Aleksandra Heitland

The novel β-defensin DEFB123 prevents lipopolysaccharide-mediated effects in vitro and in vivo

Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel β‐defensin DEFB123. A peptide comprising the β‐defensin core region was synthesized and used for our analysis. Like other β‐defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram‐positive and Gram‐negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)‐binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS‐induced tumor necrosis factor (TNF)‐alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS‐mediated MAPK induction in these cells. Protection against LPS‐mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic β‐defensin DEFB123 prevents LPS‐induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of β‐defensins may include interference with LPS‐action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.—Motzkus, D., Schulz‐Maronde, S., Heitland, A., Schulz, A., Forssmann, W.‐G., Jübner, M., and Maronde, E. The novel β ‐defensin DEFB123 prevents lipopolysaccharide‐me‐diated effects in vitro and in vivo. FASEB J. 20, E997‐E1004 (2006)

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.

Cellular localization, membrane distribution, and possible function of guanylyl cyclases A and 1 in collecting ducts of rat

BACKGROUND Natriuretic peptides regulate Na+ and H(2)O transport in the cortical collecting duct (CCD). We have shown that natriuretic peptides have no effect on ion conductances or water transport of principal cells (PC) even though a cGMP-regulated K+ channel is located in the basolateral membrane of these cells. METHODS RT-PCR was used to screen for different guanylyl cyclases (GC) in CCD and to look for the expression of GC-1 and GC-A mRNA in CCD of male and female Wistar and Sprague-Dawley rats. Polyclonal antibodies were raised against the detected GC. BCECF was used to investigate the effects of ANP on intracellular pH in intercalated cells (IC). RESULTS GC-A and GC-1 were detected. GC-A was immunolocalized in the luminal membrane of IC while GC-1 was mainly found in the luminal membrane of PC. GC-1 is expressed in Sprague-Dawley and Wistar rats except for male Sprague-Dawley rats, while GC-A is expressed in all strains. ANP (160 nM, n=11), urodilatin (140 nM, n=6), which had no effect in PC, significantly decreased pH(i) by 0.02+/-0.01 and 0.03 +/- 0.01 Units in IC, respectively. ANP as well as urodilatin and 8-Br-cGMP decreased the pH(i) recovery after acidification by 30 +/- 6% (n=12), 37 +/- 7% (n=8), and 19 +/- 3% (n=8), respectively. CONCLUSION GC-A is located in the luminal membrane of IC of rat CCD and ANP acts through this receptor when regulating pH(i) via an inhibition of the Na+/H+-exchanger. PC do not possess GC-A. GC-1 seems to be the only GC in these cells of most rat strains tested and therefore, it could be responsible for the regulation of K+ channels in the basolateral membrane via cGMP-dependent protein kinase.

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.

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.