U. Forssmann

Chemoattraction of Macrophages, T Lymphocytes, and Mast Cells Is Evolutionarily Conserved within the Human α-Defensin Family

Human defensins are natural peptide antibiotics. On the basis of the position and bonding of six conserved cysteine residues, they are divided into two families, designated α- and β-defensins. Human α-defensins are expressed predominantly in neutrophils (human neutrophil peptides (HNP) 1–4) or intestinal Paneth cells (human defensins (HD) 5 and 6). Although α-defensins have been implicated in the pathogenesis of inflammatory bowel disease, their immunomodulatory functions are poorly understood. In the present study, HNP-1, HNP-3, and HD5 were found to be potent chemotaxins for macrophages but not dendritic cells using Gαi proteins and MAPK as signal transducers. α-Defensins were also chemoattractive for the human mast cell line HMC-1 but lacked, in contrast to β-defensins, the ability to induce intracellular calcium fluxes. Furthermore, HNP-1, HNP-3, and HD5 comparably mobilized naive as well as memory T lymphocytes. Using the protein kinase C (PKC) inhibitors GF109 and Gö6976, we observed a PKC-independent functional desensitization to occur between human α-defensins, which suggests a common receptor for HNP-1, HNP-3, and HD5 on immune cells. This α-defensin receptor was subject to heterologous desensitization by the PKC activator PMA and to PKC-dependent cross-desensitization by human β-defensins. Conversely, α-defensins desensitized β-defensin-mediated migration of immune cells in a PKC-dependent manner, suggesting unique receptors for both defensin families. Taken together, our observations indicate that chemoattraction of macrophages, T lymphocytes, and mast cells represents an immunomodulatory function which is evolutionarily conserved within the human α-defensin family and tightly regulated by β-defensins.

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.

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.

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.

Intravascular inactivation of CCR5 by n-Nonanoyl-CC chemokine ligand 14 and inhibition of allergic airway inflammation

Modulation of leukocyte recruitment through intervention with chemokine receptors is an attractive, therapeutic strategy. Recently, we have shown that n‐Nonanoyl (NNY)‐CCL14 internalizes and desensitizes human (h)CCR3, resulting in the inactivation of eosinophils. In this study, we investigated the interaction of NNY‐CCL14 with CCR1 and CCR5 and the relevance of these NNY‐CCL14 receptors on its in vivo effects in allergic airway inflammation. NNY‐CCL14 has inactivating properties on CCR1+ and CCR5+ cell lines and primary leukocytes. It desensitizes hCCR1‐ and hCCR5‐mediated calcium release and internalizes these receptors from the cellular surface. Treatment of OVA‐sensitized BALB/c mice with NNY‐CCL14 resulted in reduced pulmonary inflammation. Above all, it is demonstrated that systemic treatment with NNY‐CCL14 down‐modulates CCR5 from the surface of lymphocytes in vivo. Although NNY‐CCL14 acts on murine lymphocytes and internalizes CCR5, it does not internalize CCR3 on mouse eosinophils, showing species selectivity regarding this particular receptor. Therefore, the inhibitory effects of NNY‐CCL14 in murine models of allergic airway inflammation can be assigned to its interaction with CCR5. The presented results substantiate the relevance of CCR5 as a target for allergic airway inflammation.

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.