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Psoriasis is a chronic inflammatory skin disease with a characteristic isomorphic reaction, i.e. the Köbner reaction, induced by slight epidermal trauma. In this study, the tape-stripping technique was used to induce the development of Köbner reaction in 18 subjects with psoriasis. Eight subjects developed a positive reaction. To study the early cellular changes, skin biopsies were taken at the baseline and subsequent time points of 2 h, 1 d, 3 d, and 7 d for the immunostaining of complement C3c, iC3b, and cells expressing complement receptor 3 (CD11b/CD18; a receptor of iC3b) or CD14. The results show that the positive Köbner reaction is associated with rapid (2 h-1 d) and sustained (3-7 d) increase in the expression of epidermal C3c and iC3b and dermal C3c. In addition, there was a positive correlation between CD11b+ and CD14+ cells in baseline and 2 h-1 d biopsies with a subsequent increase in CD11b+ and CD14+ cells in 3-7 d biopsies in the Köbner-positive group. In the Köbner-negative group, only a transient increase in epidermal iC3b at 2 h-1 d, as well as rapid (2 h-1 d) and sustained increase (3-7 d) in dermal iC3b and CD14+ cells, was observed. In experiments with cultured monolayer keratinocytes, a slight cell damage already at 30 mJ/cm2 ultraviolet B irradiation led to increased expression of C3c, but not iC3b. Therefore, there are marked differences between Köbner groups in respect to the expression of C3c, iC3b, and cells expressing CD11b or CD14. Of note is the rapid and sustained increase in epidermal C3c and iC3b in the positive Köbner reaction., (© The Author(s) 2024. Published by Oxford University Press on behalf of the British Society for Immunology.)

CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: C3 glomerulopathy (C3G) is caused by overactivity of the alternative pathway of complement that results in bright glomerular C3 staining with minimal or no deposition of immunoglobulins on immunofluorescence microscopy. Laser microdissection and mass spectrometry of the two subtypes, C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), have identified C3 as the predominant glomerular complement protein, although lesser amounts of C9, C5, C6, C7 and C8 are detectable. C3 plays a central role in complement activity, with its proteolytic cleavage first generating C3a and C3b, followed by inactivation of C3b generating iC3b (which includes C3α and C3β), which undergoes further breakdown yielding C3c and terminal breakdown fragment C3dg. The composition of C3 breakdown products in C3G is not known., Methods: In this study, we chose six cases each of C3GN and DDD to analyze the composition of C3 deposits. We analyzed the amino acid sequence of C3 spectra detected by mass spectrometry to determine the relative abundance of C3 fragments in C3G. Thus we were able to determine the amino acid sequences mapping to the various C3 activation products including C3dg, C3α (C3α1 and α2), and C3β that are part of C3b/iC3b/C3c., Results: C3dg is the predominant cleavage product detected with the highest amino acid coverage. The remaining amino acids map to C3α (C3α1 and α2) and C3β. Amino acids mapping to C3a and C3f are absent. Taken together, the C3α and C3β amino acids represent iC3b prior to or after C3c cleavage of C3dg. The C3 spectra for both C3GN and DDD are surprisingly similar., Conclusion: The finding of large amounts of C3dg suggests that C3b deposition in the glomerulus is an active process triggered by thioester binding of C3b to the glycocalyx overlying the glomerular endothelial cells and glomerular basement membrane. Regulatory protein-mediated inactivation of C3b results in the generation of iC3b. After additional cleavages, mostly C3dg remains., (© The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Mycobacterium leprae infection gives rise to the immunologically and histopathologically classified spectrum of leprosy. At present, several tools for the stratification of patients are based on acquired immunity markers. However, the role of innate immunity, particularly the complement system, is largely unexplored. The present retrospective study was undertaken to explore whether the systemic levels of complement activation components and regulators can stratify leprosy patients, particularly in reference to the reactional state of the disease. Serum samples from two cohorts were analysed. The cohort from Bangladesh included multi-bacillary (MB) patients with (n = 12) or without (n = 46) reaction (R) at intake and endemic controls (n = 20). The cohort from Ethiopia included pauci-bacillary (PB) (n = 7) and MB (n = 23) patients without reaction and MB (n = 15) patients with reaction. The results showed that the activation products terminal complement complex (TCC) (P ≤ 0·01), C4d (P ≤ 0·05) and iC3b (P ≤ 0·05) were specifically elevated in Bangladeshi patients with reaction at intake compared to endemic controls. In addition, levels of the regulator clusterin (P ≤ 0·001 without R; P < 0·05 with R) were also elevated in MB patients, irrespective of a reaction. Similar analysis of the Ethiopian cohort confirmed that, irrespective of a reaction, serum TCC levels were increased significantly in patients with reactions compared to patients without reactions (P ≤ 0·05). Our findings suggests that serum TCC levels may prove to be a valuable tool in diagnosing patients at risk of developing reactions., (© 2016 British Society for Immunology.)

