Ultrastructure of the membrane attack complex of complement: detection of the tetramolecular C9-polymerizing complex C5b-8.

The ultrastructure of the membrane attack complex (MAC) of complement had been described as representing a hollow cylinder of defined dimensions that is composed of the proteins C5b, C6, C7, C8, and C9. After the characteristic cylindrical structure was identified as polymerized C9 [poly(C9)], the question arose as to the ultrastructural identity and topology of the C9-polymerizing complex C5b-8. An electron microscopic analysis of isolated MAC revealed an asymmetry of individual complexes with respect to their length. Whereas the length of one boundary (+/- SEM) was always 16 +/- 1 nm, the length of the other varied between 16 and 32 nm. In contrast, poly(C9), formed spontaneously from isolated C9, had a uniform tubule length (+/- SEM) of 16 +/- 1 nm. On examination of MAC-phospholipid vesicle complexes, an elongated structure was detected that was closely associated with the poly(C9) tubule and that extended 16-18 nm beyond the torus of the tubule and 28-30 nm above the membrane surface. The width of this structure varied depending on its two-dimensional projection in the electron microscope. By using biotinyl C5b-6 in the formation of the MAC and avidin-coated colloidal gold particles for the ultrastructural analysis, this heretofore unrecognized subunit of the MAC could be identified as the tetramolecular C5b-8 complex. Identification also was achieved by using anti-C5 Fab-coated colloidal gold particles. A similar elongated structure of 25 nm length (above the surface of the membrane) was observed on single C5b-8-vesicle complexes. It is concluded that the C5b-8 complex, which catalyzes poly(C9) formation, constitutes a structure of discrete morphology that remains as such identifiable in the fully assembled MAC, in which it is closely associated with the poly(C9) tubule.     

Molecular reorganization of lipid bilayers by complement: a possible mechanism for membranolysis.

The interaction between the membrane attack complex (MAC) of complement and flat lipid bilayers was investigated. Using spin-labeled derivatives of phospholipids and cholesterol and electron paramagnetic resonance spectroscopy, we measured the penetration of the MAC into bilayers and its influence on the order of bilayers. The MAC precursor components C5b--6, C7, C8, and C9 did not exert any measurable influence on lipid membranes. Functional C5b--7 was shown to interact strongly with the bilayer surface without deep penetration into the bilayer. Formation of C5b--8 and especially C5b--9 caused a marked change in the anisotropy of spectra from probes located within the hydrocarbon phase. The spectral changes are not caused by changes in probe rotation and, in the case of the cholesterol probes, are not due to direct probe--protein interactions. For these reasons we interpret the spectral changes to be the result of reorientation of ordered bilayer lipids effected by strong binding of phospholipids to MAC proteins.     

Nucleotide sequence of cDNA and derived amino acid sequence of human complement component C9.

The nucleotide sequence coding for the ninth component of human complement (C9) has been determined and the corresponding amino acid sequence has been derived. A human liver cDNA library was screened by the colony-hybridization technique using two radiolabeled oligonucleotide probes that correspond to known regions of the C9 amino acid sequence. Two recombinant plasmids were isolated and their cDNA inserts were sequenced. The derived protein sequence consists of 537 amino acids in a single polypeptide chain. A profile of the hydropathic index versus sequence number indicates that the amino-terminal half of C9 is predominantly hydrophilic in character whereas the carboxyl-terminal section of this protein is more hydrophobic. The amphipathic organization of the primary structure of C9 is consistent with the known potential of polymerized C9 to penetrate lipid bilayers, causing the formation of transmembrane channels.     

Essential role of TNF receptor superfamily 25 (TNFRSF25) in the development of allergic lung inflammation

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation, which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo, antibody blockade of TNFSF15 (TL1A), which is the ligand for TNFR25, inhibits lung inflammation and production of Th2 cytokines such as IL-13, even when administered days after airway antigen exposure. Similarly, blockade of TNFR25 by a dominant-negative (DN) transgene, DN TNFR25, confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant, NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells, but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung, and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics.     

Modulation of perforin expression in the decidual and peripheral blood cytotoxic lymphocytes in culture.

PROBLEM: Perforin (P) is a cytolytic molecule located in intracellular granules of cytotoxic lymphocytes both in the peripheral blood and decidua of pregnancy. The aim was to analyze the kinetics of P expression during in vitro culture and modulation of P expression by adherent cells, their supernatants and mitogen (PHA) stimulation. METHOD OF STUDY: P (intracellular antigen) was detected by flow cytometry in the suspension of first trimester pregnancy peripheral blood lymphocytes (PBL) and decidual lymphocytes (DL). RESULTS: A decrease of the percentage of P+ cells was obtained after 1 hr incubation and was prevented by addition of 30% of decidual adherent cells (DAC) or their supernatants. Upregulation of P expression was obtained when, in addition to adherent cells, DL and PBL were stimulated by PHA. DAC present in the culture in physiological concentrations prevent downregulation of P expression. CONCLUSION: DAC located in the vicinity of decidual cytotoxic lymphocytes, owing to their unique ability to produce a wide range of substances on demand, contribute to the high level of P expression in the decidua of pregnancy.     

