Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired-defect of bone marrow stem cells in which the affected clones produce erythrocytes (also granulocytes and platelets) with membranes that are abnormally sensitive to complement-mediated lysis. Abnormal erythrocytes (E) from patients with PNH (PNH-E) are 3-5 times more sensitive (type II PNH-E) or 15-25 times more sensitive (type Im PNH-E) to lysis in vitro by human complement than normal -E from unaffected individuals and the functionally normal E that arise from unaffected clones in PNH patients (type I PNH-E). After complement activation by either the classical or alternative pathway, abnormal amounts of C3b are deposited on the membranes of PNH-E compared with normal E, suggesting that the PNH-E membrane cannot regulate the events responsible for C3b deposition. Two proteins that decrease the stability of the classical and alternative pathway C3 convertases on target cells have been isolated from normal human E stroma: the 70,000 Mr decay accelerating factor of stroma (DAF) and the 250,000 Mr C3b receptor (C3bR pathway (8, 9), by the alternative pathway (10, 11), or by acid treatment of C5 that results in subsequent formation of the lytic complex of complement proteins, C5-9 (12, 13). The relative contribution of each of these pathways to the lysis of PNH-E in vivo is not known.With equivalent amounts of isohemagglutinin sensitization, normal E, type II PNH-E, and type III PNH-E activate the same amount of C1 and bind equivalent amounts of C4b, but 6 times more membrane-bound C3b is deposited on PNH-E than on normal E (14). The classical C3 convertase, C4b,2a, loses its ability to cleave C3 enzymatically and deposit C3b when the C2a subunit "spontaneously" dissociates from the membrane-bound C4b subunit (15, 16). The increased ratio of C3b/ C4b bound to PNH-E suggests that these cells may lack a regulatory mechanism for C3 convertase activity.Two proteins that regulate complement C3 convertases on target cells have been isolated from the membranes of normal E: the 70,000 M, decay accelerating factor of stroma (DAF) (17, 18) and the 250,000 Mr C3b receptor (C3bR) (19,20). When DAF and the C3bR are solubilized and isolated, each can cause the accelerated decay of both membrane-bound classical (C4b,2a) and properdin-stabilized alternative complement (C3b, Bb) C3 convertases; DAF has preferential activity for the C4b,2a convertase and C3bR for the C3b,Bb convertase (18). The availability of an antiserum to each of these moieties allowed the direct determination of their presence or absence on PNH-E. MATERIALS AND METHODSThe following reagents were-obtained as noted: acrylamide, bisacrylamide, temed, 2-mercaptoethanol, NaDodSO4, hydroxyapatite, and nitrocellulose paper from Iodogen (1,3,4,

Molecular definition of the cellular receptor for the collagen domain of C1q has been elusive. We now report that C1q binds specifically to human CR1 (CD35), the leukocyte C3b/C4b receptor, and the receptor on erythrocytes for opsonized immune complexes. Biotinylated or radioiodinated C1q (*C1q) bound specifically to transfected K562 cells expressing cell surface CR1 and to immobilized recombinant soluble CR1 (rsCR1). *C1q binding to rsCR1 was completely inhibited by unlabeled C1q and the collagen domain of C1q and was partially inhibited by C3b dimers. Kinetic analysis in physiologic saline of the interaction of unlabeled C1q with immobilized rsCR1 using surface plasmon resonance yielded an apparent equilibrium dissociation constant (K[eq2]) of 3.9 nM. Thus, CR1 is a cellular C1q receptor that recognizes all three complement opsonins, namely, C1q, C3b, and C4b.

Mannan-binding lectin (MBL), a member of the collectin family, is known to have opsonic function, although identification of its cellular receptor has been elusive. Complement C1q, which is homologous to MBL, binds to complement receptor 1 (CR1/CD35), and thus we investigated whether CR1 also functions as the MBL receptor. Radioiodinated MBL bound to recombinant soluble CR1 (sCR1) that had been immobilized on plastic with an apparent equilibrium dissociation constant of 5 nM. N-acetyl-d-glucosamine did not inhibit sCR1–MBL binding, indicating that the carbohydrate binding site of MBL is not involved in binding CR1. C1q inhibited MBL binding to immobilized sCR1, suggesting that MBL and C1q might bind to the same or adjacent sites on CR1. MBL binding to polymorphonuclear leukocytes (PMNs) was associated positively with changes in CR1 expression induced by phorbol myristate acetate. Finally, CR1 mediated the adhesion of human erythrocytes to immobilized MBL and functioned as a phagocytic receptor on PMNs for MBL–immunoglobulin G opsonized bacteria. Thus, MBL binds to both recombinant sCR1 and cellular CR1, which supports the role of CR1 as a cellular receptor for the collectin MBL.

SummaryInteractions between endothelium and vascular smooth muscle cells play a major role in the biology of the blood vessel wall. Growth factors released from endothelial cells control in part the normal and pathological proliferation of vascular smooth muscle cells. Endothelial deposits of C5b-9 proteins, the membrane attack complex of complement (MAC), have been found in a variety of pathological tissues in which cell proliferation is an early characteristic abnormality, including atherosclerosis. We have explored a possible bridging role for terminal complement C5b-9 proteins in eliciting focal signals for cell proliferation by releasing growth factors from endothelial cells. We found that both bovine aortic and human umbilical vein cells respond to the MAC by releasing basic fibroblast growth factor and platelet-derived growth factor. These mitogens stimulate DNA synthesis in Swiss 3T3, vascular smooth muscle, and glomerular mesangial cells. Based on these findings, we propose that complement-induced release of mitogens from endothelial cells is a novel pathogenic mechanism for proliferative disorders.