In this paper we have extended our earlier studies of the action of increasing Factor I concentration on complement activation by using a soluble activator, lipopolysaccharide (LPS) endotoxin, and using human erythrocytes as a source of CR1 - the co-factor needed for the final clip of iC3b to C3dg by Factor I. Using this more physiological system, the results show that we can predict that a quite modest increase in Factor I concentration - 22 µg/ml of extra Factor I - will convert the activity of the highest risk sera to those of the lowest risk. Preliminary experiments have been performed with erythrocytes allotyped for CR1 number. While we have not been able to perform an adequate study of their co-factor activities in our assays, preliminary experiments suggest that when Factor I levels are increased the difference produced by different allotypes of red cells is largely overcome. This suggests that in patients with paroxysmal nocturnal haemoglobinuria (PNH) treated with eculizumab, additional treatment with Factor I may be very useful in reducing the need for blood transfusion. We have also explored the age-related allele frequency for the two polymorphisms of Factor H and the polymorphism of C3. In our population, unlike the 1975 study, we found no age variation in the allele frequency in these polymorphisms. This may, however, reflect that the Cambridge BioResource volunteers do not include many very young or very elderly patients, and in general comprise a population not greatly at risk of death from infectious disease., (© 2015 British Society for Immunology.)

The interaction between neutrophils and activation of alternative complement pathway plays a pivotal role in the pathogenesis of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). ANCAs activate primed neutrophils to release neutrophil extracellular traps (NETs), which have recently gathered increasing attention in the development of AAV. The relationship between NETs and alternative complement pathway has not been elucidated. The current study aimed to investigate the relationship between NETs and alternative complement pathway. Detection of components of alternative complement pathway on NETs in vitro was assessed by immunostain and confocal microscopy. Complement deposition on NETs were detected after incubation with magnesium salt ethyleneglycol tetraacetic acid (Mg-EGTA)-treated human serum. After incubation of serum with supernatants enriched in ANCA-induced NETs, levels of complement components in supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Complement factor B (Bb) and properdin deposited on NETs in vitro. The deposition of C3b and C5b-9 on NETs incubated with heat-inactivated normal human serum (Hi-NHS) or EGTA-treated Hi-NHS (Mg-EGTA-Hi-NHS) were significantly less than that on NETs incubated with NHS or EGTA-treated NHS (Mg-EGTA-NHS). NETs induced by ANCA could activate the alternative complement cascade in the serum. In the presence of EGTA, C3a, C5a and SC5b-9 concentration decreased from 800·42 ± 244·81 ng/ml, 7·68 ± 1·50 ng/ml, 382·15 ± 159·75 ng/ml in the supernatants enriched in ANCA induced NETs to 479·07 ± 156·2 ng/ml, 4·86 ± 1·26 ng/ml, 212·65 ± 44·40 ng/ml in the supernatants of DNase I-degraded NETs (P < 0·001, P = 0·008, P < 0·001, respectively). NETs could activate the alternative complement pathway, and might thus participate in the pathogenesis of AAV., (© 2015 British Society for Immunology.)

Atypical haemolytic uraemic syndrome (aHUS) is associated with (genetic) alterations in alternative complement pathway. Nevertheless, comprehensive evidence that the complement system in aHUS patients is more prone to activation is still lacking. Therefore, we performed a thorough analysis of complement activation in acute phase and in remission of this disease. Complement activation patterns of the aHUS patients in acute phase and in remission were compared to those of healthy controls. Background levels of complement activation products C3b/c, C3bBbP and terminal complement complex (TCC) were measured using enzyme-linked immunosorbent assay (ELISA) in ethylenediamine tetraacetic acid (EDTA) plasma. In vitro-triggered complement activation in serum samples was studied using zymosan-coating and pathway-specific assay. Furthermore, efficiencies of the C3b/c, C3bBbP and TCC generation in fluid phase during spontaneous activation were analysed. Patients with acute aHUS showed elevated levels of C3b/c (P < 0·01), C3bBbP (P < 0·0001) and TCC (P < 0·0001) in EDTA plasma, while values of patients in remission were normal, compared to those of healthy controls. Using data from a single aHUS patient with complement factor B mutation we illustrated normalization of complement activation during aHUS recovery. Serum samples from patients in remission showed normal in vitro patterns of complement activation and demonstrated normal kinetics of complement activation in the fluid phase. Our data indicate that while aHUS patients have clearly activated complement in acute phase of the disease, this is not the case in remission of aHUS. This knowledge provides important insight into complement regulation in aHUS and may have an impact on monitoring of these patients, particularly when using complement inhibition therapy., (© 2014 British Society for Immunology.)

Leptospira, the causative agent of leptospirosis, interacts with several host molecules, including extracellular matrix components, coagulation cascade proteins, and human complement regulators. Here we demonstrate that acquisition of factor H (FH) on the Leptospira surface is crucial for bacterial survival in the serum and that these spirochetes, besides interacting with FH, FH related-1, and C4b binding protein (C4BP), also acquire FH like-1 from human serum. We also demonstrate that binding to these complement regulators is mediated by leptospiral immunoglobulin-like (Lig) proteins, previously shown to interact with fibronectin, laminin, collagen, elastin, tropoelastin, and fibrinogen. Factor H binds to Lig proteins via short consensus repeat domains 5 and 20. Competition assays suggest that FH and C4BP have distinct binding sites on Lig proteins. Moreover, FH and C4BP bound to immobilized Ligs display cofactor activity, mediating C3b and C4b degradation by factor I. In conclusion, Lig proteins are multifunctional molecules, contributing to leptospiral adhesion and immune evasion.

