNA in normal PMNs. However, peritoneal PMNs from CAPD peritonitis patients expressed a strong signal for ICAM-1 mRNA, suggesting that ICAM-1 is newly synthesized when PMNs invade the peritoneal cavity. In summary, this study clearly demonstrates that peritoneal PMNs of CAPD peritonitis express high amounts of ICAM-1 receptor on the level of mRNA and on the surface. Therefore, it is tempting to speculate that peritoneal PMNs interact amongst each other between ICAM-1 and its counter receptors CD11a,b/CD18 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The allergy-associated chemokine receptors CCR3 and CCR5 can be inactivated by the modified chemokine NNY-CCL11.

Background:  CC chemokine ligand 11 (CCL11) is the outstanding member of all described CC chemokine receptor 3 (CCR3) ligands and is shown to be selective for this receptor. However, it also activates CCR5 but only in the micromolar range. The in vivo activity of CCL11 is expected to be temporally restricted, as it is degraded by specific proteases such as the dipeptidyl‐peptidase IV (DP4), also termed CD26. Based on the approach to inactivate chemokine receptors in allergic disease models as has been demonstrated for DP4‐resistant n‐nonanoyl (NNY)‐CCL14 and for amino‐oxypentane (AOP)‐CCL5, it is tempting to study similar compounds derived from CCL11.

Differential pattern of CCR1 internalization in human eosinophils: prolonged internalization by CCL5 in contrast to CCL3

Background:  Whereas recent studies underlie the fundamental importance of the CC chemokine receptor 3 (CCR3) for the recruitment of eosinophils in allergic diseases, controversial data exist about the relevance of CCR1 on eosinophils. Therefore, the purpose of this study was to investigate the expression and regulation of CCR1 on eosinophils.

Cloning, expression, and functional characterization of cynomolgus monkey (Macaca fascicularis) CC chemokine receptor 1

The CC chemokine receptor 1 (CCR1) has emerged as a relevant factor contributing to inflammatory diseases such as allergic asthma. Commonly used animal models of allergic airway inflammation, especially murine models, have certain limitations. The elaborate, nonhuman, primate models of asthma display the highest comparability with the situation in humans. These models play an important role in the understanding of the pathogenesis of asthma. To improve the understanding in cynomolgus monkey models, we identified and characterized CCR1 in this nonhuman primate. Initially, we cloned the cynomolgus monkey CCR1 (cCCR1) gene, and the sequence analysis revealed high homology at the nucleotide (92%) and amino acid (88.4%) levels with its human counterpart. Human embryonic kidney 293 cells were stably transfected with cCCR1 and used in functional assays. Among those CCR1 ligands tested, CCL14(9‐74) was most potent in the induction of intracellular Ca2+ fluxes as observed for human CCR1 (hCCR1). Complete cross‐desensitization could be achieved between CCL14(9‐74) and CCL15. However, CCL3 could not fully abrogate the response to the potent ligand CCL14(9‐74). Competition‐binding studies with radiolabeled CCL3 concordantly showed that CCL14(9‐74) has a higher affinity to cCCR1 than hCCL3. Moreover, differential tissue‐specific expression of cCCR1 was investigated by real‐time quantitative polymerase chain reaction, displaying the highest levels in spleen. This study adds basic information needed for the evaluation of the role of CCR1 in the pathophysiology of asthma using the highly relevant cynomolgus monkey model and in addition, aids in the preclinical evaluation of potential novel drugs targeting CCR1.

CCR1- and CCR5-mediated inactivation of leukocytes by a nonglycosaminoglycan (non-GAG)-binding variant of n-nonanoyl-CCL14 (NNY-CCL14).

Intervention on chemokine receptors to prevent directional leukocyte migration is a potential therapeutic strategy. NNY‐CCL14 is a CD26‐resistant lead molecule, which exerts its effects on multiple chemokine receptors (CCR1, CCR2, CCR3, and CCR5). The inhibitory effects of NNY‐CCL14 in murine models of allergic airway inflammation have been assigned to its interaction with CCR1 and CCR5. In this study, a non‐GAG‐binding variant of NNY‐CCL14 was generated by mutating basic amino acids within the identified GAG‐binding 49BBXB52 motif. This CD26‐resistant, non‐GAG binding variant, NNY‐CCL14(G,A), does not promote CCR1‐dependent cell arrest on modeled endothelium. Its biological activity tested on human and murine chemokine receptors revealed distinguishing properties to NNY‐CCL14. As suggested by EC50 values for intracellular calcium mobilization, NNY‐CCL14(G,A) demonstrated a reduced ability to activate hCCR1, but internalization and desensitization of hCCR1 were unperturbed. Surprisingly, its activity on hCCR3 was strongly reduced, and it did not internalize mCCR3. A significantly reduced chemotactic activity of eosinophils and monocytes was observed. All biological effects mediated by NNY‐CCL14(G,A) via hCCR5 and mCCR5 showed no difference to NNY‐CCL14. In mice treated i.v. with NNY‐CCL14(G,A), a sustained in vivo down‐modulation of CCR5 was achieved over 3 h. Therefore, NNY‐CCL14(G,A) inactivates leukocytes by desensitizing and internalizing multiple chemokine receptors, thus rendering them unresponsive to further stimulation by natural ligands. When administered systemically, NNY‐CCL14(G,A) may modulate leukocyte functions prior to their interaction with other endothelium‐bound chemokines expressed under pathophysiological conditions, such as allergic inflammation.

n-Nonanoyl-CCL14 (NNY-CCL14), a novel inhibitor of allergic airway inflammation is a partial agonist of human CCR2.

Background:  Modulation of leukocyte recruitment through blocking of chemokine receptors has been proposed as an attractive therapeutic strategy. We have previously demonstrated that n‐Nonanoyl‐CC chemokine ligand 14 (NNY‐CCL14), a modified analog of the naturally occurring chemokine CCL14(9‐74) internalizes and desensitizes human CCR3 resulting in the inactivation of eosinophils. However, inhibitory effects of NNY‐CCL14 in murine models of allergic airway inflammation are assigned to its interaction with CCR1 and CCR5.

Donor to donor variation of the chemokine receptor CCR1 on human eosinophils and its prolonged internalization by RANTES

Abstract Rationale Whereas recent studies underlies the fundamental importance of the CC chemokine receptor CCR3 for the recruitment of eosinophils in allergic diseases controversial data about the expression of CCR1 on eosinophils exist. Therefore, the purpose of this study was to investigate the expression and regulation of CCR1 on eosinophils. Methods Flow cytometric analysis of whole blood eosinophils or CD16-negative selected freshly isolated eosinophils from atopic and non-atopic donors using an anti-CCR1 mAb. Results We found that 32% of the donors (n=24) were positive and 68% (n=49) were negative to express CCR1. Pre-incubation of isolated CCR1-positive eosinophils for 24 h with IL-3 (p=0.021), RANTES (p Conclusions A subpopulation of human donors are able to express CCR1 on the surface of eosinophils which helps to explain the different response of eosinophils to MIP-1α in previous studies. It is tempting to speculate whether the higher positive charge of RANTES in comparison to its overall negative charge of MIP-1α is responsible for the prolonged CCR1 internalization in response to RANTES.