Hans Ulrich Weltzien

Cytolytic and membrane-perturbing properties of lysophosphatidylcholine☆

I. Int roduct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259 II. Membranelytic properties of lysophosphat ides . . . . . . . . . . . . . . . . . . . . . . . . . 261 A. Lysis of erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261 1. General features of lysophosphat ideinduced hemolysis . . . . . . . . . . . . . . . . 262 2. Effects of chemical structure on hemolyt ic reaction . . . . . . . . . . . . . . . . . . 263 3. Role o f temperature and erythrocyte species . . . . . . . . . . . . . . . . . . . . . . 271 B. Lysis of non-erythroid cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 C. Lysis of pure lipid membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 III. Membrane effects of non-lytic concentrat ions of lysophosphat idylchol ine . . . . . . . . . 277 A. Effects on cell fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 B. Effects on cell surface structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278 C. Effects on lipid miscibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 D. Effects on membrane-associated enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . 280

T cell immune responses to haptens. Structural models for allergic and autoimmune reactions

Protein-reactive chemicals, metal salts and drugs, commonly classified as immunological haptens, are major environmental noxes targeted at the immune system of vertebrates. They may not only interfere with this defense system by toxicity alone, but more often by evoking hapten-specific immune responses resulting in allergic and eventually autoimmune responses. Here, we review recent developments in the analysis of the structural basis of hapten recognition, particularly by T lymphocytes, which represent central elements in cell-mediated, as well as in IgE dependent, allergies. A break-through in this field was the finding that T cells detect haptens as structural entities, attached covalently or by complexation to self-peptides anchored in binding grooves of major histocompatibility antigens (MHC-proteins). Synthetic hapten-peptide conjugates were shown to induce hapten-specific contact sensitivity in mice, opening new routes for studying hapten-induced immune disorders.

A New Type of Metal Recognition by Human T Cells: Contact Residues for Peptide-independent Bridging of T Cell Receptor and Major Histocompatibility Complex by Nickel

In spite of high frequencies of metal allergies, the structural basis for major histocompatibility complex (MHC)-restricted metal recognition is among the unanswered questions in the field of T cell activation. For the human T cell clone SE9, we have identified potential Ni contact sites in the T cell receptor (TCR) and the restricting human histocompatibility leukocyte antigen (HLA)-DR structure. The specificity of this HLA-DR–promiscuous VA22/VB17+ TCR is primarily harbored in its α chain. Ni reactivity is neither dependent on protein processing in antigen-presenting cells nor affected by the nature of HLA-DR–associated peptides. However, SE9 activation by Ni crucially depends on Tyr29 in CDR1α, an N-nucleotide–encoded Tyr94 in CDR3α, and a conserved His81 in the HLA-DR β chain. These data indicate that labile, nonactivating complexes between the SE9 TCR and most HLA-DR/peptide conjugates might supply sterically optimized coordination sites for Ni ions, three of which were identified in this study. In such complexes Ni may effectively bridge the TCR α chain to His81 of most DR molecules. Thus, in analogy to superantigens, Ni may directly link TCR and MHC in a peptide-independent manner. However, unlike superantigens, Ni requires idiotypic, i.e., CDR3α-determined TCR amino acids. This new type of TCR–MHC linkage might explain the high frequency of Ni-reactive T cells in the human population.

The influence of alkyl-lysophospholipids and lysophospholipid-activated macrophages on the development of metastasis of 3-Lewis lung carcinoma

Alkyl-lysophospholipids are synthetic analogs of natural occurring 2-lysophosphatidylcholine. They inhibit the development of metastasis of 3-Lewis lung tumor in the lung of C57Bl/6 mice if given i.v., i.c. or even orally as demonstrated by the increase of the median survival time and the number of surviving animals. Furthermore, i.v. injections of lysophospholipid-activated bone marrow macrophages increase the number of surviving animals and cause also a prolongation of the median survival time.