Given the ability of erythrocytes to bind immune complexes (ICs), we postulated that they can serve a dual role during inflammatory or infectious processes. Erythrocytes could restrict stimulation of macrophages by free ICs by binding C3b-opsonized ICs via their complement receptor 1 (CR1). Conversely, IC-loaded erythrocytes could stimulate macrophages to produce proinflammatory cytokines such as tumour necrosis factor (TNF)-α. To test our hypothesis we selected 72 individuals with low, medium or high red cell CR1 expression and determined their IC binding capacity. We tested the in vitro ability of red cells to inhibit IC-mediated stimulation of TNF-α production by macrophages or to stimulate TNF-α production when loaded with ICs. Plain erythrocytes inhibited IC-induced TNF-α production by macrophages and low CR1 expressors showed the lowest inhibitory capacity. IC-loaded erythrocytes stimulated macrophages to release TNF-α, but the effect was not proportional to the CR1 level. These data support our hypothesis that erythrocytes can serve a dual role in regulation of cytokine responses in a setting of IC formation. Our findings suggest that individuals with low CR1 expression are ill-equipped to clear ICs and prevent IC-mediated stimulation of macrophages. In addition, IC-loaded red cells in areas of sluggish circulation such as in the spleen or in brain capillaries blocked by sequestered malaria-infected red cells may induce inflammation by stimulating monocytes and macrophages, the latter leading to the development of cerebral malaria., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

Induction of molecular proximity can mediate a discrete functional response in biological systems. Therefore, creating new and specific connectivity between non-interacting proteins is a means of imposing rational control over biological processes. According to this principle, here we use composite RNA aptamers to generate molecular adaptors that link various 'target' molecules to a common 'utility' molecule, with the utility molecule being an entry point to a pathway conscripted to process the target molecule. In particular, we created a bi-functional aptamer that simultaneously binds to the green fluorescent protein (serving as a surrogate extracellular target) and the opsonin C3b/iC3b (serving as the utility molecule). This bi-functional aptamer enabled us to commandeer the C3-based opsonization-phagocytosis pathway to selectively transport an extracellular target into the lysosome for degradation. This novel strategy has the potential for powerful therapeutic applications with extracellular proteins involved in tumor development or surface markers on cancer cells as the target molecules.

The macrophage scavenger receptor SR-AI binds to host tissue debris to perform clearance and it binds to bacteria for phagocytosis. In addition, SR-AI modulates macrophage activation through cell signaling. However, investigation of SR-AI signaling on macrophages is complicated due to its promiscuous ligand specificity that overlaps with other macrophage receptors. Therefore, we expressed SR-AI on HEK 293T cells to investigate its ligand binding and signaling. On 293Tcells, SR-AI could respond to E. coli DH5α, leading to NF-κB activation and IL-8 production. However, this requires E. coli DH5α to be sensitized by fresh serum that is treated with heat-inactivation or complement C3 depletion. Anti-C3 antibody inhibits the binding of SR-AI to serum-sensitized DH5α and blocks DH5α stimulation of SR-AI signaling. Further analysis showed that SR-AI can directly bind to purified iC3b but not C3 or C3b. By mutagenesis, The SRCR domain of SR-AI was found to be essential in SR-AI binding to serum-sensitized DH5α. These results revealed a novel property of SR-AI as a complement receptor for iC3b-opsonized bacteria that can elicit cell signaling.

Objective: To determine whether opsonization of Streptococcus pneumoniae with C3b/iC3b is impaired in serum from patients with SLE., Methods: The ability of serum samples from 30 patients with SLE, 20 with non-SLE rheumatic diseases (RA, PsA, AS, SS) and 20 healthy controls to opsonize S. pneumoniae (strains D39 and Io11697) with C3b/iC3b was assessed using a standardized FACS technique and a FACSCalibur flow cytometer. Results were compared among the three groups using analysis of variance, followed by pairwise comparisons among groups using the Mann-Whitney U-test., Results: The proportion of bacteria positive for C3b/iC3b was significantly lower in serum from patients with SLE (strain D39: 60.3% +/- s.e.m. 2.87, strain Io11697: 55.3% +/- 3.8) compared with healthy controls (strain D39: 70.6% +/- 2.0, P = 0.01; strain Io11697: 67.8% +/- 2.6; P = 0.05) and non-SLE rheumatic controls (strain D39: 69.8% +/- 3.1; P = 0.03). For the patients with SLE, there was no association between C3b/iC3b deposition and serum complement levels or measurable classical pathway activity. C3b/iC3b deposition on S. pneumoniae was significantly lower in serum from SLE patients with a past history of pneumonia (n = 3) compared with those without (n = 27; P = 0.03)., Conclusions: Opsonization of S. pneumoniae with C3b/iC3b was significantly reduced in serum from patients with SLE compared with non-SLE rheumatic disease and healthy controls. Failure to appropriately activate the immune system via complement may contribute to the increased susceptibility of SLE subjects to infections, and may correlate with a risk of pneumonia in a subgroup of SLE patients.

The human complement system plays an important role in the control of Staphylococcus aureus infection. For instance, we previously demonstrated that the central complement component deposited on the organism's surface, C3b, can be cleaved by the host complement control protein, factor I, resulting in diminished phagocytosis of S. aureus. In the present study, we have identified clumping factor A (ClfA) from cell wall proteins of S. aureus as a specific protein bound by factor I. Recombinant ClfA (rClfA) containing the full-length A region (peptides 40-559) also bound factor I. We identified an 50-kDa fragment of ClfA that is shed by S. aureus into growth medium. The shed ClfA fragment was derived from the A region of ClfA and bound factor I. rClfA and the shed ClfA fragment increased factor I cleavage of C3b into inactive C3b. Our findings describe a new S. aureus mechanism for modification of host complement activities.

