C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella montevideo

We studied the population of LPS molecules on Salmonella montevideo that bind C3 during alternative pathway activation in serum. LPS molecules of Salmonella are composed of lipid A:core oligosaccharide (one copy per molecule), substituted by an O-polysaccharide (O-PS) side chain, which is a linear polymer of 0 to greater than 60 O-antigen repeat units containing mannose. A mutant of S. montevideo called SL5222 that inserts galactose only into core oligosaccharide and mannose only into O-antigen subunits was grown with [3H]mannose and [14C]galactose, so that LPS molecules bearing large numbers of O-antigen subunits have high 3H to 14C ratios, whereas molecules with few O-antigen subunits have lower 3H to 14C ratios. Double-labeled SL5222 was incubated in C8-deficient (C8D) serum or C8D serum with 2 mM Mg++Cl2 and 10 mM ethylene glycoltetraacetic acid (MgEGTA C8D). LPS molecules with covalently attached C3 were identified by binding to anti-C3. LPS molecules that bound C3 under both incubation conditions had O chains seven to eight times longer than the average LPS molecule. SL5222 was then grown in suboptimal concentrations of mannose in order to decrease the number of LPS molecules with long O-PS side chains. C3 attached to progressively shorter chain molecules of LPS as the mannose input was lowered, but still chose the longest available molecules. This finding and recently published observations indicate that C3 can bind to LPS molecules with short O-PS side chains. We postulate that preferential attachment of C3 to long-chain LPS in SL5222 results because long-chain LPS molecules sterically hinder shorter chain LPS molecules from macromolecules. This study provides direct proof that the O-PS of LPS sterically hinders access of large molecules to the outer membrane and indicates that the LPS coat of these bacteria functions as a barrier against large protein molecules.     

Regulation of the alternative pathway of T cell activation by anti-T3 monoclonal antibody

T cell activation may be triggered either through the T3-Ti antigen receptor complex or via an alternative macrophage-independent pathway involving the 50KD T11 sheep erythrocyte-binding glycoprotein. Monoclonal antibodies anti-T11(2) and anti-T11(3), directed at distinct epitopes of the T11 molecule, trigger mature T cells to proliferate and express their functional programs, and induce expression of IL 2 receptors on both T3+ and T3- thymocytes. We now show that a non-mitogenic anti-T3 antibody blocks activation via the T11 pathway of not only peripheral blood T cells, but also T3+ thymocytes. Anti-T3 does not affect surface expression of T11 or the rapid augmentation of T11(3) expression after incubation of cells with anti-T11(2). However, anti-T3 inhibits generation of IL 2 receptors and production of IL 2 by T lineage cells cultured with anti-T11(2) plus anti-T11(3). In contrast, modulation of the T11 molecule by a non-mitogenic anti-T11 antibody does not inhibit activation of T cells by a mitogenic anti-T3 antibody. The ability of anti-T3 to block expression of IL 2 receptors on both thymocytes and mature T cells activated by the T11 pathway suggests that a regulatory interaction may be important during T cell ontogeny to provide a mechanism for inhibiting expansion of autoreactive clones.     

Formation of classical C3 convertase during the alternative pathway of human complement activation

The fluid phase C3 convertase of the alternative pathway of human complement activation has been constructed from the isolated C3 component and from purified factors B and D. The enzyme was able to activate the isolated components C4 and C2 in the presence of C4 but had no effect on C2 in the absence of C4. The C4 and C2 activation was monitored by the loss of their hemolytic activity during the incubation with the alternative fluid phase C3 convertase. The activation of C4 and C2 components by the membrane-bound alternative C3 convertase formed on red cells (EC3bBb) was followed by the formation of C3 convertase of the classic pathway--EC4b2a. This resulted in the enhancement of hemolysis.     