T Cell Recognition of Haptens, a Molecular View

Our review attempts to summarize the present knowledge on how T lymphocytes recognize chemically modified autologous cells. Concerning the broad spectrum of chemically and drug-induced allergic and autoimmune diseases, the molecular mechanisms of hapten recognition by T cells are clearly of more than academic interest. The past few years revealed that in contrast to the expectations of many researchers, major histocompatibility complex (MHC)-restricted hapten-specific T cell receptors in their majority do not react to covalently modified MHC molecules, but to haptenized peptides associated with the MHC peptide-binding groove. This finding allowed the introduction of synthetic hapten-peptide conjugates, the MHC specificity of which may be predetermined by allele-specific peptide sequence motifs. Thus, it has now become feasible to selectively hapten-modify defined sets of MHC molecules on living cells, and to study their immunological properties. In that way two major types of hapten-specific T cell receptors were identified: one reacting to hapten without caring for the chemical composition of the carrier peptide, and the other contacting hapten and peptide by two apparently independent contact sites. The consequences of these findings for hapten-specific allergies and autoimmunities, but also for our molecular understanding of antigen recognition by T cells are discussed.

Components of the Ligand for a Ni++ Reactive Human T Cell Clone

The major histocompatibility complex (MHC) restriction element for a human Ni2+ reactive T cell, ANi-2.3, was identified as DR52c. A series of experiments established that the functional ligand for this T cell was a preformed complex of Ni2+ bound to the combination of DR52c and a specific peptide that was generated in human and mouse B cells, but not in fibroblasts nor other antigen processing–deficient cells. In addition, ANi-2.3 recognition of this complex was dependent on His81 of the MHC β chain, suggesting a role for this amino acid in Ni2+ binding to MHC. We propose a general model for Ni2+ recognition in which βHis81 and two amino acids from the NH2-terminal part of the MHC bound peptide coordinate Ni2+ which then interacts with some portion of the Vα CDR1 or CDR2 region.

Metal-Protein Complex-Mediated Transport and Delivery of Ni2+ to TCR/MHC Contact Sites in Nickel-Specific Human T Cell Activation

Nickel allergy clearly involves the activation of HLA-restricted, skin-homing, Ni-specific T cells by professional APCs. Nevertheless, knowledge concerning the molecular details of metal-protein interactions underlying the transport and delivery of metal ions to APC during the early sensitization phase and their interactions with HLA and TCRs is still fragmentary. This study investigates the role of human serum albumin (HSA), a known shuttling molecule for Ni2+ and an often-disregarded, major component of skin, in these processes. We show that Ni-saturated HSA complexes (HSA-Ni) induce and activate Ni-specific human T cells as potently as Ni salt solutions when present at equimolar concentrations classically used for in vitro T cell stimulation. However, neither HSA itself nor its Ni-binding N-terminal peptide are involved in determining the specificity of antigenic determinants. In fact, HSA could be replaced by xenogeneic albumins exhibiting sufficient affinity for Ni2+ as determined by surface plasmon resonance (Biacore technology) or atomic absorption spectroscopy. Moreover, despite rapid internalization of HSA-Ni by APC, it was not processed into HLA-associated epitopes recognizable by Ni-specific T cells. In contrast, the presence of HSA-Ni in the vicinity of transient contacts between TCR and APC-exposed HLA molecules appeared to facilitate a specific transfer of Ni2+ from HSA to high-affinity coordination sites created at the TCR/HLA-interface.

Quantitative studies on lysolecithin-mediated hemolysis. Use of ether-deoxy lysolecithin analogs with varying aliphatic chain-lengths