Human oviductal epithelial (OE) cells produce complement protein 3 (C3) and its derivatives, C3b and inactivated complement-3b (iC3b). Among them, iC3b is the most potent embryotrophic molecule. We studied the production of iC3b in the oviductal cell/embryo culture system. In the immune system, C3 convertase converts C3 into C3b, and the conversion of C3b to iC3b requires factor I (fI) and its cofactors, such as factor H or membrane cofactor protein. Human oviductal epithelium and OE cells expressed mRNA and protein of the components of C3 convertase, including C2, C4, factor B, and factor D. The OE cell-conditioned medium contained active C3 convertase activity that was suppressed by C3 convertase inhibitor, H17 in a dose and time-dependent manner. Although the oviductal epithelium and OE cells produced fI, the production of its cofactor, factor H required for the conversion of C3b to iC3b, was weak. Thus, OE cell-conditioned medium was inefficient in producing iC3b from exogenous C3b. On the contrary, mouse embryos facilitated such conversion to iC3b, which was taken up by the embryos, resulting in the formation of more blastocysts of larger size. The facilitatory activity was mediated by complement receptor 1-related gene/protein Y (Crry) with known membrane cofactor protein activity on the trophectoderm of the embryos as anti-Crry antibody inhibited the conversion and embryotrophic activity of C3b in the presence of fI. In conclusion, human oviduct possesses C3 convertase activity converting C3 to C3b, and Crry of the preimplantation embryos may be involved in the production of embryotrophic iC3b on the surface of the embryos.

The effect of coupling C3bi to immunoglobulin G (IgG) immune complexes (IC) on their ability to produce protein tyrosine phosphorylation and activation of the mitogen-activated protein kinase (MAPK) and the Akt/protein kinase B (PKB) routes was assessed in human monocytes. Cross-linking Fc receptors for IgG activated the protein tyrosine kinase Syk, phospholipases Cgamma1 and Cgamma2, the MAPK cascade, and the Akt/PKB route. Linkage of C3bi to the gamma-chain of IgG produced a decrease of the protein bands displaying tyrosine phosphorylation, whereas the MAPK cascades and the Akt/PKB route remained almost unaffected. Zymosan particles, which because of their beta-glucan content mimic the effect of fungi, produced a limited increase of tyrosine-phosphorylated protein bands, whereas treatment of zymosan under conditions adequate for C3bi coating increased its ability to induce protein tyrosine phosphorylation. Noteworthy, this was also observed under conditions where other components of serum might be bound by zymosan particles, for instance, serum IgG, thereby suggesting their potential involvement in Syk activation. The induction of cytokines showed a changing pattern consistent with the changes observed in the signaling pathways. IC induced monocyte chemoattractant protein-1 (MCP-1)/CC chemokine ligand 2 (CCL2), interleukin (IL)-1beta, and eotaxin-2/CCL24, which were not observed with C3bi-coated IC. Zymosan induced the expression of tumor necrosis factor alpha (TNF-alpha), TNF-beta, IL-10, IL-6, and MCP-2/CCL8, whereas the cytokine signature of C3bi-coated zymosan also included interferon-inducible protein 10/CXC chemokine ligand 10, platelet-derived growth factor-BB, and I-309/CCL1. Taken together, these findings indicate that C3bi targets the phagocytic cargo, and engagement or diversion of the Syk route determines the phagocyte response.

Mannose-binding lectin (MBL) is a serum collectin believed to be of importance in innate immunity. We have investigated the role of MBL in the first-line defense against Candida albicans, an opportunistic fungal pathogen. MBL bound C. albicans via its lectin domain, resulting in agglutination of the organisms upon their outgrowth of hyphae. In a human in vitro MBL system, deposition of C4 fragments on C. albicans was increased when exogenous MBL was added to serum samples from MBL-deficient individuals. Similar enhancement of deposition of iC3b also was observed. MBL and enhanced opsonic C3 fragments mediated by MBL did not facilitate opsonophagocytosis of the organisms by monocyte-derived dendritic cells (DCs). However, MBL was found to inhibit the growth of C. albicans independently of complement activation, although, with complement activation, further inhibition was observed. We concluded that MBL plays an important role in the first-line defense against C. albicans without the need for opsonophagocytosis by DCs, in which a direct interaction of MBL with C. albicans results in agglutination and accelerated complement activation via the lectin pathway, leading to inhibition of growth.