Effect of membrane phospholipids on activation of the alternative complement pathway

Although some of the membrane glycoproteins that serve as activators or regulators of C activation have been identified, the influence of membrane lipids has not been studied extensively. A model of alternative C pathway activation was established using liposomes composed of cholesterol and synthetic phospholipids. Liposomes containing phosphatidylcholine (PC) as the sole phospholipid did not activate C as measured by C3 binding after incubation in normal human serum containing 2.5 mM MgCl2 and 10 mM EGTA. When phosphatidylethanolamine (PE) was included as 20% or more of the phospholipid, C3 binding was observed. C3 binding to liposomes was inhibited by salicylhydroxamic acid indicating binding through the C3 thioester bond. The phospholipid composition did not influence C3 binding to liposomes in an unregulated system of C3, B, D, and P indicating equivalent C3b binding sites on activating and nonactivating liposomes. When the regulatory proteins H and I were added to the other components, liposomes containing PE bound three times more C3 than PC liposomes suggesting that the phospholipid affects C3 regulation. This was tested directly in a radiolabeled H binding assay. In the presence of equal amounts of C3b, PC liposomes showed a greater number of high affinity H binding sites than PE liposomes. Using different PE derivatives, C activation could be directly related to the phospholipid polar head group. Liposomes containing PE, trinitrophenyl-PE or monomethyl-PE did activate the alternative C pathway, whereas those containing dimethyl-PE, PC, or phosphatidylserine did not. These studies provide evidence that primary and secondary amino groups on lipid membranes can decrease the interaction between H and C3b and provide sites for alternative pathway activation.     

Potentiation of antiboty-dependent cell-mediated cytotoxicity by target cell-bound C3b.

Antibody-dependent cytolytic effector lymphocytes are known to possess, in part, receptors for activated C3. Employing a model system consisting of 51Cr-labeled chicken erythrocytes and purified human peripheral lymphocytes, we investigated the effect of target cell bound C3b on antibody-dependent cellular cytotoxicity (ADCC). At concentrations of anti-target cell antibody too low to cause effective ADCC, target cell bound C3b cooperated with antibody to produce marked target cell lysis. In the presence of a 1/6.25 X 10(6) dilution of anti-chicken erythrocyte rabbit IgG, cell lysis increased from 20% to 65% by the attachment of 18,000 C3b molecules per cell. C3b-dependent enhancement of ADCC was dose dependent. It was augmented by attachment of activated properdin (P) to the C3b-bearing target cells.     

The alternative pathway C3/C5 convertase: chemical basis of factor B activation.

The structural basis of activation of the alternative pathway C3 convertase was explored. For this purpose a modified isolation procedure of the activating enzyme, Factor D, was elaborated. The procedure affords a 70,000-fold purification of the enzyme with a 20% yield. A simple assay was designed for the quantitation of both Factor D and Factor B activity. On the basis of activity measurements and amino acid analysis, Factor D concentration in plasma was estimated to be 1 microgram/ml. Highly purified Factor D was used to activate Factor B in the presence of C3b and Mg++. The resulting fragments, Ba and Bb, were characterized with respect to their circular dichroism spectra, amino acid compositions, reactive sulfhydryl groups, and partial amino- and carboxy-terminal sequences. The results indicate that the Ba fragment constitutes the amino-terminal region and the Bb fragment the carboxy-terminal region of Factor B. The bond in Factor B that is cleaved by Factor D is proposed to be an arginyl-lysine bond.     

The influence of membrane components on regulation of alternative pathway activation by decay-accelerating factor.

Decay-accelerating factor (DAF) is a C regulatory protein which functions in membranes to inhibit autologous C activation on cell surfaces. A liposome model was used to study the mechanism of DAF action and examine the effects of membrane-bound glycophorin and LPS on the regulatory activity of DAF. Liposomes were incubated in MgEGTA-treated human serum and activation of the alternative pathway measured by C3b binding. Liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol activated the alternative pathway in proportion to their content of PE. Incorporation of 10(-7) mol/mol phospholipid of either human E or HeLa cell-derived DAF inhibited C activation by liposomes containing 40% phosphatidylethanolamine by 50%, an efficiency comparable to that observed in intact E. HeLa DAF that had been treated with phosphatidylinositol-specific phospholipase C to remove its glycolipid anchor had no effect on C activation by liposomes at concentrations as high as 10(-5) mol/mol phospholipid. Incorporation of DAF into liposomes prepared with bound C3b inhibited the deposition of additional C3b by C3bBbP. However, the incorporated DAF increased the amount of Bb generated from B in the presence of D indicating that accelerated decay of the convertase was the primary effect of DAF. Similarly, treatment of intact human E with anti-DAF decreased the amount of Bb generated by the alternative pathway convertase. To study the effects of other membrane components on DAF activity, liposomes were prepared with purified human glycophorin A or LPS. In glycophorin liposomes the presence of PE was required to activate the alternative pathway and DAF inhibited this activation. In contrast, LPS liposomes bound C3b independently of PE and the incorporation of DAF had no effect. These results demonstrate that within a membrane, DAF's inhibitory activity on the alternative pathway C3 convertase is mediated independently of other membrane proteins, that in this model the major activity of DAF is to accelerate convertase decay, and that the presence of other membrane molecules that may serve as C3 acceptors can circumvent DAF function.     

