Septischer Schock: Molekulare Pathophysiologie und therapeutische Ansaetze. Teilprojekt A6: Mechanismen der antibakteriellen Wirkung polykationischer Proteine aus Granulozyten Schlussbericht

Because of the presently insufficient therapeutic possibilities of Gram-negative sepsis and the increasing number of antibiotic resistant bacterial strains, there is an urgent need for the development of new antibacterial drugs. Polycationic antibacterial peptides/proteins from neutrophils represent an important class of leading substances. By means of biophysical test systems, among others a reconstitution model of the lipid matrix of the outer membrane of Gram-negative bacteria, we have investigated the molecular mechanisms of interaction of some of these substances with the outer membrane. With our results we could substantially contribute to the elucidation of the molecular mechanisms responsible for the antibacterial properties of the bactericidal/premeability-increasing protein BPI. We have, furthermore, developed a model for the interaction of the polycationic antibiotic polymyxin B (PMB) with the outer membrane of Gram-negative bacteria, which can serve as a basis for the development of analogous antibacterial model substances not exhibiting the toxic properties of PMB. Also, informations on the reasons for the different actions of BPI and the lipopolysaccharide (LPS)-binding protein (LBP) with respect to the LPS-induced cytokine production were obtained, and the interaction of defensins with membranes was investigated. (orig.)SIGLEAvailable from TIB Hannover: F98B1895 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Septischer Schock: Molekulare Pathophysiologie und therapeutische Ansaetze. Teilprojekt A6: Mechanismen der antibakteriellen Wirkung polykationischer Proteine aus Granulozyten Schlussbericht

SIGLEAvailable from TIB Hannover: F02B1395 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung und Forschung, Berlin (Germany); DLR Deutsches Zentrum fuer Luft- und Raumfahrt e.V., Bonn (Germany)DEGerman

The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent.

Bacterial lipopolysaccharide (LPS, endotoxin) is a ubiquitous component of dust and air pollution and is suspected to contribute after inhalation to an activation of eosinophils in bronchial tissues of asthmatic patients, provoking inflammatory and allergic processes. We were therefore interested in the interaction of eosinophil granulocytes with LPS and have examined the activation of and uptake to human peripheral blood eosinophils by LPS. Eosinophils were stimulated by LPS and the endotoxic component lipid A and the release of tumor necrosis factor alpha (TNF-α) and of the eosinophil-specific granule protein eosinophil cationic protein (ECP) was estimated. The results show induction of TNF-α and ECP-release by LPS and lipid A in a dose-dependent manner. Anti-CD14 monoclonal antibody (moAb) (clone MEM-18) and the synthetic lipid A partial structure 406 blocked the release of TNF-α and ECP by LPS-stimulated eosinophils. Studies with radioactively labeled LPS showed dose-dependent uptake of3H-LPS to eosinophils. The 3H-LPS uptake was found to be specific because preincubation with unlabeled LPS, compound 406 and also anti-CD14 antibodies inhibited uptake of3H-LPS to eosinophil granulocytes. By flow cytometry using anti-CD14 moAb and by reverse transcriptase-polymerase chain reaction (RT-PCR) technique, CD14 expression was detectable. Furthermore, messenger RNA (mRNA) expression of Toll-like receptors (TLR) 2 and TLR 4 was detected, indicating the presence of these CD14 coreceptors. The results indicate that eosinophils can take up LPS and can be stimulated by LPS in a CD14-dependent manner. Hence, in addition to allergens, eosinophils interact with endotoxin, a process that possibly exacerbates ongoing inflammatory and allergic processes

Induction of tolerogenic vs immunogenic dendritic cells (DCs) in the presence of GM-CSF is regulated by the strength of signaling from monophosphoryl lipid A (MPLA) in association with glutathione and fetal hemoglobin gamma-chain.

Previous studies showed a fetal sheep liver extract (FSLE), in association with monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS), could interact to induce the development of dendritic cells (DCs) which regulated production of Foxp3+ Treg. This interaction was associated with an altered gene expression both of distinct subsets of TLRs and of CD200Rs. Prior studies had suggested that major interacting components within FSLE were gamma-chain of fetal hemoglobin (Hgbgamma) and glutathione (GSH). We investigated whether differentiation/maturation of DCs in vitro in the presence of either GM-CSF or Flt3L to produce preferentially either immunogenic or tolerogenic DCs was itself controlled by an interaction between MPLA, GSH and Hgbgamma. At low (approximately 10 microg/ml) Hgbgamma concentrations, DCs developing in culture with GSH and MPLA produced optimal stimulation of allogeneic CTL cell responses in vitro (and enhanced skin graft rejection in vivo). At higher concentrations (>40 microg/ml Hgbgamma) and equivalent concentrations of MPLA and GSH, the DCs induce populations of Treg which can suppress the induction of allogeneic CTL and graft rejection in vivo. These different populations of DCs express different patterns of mRNAs for the CD200R family. Addition of anti-TLR or anti-MD-1 mAbs to DCs developing in this mixture (Hgbgamma+GSH+MPLA), suggests that one effect of (GSH+Hgbgamma) on MPLA stimulation may involve altered signaling through TLR4

A role for altered TLR gene expression in association with increased expression of CD200R in the induction of mucosal tissue CD4(+) Treg in aged mice following gavage with a liver extract along with intramuscular monophosphoryl lipid A (MPLA) injection.

Previous studies showed a fetal sheep liver extract (FSLE), in association with LPS, injected into aged (>20 months) mice reversed the altered polarization (increased IL-4 and IL-10 with decreased IL-2 and IFN-gamma) in cytokine production seen from ConA stimulated lymphoid cells of those mice. Aged mice show a >60% decline in numbers and suppressive function of both CD4(+)CD25(+)Foxp3(+)Treg and so-called Tr3 (CD4(+)TGFbeta(+)). Their number/function is restored to levels seen in control (8-week-old) mice by FSLE. We have reported at length on the ability of a novel pair of immunoregulatory molecules, members of the TREM family, namely CD200:CD200R, to control development of dendritic cells (DCs) which themselves regulate production of Foxp3(+) Treg. The latter express a distinct subset of TLRs which control their function. We report that a feature of the altered Treg expression following combined treatment with FSLE and monophosphoryl lipid A, MPLA (a bioactive component of lipid A of LPS) is the altered gene expression both of distinct subsets of TLRs and of CD200Rs. We speculate that this may represent one of the mechanisms by which FSLE and MPLA alter immunity in aged mice