Macrophage-stimulating protein (MSP) promotes the phagocytosis of C3bi-coated erythrocytes by resident peritoneal macrophages, although the mechanism by which this occurs is largely unknown. We show that MSP-induced complement-mediated phagocytosis requires the RON receptor tyrosine kinase and the alphaMbeta2 integrin, as evidenced by the inability of RON-/- and alphaM-/- peritoneal macrophages to augment phagocytosis of complement-coated sheep erythrocytes in response to MSP. MSP stimulation of macrophages results in tyrosine phosphorylation and AKT activation, and inhibitor studies demonstrate a phagocytic requirement for tyrosine kinase and phosphatidylinositol 3-kinase (PI-3K) activity as well as activity of the atypical protein kinase C (PKC) isoform zeta, which localizes to MSP-induced phagosomes containing complement-coated beads. Additionally, MSP augments the ability of peritoneal macrophages to bind to intercellular adhesion molecule-1 (ICAM-1) via the alphaMbeta2 integrin. MSP-induced ICAM-1 adhesion is also dependent on tyrosine kinase activity, PI-3K, and PKC zeta, indicating that these signaling requirements are upstream of complement receptor 3 activation.

Human C4b-binding protein (C4bp) facilitates the factor I-mediated proteolytic cleavage of the active forms of complement effectors C3b and C4b into their inactive forms. C4bp comprises a disulfide-linked heptamer of alpha-chains with complement (C) regulatory activity and a beta-chain. Each alpha-chain contains 8 short consensus repeat (SCR) domains. Using SCR-deletion mutants of recombinant multimeric C4bp, we identified the domains responsible for the C3b/C4b-binding and C3b/C4b-inactivating cofactor activity. The C4bp mutant with deletion of SCR2 lost the C4b-binding ability, as judged on C3b/C4b-Sepharose binding assaying and ELISA. In contrast, the essential domains for C3b-binding extended more to the C-terminus, exceeding SCR4. Using fluid phase cofactor assaying and deletion mutants of C4bp, SCR2 and 3 were found to be indispensable for C4b cleavage by factor I, and SCR1 contributed to full expression of the factor I-mediated C4b cleaving activity. On the other hand, SCR1, 2, 3, 4, and 5 participated in the factor I-cofactor activity for C3b cleavage, and SCR2, 3, and 4 were absolutely required for C3b inactivation. Thus, different sets of SCRs participate in C3b and C4b inactivation, and the domain repertoire supporting C3b cofactor activity is broader than that supporting C4b inactivation by C4bp and factor I. Furthermore, the domains participating in C3b/C4b binding are not always identical to those responsible for cofactor activity. The necessity of the wide range of SCRs in C3b inactivation compared to C4b inactivation by C4bp and factor I may reflect the physiological properties of C4bp, which is mainly directed to C4b rather than C3b.

CD46, a complement regulatory protein widely expressed on human cells, serves as an entry receptor for measles virus (MV). We have previously shown that the expression of human CD46 in mouse macrophages restricts MV replication in these cells and enhances the production of nitric oxide (NO) in the presence of gamma interferon (IFN-gamma). In this study, we show that crosslinking human CD46 expressed on the mouse macrophage-like cell line RAW264.7 with purified C3b multimer but not monomer enhances NO production. The enhanced production of NO in response to IFN-gamma was observed again with C3b multimer but not monomer. The augmentation of NO production is human CD46-dependent with a CYT1>CYT2 profile. Thus, the reported MV-mediated NO production, irrespective of whether it is IFN-gamma-dependent or -independent, should be largely attributable to CD46 signaling but not to MV replication. Similar CYT1-dependent augmentation of NO production was reproducible with two CD46 ligating reagents, CD46-specific monoclonal antibodies (mAb) or their F(ab')(2) and MV hemagglutinin (H) and fusion (F) glycoproteins. Co-cultivation of mouse macrophages bearing human CD46 with Chinese hamster ovary (CHO) cells expressing MV H and F enhanced IFN-gamma-induced NO production. Yet, the NO levels induced by F(ab')(2) against CD46 or MV H/F on CHO cells were much lower than those induced by CD46-crosslinking mAb with Fc or MV infection. Removing the cytoplasmic tails of CD46 abrogated the augmentation of NO production triggered by all three stimulators. Thus, the CD46 CYT1 and CYT2 isoforms functionally diverge to elicit innate immune responses, which can be modulated by purified C3b multimer or anti-CD46 mAbs.

The production of nitric oxide (NO) within neutrophils is an important element of the innate immune response. We have previously shown that cytokines (IL-1alpha, tumour necrosis factor-alpha and interferon-gamma) induce human neutrophils in buffy coat preparations to produce iNOS. In order to define better the exact requirements for iNOS production within human neutrophils, we have studied the conditions needed for the production of iNOS in purified neutrophils. In contrast to buffy coat preparations, purified neutrophils in suspension did not produce an increase in iNOS following addition of cytokines. However, when purified neutrophils were allowed to adhere to glass surfaces either uncoated or coated with fetal calf serum (FCS), plasma, fibronectin or laminin, there was an increase in the percentage of iNOS-positive cells. The addition of cytokines during adhesion of these cells increased this proportion further. This was most marked for glass alone and FCS-coated glass on which the proportion of iNOS-positive cells increased to 22.7% and 35.5%, respectively, a significant increase compared with cytokine-treated neutrophils in suspension. Neither transmigration through activated endothelial monolayers nor the addition of soluble intercellular adhesion molecule-1 to purified neutrophil suspensions increased the percentage of iNOS-positive cells following cytokine stimulation. Adhesion of neutrophils to surfaces coated with IgG or complement also failed to increase cytokine-induced iNOS production. We conclude that iNOS production in human neutrophils requires not only cytokine stimulation, but also additional stimuli from adhesion to a surface.