Complement activation by measles virus cytotoxic antibodies: alternative pathway C activation by hemagglutination-inhibition antibodies but classical activation by hemolysin antibodies.

Antibodies against measles virus hemagglutinating (HA particles and hemolysin were shown to activate C differently. HA antibodies of rabbit or human origin activated C via the alternative pathway in cytotoxicity against chronically measles-infected cells. This cytotoxicity was expressed in C-4 deficient guinea-pig C or in rabbit C in the presence of 3 mM EGTA (ethylene-glycol-tetraacetic-acid) but not in 3 mM EDTA (ethylene-diamine-tetraacetic-acid). In contrast, human hemolysin antibodies activated C only via the classical way. F (ab')2 fragments from rabbit or human anti-HA IgG antibodies were as efficient in C activation via the alternative pathways as intact IgG antibodies with a corresponding hemagglutination-inhibition titer.     

The binding of complement component C3 to antibody-antigen aggregates after activation of the alternative pathway in human serum.

Preformed immune aggregates, containing antigen and either IgG (immunoglobulin G) or F(ab')2 rabbit antibody, were incubated with normal human serum under conditions allowing activation of only the alternative pathway of complement. Both the IgG and F(ab')2 immune aggregates bound C3b, the activated form of the complement component C3, in a similar manner, 2-3% of the C3 available in the serum being bound to the aggregates as C3b, and the rest remaining in the fluid phase as inactive C3b or uncleaved C3. It was found that the C3b was probably covalently bound to the IgG in the aggregates, since C3b-IgG complexes could be demonstrated on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, after repeated washing with buffers containing high salt or boiling under denaturing conditions. Incubation of the C3b-antibody-antigen aggregates in buffers known to destroy ester linkages had little effect on the C3b-IgG complexes, which suggested that C3b and IgG might be linked by an amide bond. Two main types of C3b-IgG complexes were found that had apparent mol.wts. of 360000 and 580000, corresponding to either one to two C3b molecules respectively bound to one molecule of antibody. On reduction of the C3b-IgG complexes it was found that the beta-chain, but not the alpha'-chain, of C3b was released along with all the light chain of IgG but only about half or less of the heavy chain of IgG. These results indicate that, during activation of the alternative pathway of complement by immune aggregates containing IgG antibody, the alpha'-chain of C3b may become covalently bound at one or two sites in the Fd portion of the heavy chain of IgG.     

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.     

Regulation by glycophorin of complement activation via the alternative pathway

The effect of glycophorin on complement activation via the alternative pathway was examined by incorporating it into the liposome membrane with trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine (TNP-Cap-DPPE). Liposomes having incorporated TNP-Cap-DPPE onto the membrane activate the alternative complement pathway of guinea pig as reported previously, and the additional insertion of glycophorin was found to reduce their activating capacity on the alternative complement pathway. This inhibitory effect was cancelled by pretreatment of the glycophorin-containing liposomes with neuraminidase indicating that the sialic acid in glycophorin is playing a role in the regulation of alternative complement pathway-activation on the biological membrane.     