Most isolates of Staphylococcus aureus produce a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide. To investigate whether CP5 and CP8 differ in their biological properties, we created isogenic mutants of S. aureus Reynolds that expressed CP5, CP8, or no capsule. Biochemical analyses of CP5 and CP8 purified from the isogenic S. aureus strains were consistent with published structures. The degree of O acetylation of each polysaccharide was similar, but CP5 showed a greater degree of N acetylation. Mice challenged with the CP5 ؉ strain showed a significantly higher bacteremia level than mice challenged with the CP8 ؉ strain. Similarly, the CP5 ؉ strain survived preferentially in the bloodstream and kidneys of infected mice challenged with a mixed inoculum containing both strains. The enhanced virulence of the CP5 ؉ strain in vivo correlated with its greater resistance to in vitro killing in whole mouse blood. Likewise, in vitro opsonophagocytic killing assays with human neutrophils and sera revealed greater survival of the Reynolds (CP5) strain, even though the kinetics of opsonization by C3b and iC3b was similar for both the CP5 ؉ and CP8 ؉ strains. Electron micrographs demonstrated C3 molecules on the cell wall beneath the capsule layer for both serotype 5 and 8 strains. Purified CP5 and CP8 stimulated a modest oxidative burst in human neutrophils but failed to activate the alternative complement pathway. These results indicate that CP5 and CP8 differ in a number of biological properties, and these differences likely contribute to the relative virulence of serotype 5 and 8 S. aureus in vivo.Staphylococcus aureus is a major bacterial pathogen that causes a wide spectrum of clinical infections, ranging from localized soft-tissue infections to life-threatening bacteremia and endocarditis (25). Many virulence factors contribute to the pathogenesis of staphylococcal infections, including surfaceassociated adhesins and secreted exoproteins and toxins (35). Like many invasive bacterial pathogens, S. aureus produces a capsular polysaccharide (CP) that enhances its resistance to clearance by host innate immune defenses. Most clinical isolates of S. aureus are encapsulated, and serotype 5 and 8 strains predominate (2,11,40). The type 5 (CP5) and type 8 (CP8) capsular polysaccharides have similar trisaccharide repeating units comprised of N-acetyl mannosaminuronic acid, N-acetyl L-fucosamine, and N-acetyl D-fucosamine (9, 28, 43). CP5 and CP8 are serologically distinct, and this can be attributed to differences in the linkages between the sugars and in the sites of O acetylation.Previous studies have correlated S. aureus capsule production with resistance to in vitro phagocytic uptake and killing (13, 41). Human neutrophils phagocytose capsule-negative mutants in the presence of nonimmune serum with complement activity, whereas serotype 5 isolates require both capsulespecific antibodies and complement for optimal opsonophagocytic killing (4, 41). Nilsson et al. (29) reported that peritoneal macrophages from mice phagocy...

The native capsular polysaccharide antigen of type III, group B Streptococcus contains a terminal sialic acid residue on each repeating unit that masks all end-group galactopyranose residues and prevents alternative pathway complement activation by adult human sera in the absence of type-specific antibody. The critical role of the sialic acid residues in allowing the organism to evade activating the alternative complement pathway was shown when neuraminidase treatment of the organism converted the bacteria to activators of the alternative pathway as assessed in agammaglobulinemic serum. The requirement for specific antibody in permitting alternative pathway activation by the fully sialated bacteria was shown when sera that contained low levels of specific antibody failed to activate this pathway, and when prior absorption of serum that contained higher type-specific antibody levels with the capsular antigen failed to activate this pathway. The use of C2-deficient sera showed that the calssical pathway was not required for antibody-dependent alternative pathway activation. The use of isotonic, pH 7.5, veronal-NaCl buffer that contained 1% gelatin and that was supplemented to 4 mM Mg++ and 16 mM EGTA and adjusted to pH 7.5 (MgEGTA) ruled out the participation of the C1-bypass pathway. The presence of sialic acid on the bacterial surface is one means of evading an important mechanism of natural immunity, namely activation of complement by the alternative pathway. Only specific antibody, i.e., acquired immunity, can overcome this virulence factor.

The terminal complement complexes C5b-7, C5b-8 and C5b-9 are able to generate nonlethal cell signals. One universal consequence of a cell being targeted by C5b-8 or C5b-9 is an influx of Ca2+. In addition, other second messengers, including cAMP, inositol phosphate intermediates and arachidonate metabolites, are generated by the terminal complement complexes in specific cell types. In vivo, terminal complement complexes have been found in a wide variety of inflammatory processes in humans and in experimental animal models. Some of these models of inflammation putatively induced by terminal complement complexes have been tested in complement-deficient animals, and indeed no inflammation results, which supports the critical role of the terminal complement complexes in the pathogenesis of the lesion.

Summary Epstein-Barr virus (EBV) attachment to primary B-cells initiates virus entry. While CD21 is the only known receptor for EBVgp350/220 a recent report documents EBV infects B-cells from a patient genetically deficient in CD21. On normal resting B-cells CD21 forms two membrane complexes, one with CD19 another with CD35. Whereas the CD21/CD19 complex is widely retained on immortalized and B-cell tumor lines, the related complement-regulatory protein CD35 is lost. To determine the role(s) of CD35 in initial infection, we transduced a CD21-negative pre-B-cell and myeloid leukemia line with CD35, CD21 or both. Cells expressing CD35 alone bound gp350/220 and became latently infected when the fusion receptor HLA II was co-expressed. Temporal, biophysical and structural characteristics of CD35-mediated infection were distinct from CD21. Identification of CD35 as an EBV receptor uncovers a salient role in primary infection, addresses unsettled questions of virus tropism, and underscores the importance of EBVgp350/220 for vaccine development.