No abstract

Various inflammatory diseases are characterized by tissue infiltration of neutrophils. Chemokines recruit and activate leukocytes, but neutrophils are traditionally known to be restricted in their chemokine receptor (CR) expression repertoire. Neutrophils undergo phenotypic and functional changes under inflammatory conditions, but the mechanisms regulating CR expression of infiltrated neutrophils at sites of chronic inflammation are poorly defined. Here we show that infiltrated neutrophils from patients with chronic inflammatory lung diseases and rheumatoid arthritis highly express CR on their surface that are absent or only marginally expressed on circulating neutrophils, i.e., CCR1, CCR2, CCR3, CCR5, CXCR3, and CXCR4, as measured by flow cytometry, immunohistochemistry, and confocal microscopy. The induction of CR surface expression on infiltrated neutrophils was functionally relevant, because receptor activation by chemokine ligands ex vivo modulated neutrophil effector functions such as respiratory burst activity and bacterial killing. In vitro studies with isolated neutrophils demonstrated that the surface expression of CR was differentially induced in a cytokine-mediated, protein synthesis-dependent manner (CCR1, CCR3), through Toll-like (CXCR3) or NOD2 (CCR5) receptor engagement, through neutrophil apoptosis (CCR5, CXCR4), and/or via mobilization of intracellular CD63+ granules (CXCR3). CR activation on infiltrated neutrophils may represent a key mechanism by which the local inflammatory microenvironment fine-tunes neutrophil effector functions in situ. Since the up-regulation of CR was exclusively found on infiltrated neutrophils at inflammatory sites in situ, the targeting of these G protein-coupled receptors may have the potential to site-specifically target neutrophilic inflammation.

No abstract

The pathogenicity of Clostridium difficile is primarily linked to secretion of the intracellular acting toxins A (TcdA) and B (TcdB) which monoglucosylate and thereby inactivate Rho GTPases of host cells. Although the molecular mode of action of TcdA and TcdB is well understood, far less is known about toxin binding and uptake. It is acknowledged that the C-terminally combined repetitive oligopeptides (CROPs) of the toxins function as receptor binding domain. The current study evaluates the role of the CROP domain with respect to functionality of TcdA and TcdB. Therefore, we generated truncated TcdA devoid of the CROPs (TcdA1–1874) and found that this mutant was still cytopathic. However, TcdA1–1874 possesses about 5 to 10-fold less potency towards 3T3 and HT29 cells compared to the full length toxin. Interestingly, CHO-C6 cells even showed almost identical susceptibility towards truncated and full length TcdA concerning Rac1 glucosylation or cell rounding, respectively. FACS and Western blot analyses elucidated these differences and revealed a correlation between CROP-binding to the cell surface and toxin potency. These findings refute the accepted opinion of solely CROP- mediated toxin internalization. Competition experiments demonstrated that presence neither of TcdA CROPs nor of full length TcdA reduced binding of truncated TcdA1–1874 to HT29 cells. We assume that toxin uptake might additionally occur through alternative receptor structures and/or other associated endocytotic pathways. The second assumption was substantiated by TER measurements showing that basolaterally applied TcdA1–1874 exhibits considerably higher cytotoxic potency than apically applied mutant or even full length TcdA, the latter being almost independent of the side of application. Thus, different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of C. difficile.