The beta2 (CD18) leukocyte integrins play a key role in normal and inflammatory immune responses. In resting leukocytes, these receptors do not bind ligands. However, when leukocytes are exposed to an appropriate agonist, high-affinity ligand binding is achieved, presumably as a result of conformational changes in the integrin. In this study, we describe a novel monoclonal antibody, mAb 6C1, directed against the alphaM subunit, which directly induces adhesion of alphaMbeta2-transfected CHO cells to fibrinogen, ICAM-1, and iC3b. Induction of binding could also be accomplished by monovalent Fab fragments of mAb 6C1 at concentrations similar to that observed with intact IgG, demonstrating stimulation of adhesion was not because of receptor cross-linking at the cell surface. The binding of mAb 6C1 induces conformational changes in the receptor, as evidenced by the expression of an "activation reporter" epitope recognized by mAb 24. The binding of mAb 6C1 is modulated by divalent cations. Mn2+ promoted high levels of 6C1 binding, and Mg2+ supported low levels of binding, however Ca2+ failed to support binding. A unique distinction of mAb 6C1 is localization of its epitope to the alphaM I domain. The alphaM I domain is essential for ligand binding, can directly bind divalent cations, and participates in the regulation of alphaMbeta2 ligand-binding affinity. Thus, these studies have identified a novel alphaM I domain activation epitope of alphaMbeta2 and support the idea that the I domain modulates the activational state of the beta2 integrins.

The aetiopathogenesis of chronic otitis media with effusion (OME) in children is not yet fully understood. OME is characterized by metaplasia of the epithelium and accumulation of sticky, glue-like effusion in the middle ear containing different mediators of inflammation, including activation fragments of the complement system. Here we examined whether the fluid phase complement activation is reflected in the middle ear mucosa and how the mucosa is protected against the cytolytic activity of complement. Mucosal biopsies from 18 middle ears of children with a history of chronic OME were taken. The biopsies were analysed by immunofluorescence microscopy after staining for complement fragments iC3b/C3c, C3d and C9, and regulators membrane cofactor protein (MCP; CD46), decay-accelerating factor (DAF; CD55) and protectin (CD59). There was a strong staining for iC3b/C3c, and a weaker one for C3d and C9 on the surface of the middle ear epithelial cells of OME patients but not in controls without OME. MCP was expressed on the hyperplastic three to four outer cell layers of the epithelium, while CD59 was expressed throughout the middle ear mucosa. The results suggest a strong ongoing complement activation and consequent inflammation in the middle ear cavity. Unrestricted complement damage of the epithelial lining is prevented by the strong expression of MCP and CD59.

Local inflammation elicited by Neisseria gonorrhoeae correlates closely with sensitivity to killing by normal human serum. Serum-sensitive (SS) isolates are rendered resistant in vitro by lipooligosaccharide sialylation. Differences in C3b processing on N. gonorrhoeae in vitro were found to match findings at the cervical level in vivo. Nonsialylated SS gonococci bound 5-fold more C3b than did stably serum-resistant (SR) gonococci; most was processed to iC3b, yet significant C3b persisted. Sialylated SS gonococci bound 4-fold less total C3 antigen than did SR gonococci, which was promptly converted to iC3b. C3b bound later on stably SR gonococci but again was processed swiftly to iC3b. In vivo, the iC3b/C3 ratio of SS isolates more closely resembled nonsialylated SS isolates in vitro, implying heterogeneous sialylation or desialylation in vivo. In vitro, total IgM bound was unchanged by sialylation of SS isolates, but total C4 bound decreased by 75%, suggesting that sialylation may indirectly regulate the classical complement pathway.

The beta2 leukocyte integrins comprise a group of closely related adhesion receptors that mediate critical events during normal and inflammatory immune responses. Central to the understanding of beta2 integrin function is the basis of ligand recognition. Results from our laboratory and others indicate the presence of multiple ligand contact points in both the alpha and beta subunit. As an approach to identify and characterize regulatory domains of the beta2 subunit, we have generated two different subdomains of the beta2 subunit for expression on the surface of mammalian cells through a phosphatidyl-inositol glycan anchor. The first subdomain contains the putative beta2 MIDAS motif implicated in ligand binding [beta2(LB)], whereas the second beta2 subdomain contains the cysteine-rich region [beta2(CR)]. Cells expressing alphaM and beta2 constructs singly or cotransfected transiently in COS-7 cells were tested for the ability to bind to immobilized iC3b. Cells bearing the recombinant alphaMbeta2(LB) were capable of adhering to iC3b in a manner similar to that observed with the complete alphaMbeta2 heterodimer. In contrast, cells expressing alphaMbeta2(CR) failed to adhere to immobilized iC3b. Moreover, cells bearing singly transfected alpha or beta chains alone failed to adhere to immobilized iC3b. These results indicate that along with alphaM, the beta2(LB) subdomain contains the sufficient components within the beta2 subunit essential for ligand recognition. These findings support the hypothesis that the beta2 subunit cooperates with site(s) within the alphaM subunit in a receptor/cation/ligand complex resulting in high-affinity ligand interaction.