The process of lysolecithin-mediated hemolysis has been investigated by use of various ether-deoxy lysolecithin analogs (1-alkyl-propanediol-3-phosphorylcholine) with alkyl residues of 10-22 carbon atoms. Hemolytic activities were defined either as molar amounts to be added for 50% lysis (L50) or as cell-bound amounts at 50% lysis (A50). It was found, that in contrast to L50, A50 values are independent of experimental conditions. Moreover, L50 values primarily reflect the binding affinities, while A50 values give more accurate information on the actual membrane-disturbing potential. The strongest hemolytic C16-lysolecithin analog required 2 - 10(7) or 5 - 10(7) molecules bound per cell for 50% lysis at 0 or 37degrees C, respectively, corresponding to about 10 or 25% of the total membrane phospholipids. Evidence is presented, indicating that (a) lysophosphatides bind to cells below their critical micelle concentration, (b) micelles themselves are not generally necessary for cell lysis. Red cells of different species (man and cattle) as well as at varying temperatures exhibit significantly different sensitivities in terms of L50 and A50 values. These differences, however, depend on the degree of hydrophobicity of the lysolecithins and disappear in the case of lysolipids having C10 or C12 aliphatic residues. The data are in agreement with our hypothesis that cellular sensitivity to lysolecithin lysis may be determined by the degree of segregation of lysolecithin-rich areas within the membrane lipid phase.

A physical characterization of some detergents of potential use for membrane protein crystallization

Micellar solutions of lauryldimethylamine oxide, n‐dodecyl‐β‐D‐maltoside and 1‐dodecanoylpropanediol‐3‐phosphorylcholine were studied by use of small‐angle neutron scattering. These detergents have been selected due to their use as solubilizing agents for membrane proteins. LDAO was found to form a homogeneous, approximately spherical micelle with a radius of 20.7 Å and an M r of 16 000. N‐Dodecyl‐β‐D‐maltoside forms an inhomogeneous micelle with a core of low scattering density surrounded by a shell of high scattering density. The data are in accord with a micelle forming an oblate ellipsoid and the disaccharide group pointing outward radially from the hydrophobic group. The semi‐axes are 16.8 and 25.5 Å and the M r is 66 000. 1‐Dodecanoylpropanediol‐3‐phosphorylcholine forms a rather homogeneous, roughly spherical micelle. The radius is 24 Å, the M r being 28 700. The data indicate a tangential packing of the phosphorylcholine head groups into a polar layer of 3–4 Å surrounding the micelle core. The use of these detergents as solubilizing agents during membrane protein crystallization is discussed.

Effects of a synthetic lysolecithin analog on the phase transition of mixtures of phosphatidylethanolamine and phosphatidylcholine.

Lysolecithin, i.e. 1-acyl-sn-glycero-3-phosphorylcholine, is a widely distributed surface-active and cytolytic phospholipid [l-3] , which has previously been shown to induce cell fusion [4,5] as well as enhanced immune reactions [6]. Despite various efforts [ l-3,7] , however, the mechanism of action of lysolecithin on natural and model membrane is still poorly understood. So far, all attempts have failed to demonstrate an increased molecular mobility of membrane lipids upon addition of low amounts of lysolecithin, and Klopfenstein et al. [8] have shown that lysolecithin even at a molar ratio higher than 1 : 1 hardly affects the phase transition temperature of dipalmitoyl-lecithin. The present study deals with the effects of a synthetic ether-desoxylysolecithin analog [l] on the miscibility of mixed lipid phases using phosphatidylethanolamine-phosphatidylcholine mixtures and differential scanning calorimetry. purchased from Fluka, Neu-Ulm. Impurities detectable by thin-layer chromatography were below 2%. These lipids were used without further purification. 1 -Hexadecyl-propanediol3-phosphorylcholine (ether-desoxylysolecithin) was synthetized as described elsewhere 111. The lipid mixtures were prepared by dissolving the desired amounts of the dry lipids (30-40 mg) in chloroform. After removal of the solvent by a stream of nitrogen, 25 ml of aqueous 0.05 M Tris-HCI buffer at pH 7.5 were added. The lipids were dispersed ultrasonically at a temperature above the transition temperature of the mixture. The lysolecithin was either mixed with the other lipids in the chloroform phase, or added to the dispersion as aqueous micellar solution. The calorimetric measurements were made using an adiabatic differential scanning calorimeter as described before [9, lo] . The heating rate was 13.5”C/h. To test the reversibility of the system, three runs were made with each lipid mixture.