Properdin binds to late apoptotic and necrotic cells independently of C3b and regulates alternative pathway complement activation

Cells that undergo apoptosis or necrosis are promptly removed by phagocytes. Soluble opsonins such as complement can opsonize dying cells, thereby promoting their removal by phagocytes and modulating the immune response. The pivotal role of the complement system in the handling of dying cells has been demonstrated for the classical pathway (via C1q) and lectin pathway (via mannose-binding lectin and ficolin). Herein we report that the only known naturally occurring positive regulator of complement, properdin, binds predominantly to late apoptotic and necrotic cells, but not to early apoptotic cells. This binding occurs independently of C3b, which is additional to the standard model wherein properdin binds to preexisting clusters of C3b on targets and stabilizes the convertase C3bBb. By binding to late apoptotic or necrotic cells, properdin serves as a focal point for local amplification of alternative pathway complement activation. Furthermore, properdin exhibits a strong interaction with DNA that is exposed on the late stage of dying cells. Our data indicate that direct recognition of dying cells by properdin is essential to drive alternative pathway complement activation.     

Assembly of the membrane attack complex promotes decay of the alternative pathway C3 convertase on Neisseria gonorrhoeae

C3, C4, factor B, properdin, and C2 binding to serum-sensitive and serum-resistant gonococci was quantitated in C8-deficient and normal human serum by using fluorescein-conjugated antibodies and 3H-labeled components. Organism and serum-specific differences were noted, the most striking of which involved factor B and properdin binding to the serum-sensitive strains in the different sera. C3 binding to these organisms was quantitatively and kinetically equivalent in C8-deficient and normal human serum. In contrast, factor B and properdin binding reached a plateau after 5 min in C8-deficient serum but peaked and fell to control values in normal human serum. Identical results were obtained with normal human serum immunochemically depleted of C8. Between 7 and 15% of the bound C3 participated in formation of the alternative pathway convertase C3bBb/P. Reconstitution of the C cascade by adding purified C8 to C8-deficient serum led to the loss of factor B previously bound to the organisms. Factor B loss occurred coincident with bacterial killing and membrane disruption as observed by electron microscopy. Prevention of membrane disruption by depleting normal human serum of lysozyme had no effect on killing and failed to prevent factor B loss. Stabilization of the C3bBb complex with Ni2+ prevented factor B loss as well as gross membrane disruption but not bacterial killing. C2 (the classical pathway analog of factor B) binding to gonococci was equivalent in C8-deficient and normal human serum peaking within 2.5 min and falling to control values in both sera thereafter. We conclude that the assembly of the membrane attack complex promotes decay of C3bBb/P with release of factor B and properdin but not C3 from the organism surface. Membrane disruption does not appear to be required for this effect. This activity may represent a mechanism to limit continued C consumption.     

CR2 is a complement activator and the covalent binding site for C3 during alternative pathway activation by Raji cells

Antibody-independent activation of the alternative C pathway by human lymphoblastoid cell lines latently infected with EBV has been recognized for some time, although the mechanisms involved and the specific cell surface molecule(s) recognized by the C system have not been identified. The present studies, carried out with the purified proteins of the alternative pathway have addressed these questions. Activation of the purified proteins of the alternative pathway by Raji lymphoblastoid cells was found to be antibody independent, confirming earlier findings with serum. Surprisingly, activation was highly dependent on properdin. In other models properdin has been found to augment alternative pathway activation and to be required for lysis of virus infected cells. Molecules which activate the alternative pathway provide binding sites on which C3 breakdown by regulatory proteins is impeded; therefore intact C3b accumulates on the activator. Immunoprecipitation studies with either anti-CR2 or anti-C3 have identified CR2, the R for C3d,g and EBV, as a major covalent and noncovalent binding site for C3 deposition on Raji cells during alternative pathway activation. Covalently bound C3b was dissociated from CR2 by hydroxylamine, indicating attachment via an ester bond. C3b binding after activation was not reduced by an anti-CR2 mAb which blocks CR2 R function, indicating that it was probably not mediated by C3d,g R epitopes on CR2. Direct confirmation of the ability of CR2 to trigger the alternative pathway came from studies with purified CR2 which was found to activate the alternative C pathway in serum or in mixtures of the purified proteins of the pathway. This work provides conclusive evidence that CR2 is a C activator and functions in this capacity on Raji cells.     