Treatment with i.v.Ig can, on rare occasions, lead to detrimental effects such as enhanced erythrocyte sequestration and an increase in serum immune complexes with inflammatory sequellae such as exacerbation of glomerular nephritis. In this study, i.v.Ig (Sandoglobin) was examined for complement binding moieties which resemble immune complexes and can mediate the binding of IgG and C'3b to human erythrocytes via CR1 and enhance erythrocyte susceptibility to sequestration. Sephacryl S-200 HR separated i.v.Ig into two fractions: monomeric IgG (74%) and larger complexes of the molecular weight of an IgG dimer or greater (> or = 300 kD) (26%). In the presence of complement, the 'dimers' bound to human erythrocytes, rendering them susceptible to phagocytosis in vitro. Removal of erythrocyte-specific isoantibodies from the i.v.Ig had no effect on 'dimer' binding to the erythrocytes. Monomeric IgG contained virtually no complement-activating, erythrocyte-binding activity. Erythrocyte binding of complement-bearing IgG 'dimers' and subsequent phagocytosis resembles the binding of complement-bearing immune complexes to erythrocyte CR1. Exposure to Factor I leads to the release of complement-bearing IgG 'dimers' from erythrocyte CR1 and to the abrogation of erythrophagocytosis. Binding of complement-bearing IgG 'dimers' to the erythrocyte is blocked by To5, a CR1-specific monoclonal antibody.

We have investigated the interaction of complement-coated erythrocytes (E) with neutrophils, in vitro, to determine the effects of erythrocyte CR1. Complement-coating in vitro, to mimic immune complex uptake, caused a reduction in E-CR1, but subsequent interaction with neutrophils effected a removal of E-C3b and a return of E-CR1 to levels approximating that of uncoated erythrocytes. These data indicate that C3b, associated with immune complexes, may bind to E-CR1 in a reversible manner and that subsequent interaction with phagocytes need not necessarily result in cleavage (and loss of) E-CR1. Further, we conclude that E-CRI can apparently be reduced by epitope masking.

The murine CD18 monoclonal antibody (mAb) M18/2 was reported to inhibit lymphoma metastasis [Zahalka, M. A. et al. (1993) J. Immunol. 150, 4466]. To identify the pathways potentially blocked, we studied the effects of M18/2 compared with two new mAb against murine CD18, GAME-46, and -245. Whereas the GAME mAb blocked most Mac-1-mediated interactions, M18/2 had no effect, or even stimulated. The same was true for adhesion of LFA-1 to ICAM-1. To test effects on interactions with different ICAMs, we used L cells transfected with human ICAM-1, -2, and -3. As previously described, mouse LFA-1 does not bind to human ICAM-1 but we show here that mouse LFA-1 does bind to human ICAM-2 and -3. Again, the GAME mAb blocked completely, but M18/2 did not. These results indicate that the LFA-1 binding sites for ICAM-1 and ICAM-2 and -3, although in close vicinity, are distinct. Furthermore, effects of M18/2 on metastasis cannot be ascribed to blocking of any known beta2-integrin activity.

The third component of complement (C3) is central to both the classical and alternative pathways in complement activation. In this study, involvement of C3 activation in the mucosal injury of UC was investigated. We examined the distribution of activated (C3b) and degraded fragments (iC3b/C3dg) of C3, terminal complement complex (TCC), and complement regulatory proteins in normal and diseased colonic mucosa including UC and other types of colitis using immunohistochemical techniques at the level of light and electron microscopy. While C3b and iC3b/C3dg staining was negligible in the normal mucosa, iC3b/C3dg and, to a lesser extent, C3b were deposited in UC mucosa along the epithelial basement membrane. The deposition was enhanced in relation to the severity of mucosal inflammation (C3b, P less than 0.05; iC3b/C3dg, P less than 0.01). Epithelial deposition of TCC was not observed in most UC mucosa. Immunoelectron microscopy showed that C3b and iC3b/C3dg were distributed mainly along the epithelial basement membrane and the underlying connective tissue in a granular, studded manner, and weakly present along the basolateral surface of epithelial cells. These C3 fragments were also deposited in inflammatory control mucosa such as ischaemic and infectious colitis. Our findings suggest that deposition of the C3 fragments occurs in inflamed colonic mucosa of diverse etiologies, including UC, but to define a role of the deposition in the development of mucosal injury in UC awaits direct study.

Complement activation generally does not occur on homologous cells. We observed C3 deposition on cultured human umbilical vein endothelial cells (HUVEC) when those which had died of apoptosis were treated with human serum. The C3 deposition on apoptotic HUVEC required Mg2+ but not Ca2+. In addition, the incubation of apoptotic HUVEC with purified C3, B, and D in the presence of Mg2+ resulted in C3 deposition. These results indicated that the C3 deposition on apoptotic HUVEC is mediated by the activation of the alternative complement pathway. C3 contains an intrachain thioester bond in the alpha chain (110 kDa) and upon activation to C3b, binds with membrane molecules by forming an ester or amide bond. Western blotting of reduced C3b-membrane molecule complexes, isolated from serum-treated apoptotic HUVEC by immunoprecipitation with anti-C3, revealed the covalent binding of C3b to several membrane molecules. Most of the C3b-membrane molecule complexes were cleaved by hydroxylamine, suggesting covalent binding via an ester bond. The molecular mass of the major alpha chain fragment released by hydroxylamine treatment was not 105 kDa but 68 kDa, which corresponds to the alpha 1 fragment of iC3b. These results indicate that most of the C3b on HUVEC was cleaved at its alpha' chain to yield iC3b, which consists of three disulfide-linked polypeptide chains and is a ligand of the complement receptor type 3 (CR3) of phagocytes. These results suggest that apoptotic HUVEC can activate the alternative pathway of the homologous complement and that the complement is related to the clearance of apoptotic cells by phagocytes.