Murine cytomegalovirus retinitis during retrovirus-induced immunodeficiency (MAIDS) in mice: interleukin-2 immunotherapy correlates with increased intraocular levels of perforin mRNA

Mice with a retrovirus-induced immunosuppression (MAIDS) are susceptible to experimental murine cytomegalovirus (MCMV) retinitis, but can be rendered resistant to retinitis by systemic interleukin-2 (IL-2) immunotherapy. Experiments were performed to explore the mechanism by which IL-2 treatment during MAIDS might restore resistance to MCMV retinitis. Whereas 80% of untreated MAIDS mice were susceptible to MCMV retinitis, none (0%) of IL-2-treated MAIDS mice developed necrotizing retinitis. In comparison, 100% of both untreated and IL-2-treated perforin knockout mice (PKO mice) were susceptible to MCMV retinitis, and severity of retinitis and amounts of infectious intraocular MCMV in IL-2-treated PKO mice were equivalent to that in untreated PKO mice. A competitive quantitative RT-PCR assay was used to measure the levels of perforin mRNA within MCMV-infected eyes of immunologically normal mice, untreated MAIDS mice, and IL-2-treated MAIDS mice. Although the level of perforin mRNA within MCMV-infected eyes of untreated MAIDS mice susceptible to retinitis was significantly reduced when compared to the high level found within MCMV-infected eyes of normal mice resistant to retinitis, systemic treatment of MAIDS mice with IL-2 increased perforin mRNA within MCMV-infected eyes to levels found in normal mice. The ability of IL-2 treatment to increase intraocular levels of perforin mRNA diminished with the progression of MAIDS. Our findings support the hypothesis that systemic IL-2 immunotherapy during MAIDS provides protection against MCMV retinitis by upregulation of perforin-mediated cytotoxicity used by cytotoxic lymphocytes to kill virus-infected cells.     

Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation

The mechanism underlying perforin (PFN)-dependent delivery of apoptotic granzymes during cytotoxic cell granule-mediated death remains speculative. Granzyme B (GrB) and perforin were found to coexist as multimeric complexes with the proteoglycan serglycin (SG) in cytotoxic granules, and cytotoxic cells were observed to secrete exclusively macromolecular GrB-SG. Contrary to the view that PFN acts as a gateway for granzymes through the plasma membrane, monomeric PFN and, strikingly, PFN-SG complexes were shown to mediate cytosolic delivery of macromolecular GrB-SG without producing detectable plasma membrane pores. These results indicate that granule-mediated apoptosis represents a phenomenon whereby the target cell perceives granule contents as a multimeric complex consisting of SG, PFN, and granzymes, which are, respectively, the scaffold, translocator, and targeting/informational components of this modular delivery system.     

CD10 and Bcl10 expression in diffuse large B-cell lymphoma: CD10 is a marker of improved prognosis

AIMS: Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically and pathologically heterogeneous. The Bcl10 gene was recently isolated from the breakpoint region of t(1;14)(p22;q32) in mucosa-associated lymphoid tissue (MALT) lymphomas, and is considered to be an apoptosis-associated gene. CD10 is considered to be a marker of follicular centre B-cell differentiation. To assess the clinical significance and roles of CD10 and Bcl10 in DLBCL, we analysed 138 cases, using immunohistochemical methods. METHODS AND RESULTS: CD10 expression was limited to the cytoplasm, whereas Bcl10 expression was detected in the cytoplasm and/or nuclei. CD10 expression was detected in 39 of 138 cases (28.2%), cytoplasmic Bcl10 in 68 cases (49.2%), and nuclear Bcl10 in 34 cases (24.6%). Nuclear Bcl10 was detected in 14 of 28 cases (50%) of extranodal DLBCL, but only 20 of 110 cases (18.2%) of nodal DLBCL. Cytoplasmic Bcl10 was detected in 19 of 28 cases (67.8%) of extranodal DLBCL and 49 of 110 cases (44.5%) of nodal DLBCL. CD10 expression closely correlated with improved survival (68% overall survival (OS) vs. 48% OS), but not with site of disease. A high International Prognostic Index (IPI) was considered to be a poor prognostic factor associated with a shorter OS. CD10 expression was detected in 27 of 84 cases (32.1%) with low-risk IPIs, and in 12 of 54 cases (22.2%) with high-risk IPIs. In the low-risk group, cases expressing CD10 carried a better prognosis than CD10- cases (93% OS vs. 71% OS), whereas this was not the case in the high-risk group (25% vs. 20%). CONCLUSIONS: Bcl10 expression was associated with extranodal DLBCL, but not with prognosis. CD10 expression was closely associated with improved survival, but not with risk as predicted by IPI. Overall, our results suggest that CD10 expression may be useful, in combination with clinical parameters, for determining the prognosis of DLBCL.     

Membrane attack by complement

Membrane attack by complement involves the self-assembly on membranes of five hydrophilic proteins (C5b, C6, C7, C8 and C9) to an amphiphilic tubular complex comprising approximately 20 subunits. The hydrophilic-amphiphilic transition of the precursor proteins is achieved by restricted unfolding and exposure of previously hidden hydrophobic domains. Restricted unfolding, in turn, is driven by high-affinity protein-protein interactions resulting in the formation of amphilic complexes. Circular polymerization of C9 to a tubular complex (poly C9) constitutes the molecular mechanism for transmembrane channel assembly and formation of ultrastructural membrane lesions.