Cutting edge: NKG2D-dependent cytotoxicity is controlled by ligand distribution in the target cell membrane

Although the importance of membrane microdomains in receptor-mediated activation of lymphocytes has been established, much less is known about the role of receptor ligand distribution on APC and target cells. Detergent-resistant membrane domains, into which GPI-linked proteins partition, are enriched in cholesterol and glycosphingolipids. ULBP1 is a GPI-linked ligand for natural cytotoxicity receptor NKG2D. To investigate how ULBP1 distribution on target cells affects NKG2D-dependent NK cell activation, we fused the extracellular domain of ULBP1 to the transmembrane domain of CD45. Introduction of this transmembrane domain eliminated the association of ULBP1 with the detergent-resistant membrane fraction and caused a significant reduction of cytotoxicity and degranulation by NK cells. Clustering and lateral diffusion of ULBP1 was not affected by changes in the membrane anchor. These results show that the partitioning of receptor ligands in discrete membrane domains of target cells is an important determinant of NK cell activation.     

Antibody-mediated alternative complement pathway activation resists inhibition by sialoglycolipids

Although sialoglycolipids on liposome membranes were able to inhibit the activation of the alternative complement pathway (ACP) of guinea pigs mediated by trinitrophenylaminocaproyldipalmitoylphosphatidylethanolamine, they scarcely inhibited the ACP activation mediated by natural antibody to paragloboside (PG) inserted into the liposome membranes. Therefore, ACP activation was able to proceed regardless of the presence of sialoglycolipids on heterologous cell membranes when antibodies to constituents of the membranes were available. On the other hand, sialoglycolipids effectively inhibit undesirable ACP activation on self cell membranes, because little if any antibody is reacting on the self cell surface. Thus, the natural antibody reaction may effectively discriminate between self and non-self cell surface by cooperation with complement inhibitors on cell membranes such as sialoglycolipids.     

Murine antibody-dependent cellular cytotoxicity to herpes simplex virus-infected target cells.

Freshly collected peritoneal cells (PC) and cultured spleen cells (SC) (but not fresh SC) from nonimmune mice could mediate antibody-dependent cellular cytotoxicity (ADCC) against herpes simplex virus (HSV)-infected cells in the presence of mouse or human sera containing antibody to HSV. PC also demonstrated variable natural killer cell cytotoxicity to infected cells. Both PC and cultured SC required high concentrations of antibody and high effector to target cell ratios for optimal ADCC. The time kinetics of the reaction appeared to depend on the state of activation of the effector cells. In both PC and SC populations, ADCC activity was limited to adherent cells, and was profoundly inhibited by particulate latex or silica. The murine effector cell found in PC and SC able to mediate ADCC to HSV-infected cells appears to be a macrophage.     

Interaction of C3b, B, and D in the alternative pathway of complement activation.

The interaction of ZXd2, an insoluble intermediate of the alternative pathway on zymosan (Z5), with factor B and the enzyme D proceeds in a two-step reaction: 1) B binds in the presence of Mg++ to ZXd2 to form the intermediate ZXd2B, 2) B bound to ZXd2 is subsequently activated enzymatically by D to yield the complex ZXd2B which cleaves C3. Evidence was obtained that C3b, which is present on ZXd2, is required for ZXd2B formation. Studies of the functional role of C3b for ZXd2B formation revealed that C3b is involved in the first reaction step i.e., binding of B to ZXd2 to yield ZXd2B. Formation of ZXd2B is inhibited by pretreatment of ZXd2 with either anti-C3 Fab or with C3b-INA. Low ionic strength of about 2 mS was found to favor the interaction of the C3b with B. Mg++ concentrations from 1 to 31 mM as well as variation of pH in the range from 6.2 to 8.5 did not greatly influence the reaction of B with ZXd2. For the enzymatic activation of B only C3b on ZXd2 and factor D are required. This is concluded from the finding that fluid phase C3b is sufficient for the activation of B in the presence of D. This does not exclude the fact that other proteins present on ZXd2 may help to stabilize the intermediate ZXd2B or the enzymatically active complex AXd2B, or both of them.