Mesophilic Aeromonas spp. strains (serotype O:34) showed sensitivity to complement-mediated killing when they were cultivated at 37 degrees C (serum-sensitive) but not when they were cultivated at 20 degrees C (serum-resistant). These strains produced smooth lipopolysaccharide when they were grown at 20 degrees C and rough lipolysaccharide when cultivated at 37 degrees C. The reason for the resistance to complement-mediated killing could be that C3b is rapidly degraded (possibly because it is bound far from the cell membrane), consequently the lytic complex (C5b-9) is not formed.

In this study we have investigated the ability of lipopolysaccharide (LPS) to suppress binding and phagocytosis of erythrocytes via various receptors on mouse macrophages. Thioglycollate-elicited peritoneal macrophages were treated in vitro with LPS and the ability to bind and phagocytose radiolabeled sheep red blood cells was determined. We show that LPS can directly suppress phagocytosis of immunoglobulin G-opsonized and nonopsonized sheep red blood cells (SRBCs) by inflammatory macrophages. Suppression was dose dependent and was observed after 4 h of exposure. This effect lasted for at least 24 h following the removal of LPS. LPS suppressed the binding, rate, and absolute level of phagocytosis via Fc receptors. Phagocytosis via all Fc receptors (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) was suppressed by LPS. Furthermore, suppression was not limited to Fc receptor-mediated phagocytosis because binding and uptake of C3bi-opsonized SRBCs to CR3 receptors was also decreased following LPS treatment. LPS did not exert its effects via the production of interleukin-1 (IL-1), IL-6, tumor necrosis factor alpha, or interferon alpha/beta, because antibodies to these cytokines did not abrogate the effect. The ability of LPS to suppress binding and phagocytosis of microorganisms may contribute to the toxic effects of LPS during gram-negative sepsis by preventing or delaying elimination of bacteria by host macrophages.

HA-1A has been shown clinically to decrease mortality in septic patients with gram-negative bacteremia. In this study, the ability of HA-1A to augment the serum complement-dependent immune adherence of 125I-labeled Escherichia coli J5 lipopolysaccharide (LPS) to human erythrocytes (RBC) and polymorphonuclear leukocytes (PMNL) was evaluated. In vitro studies indicated three things: HA-1A mediates immune adherence of 125I-J5 LPS to human RBC and PMNL in a dose-dependent manner; under these conditions, high concentrations of LPS (400 ng/mL) could be specifically bound. Immune adherence occurs via the classical complement pathway as demonstrated by its calcium dependence; HA-1A-J5 LPS-C' immune complexes bound to CR1 on human RBC and PMNL. PMNL binding and internalization of immune complexes was demonstrated by trypsin stripping of externally bound immune complexes. These studies support the proposal that HA-1A can lower the bioavailability of endotoxin by mediating binding and potential clearance of LPS via human RBC through the reticuloendothelial system or via direct internalization by peripheral blood PMNL.

A major protein secreted by vaccinia virus-infected cells has structural similarity to the super-family of complement-control proteins. This vaccinia complement-control protein (VCP) was studied to determine how it regulates complement activation. VCP was bound by C4b and C3b and served as a cofactor with factor I in cleaving these two molecules. VCP inhibited the formation and accelerated the decay of the classical C3 convertase. It also accelerated decay of the alternative pathway convertase, although higher concentrations were apparently needed. In vitro, therefore, VCP interfered with the classical and alternative complement pathways at several steps. In vivo, this interference may increase the virulence of vaccinia virus by enabling it to escape attack by the host's complement system.

The present study investigated the rate of catabolism of CR1 (the C3b receptor, CD35) on erythrocytes (E) in vivo, in relationship with the expressed number of CR1/E, the CR1.1 HindIII quantitative CR1 polymorphism, and cell age. The relationship between the number of CR1/E and cell age was analysed by measuring G6PDH activity in E that had been sorted according to high or low expression of CR1 (CD35), by assessing the expression of CR1 (CD35) on E separated according to cell density, and by comparing the number of CR1 (CD35) antigenic sites on reticulocytes and on E. A physiological catabolism of CR1 (CD35) manifested by a reduction in the number of CR1 (CD35) antigenic sites/E with cell ageing was consistently observed in healthy individuals. The number of CR1/E decreased with ageing of E according to a complex pattern that associated an exponential decay and an offset. Calculated half-lives of CR1 (CD35) ranged between 11 and 32 days in healthy individuals. A more rapid loss of CR1 (CD35) with cell ageing occurred on cells from individuals expressing high numbers of CR1/E. In patients with systemic lupus erythematosus (SLE), half-lives of CR1 (CD35) on E were in the same range as those of healthy individuals with a similar quantitative CR1 genotype; the number of CR1 (CD35) on reticulocytes was reduced and linearly related to the number of CR1/E, independently of the patients' quantitative CR1 genotype. Transfusion experiments with E bearing high or low amounts of CR1/E indicated the lack of preferential removal of E bearing high numbers of CR1 (CD35) in patients with SLE. These results indicate that the rate of loss of CR1 (CD35) from E with cell ageing is directly related to the quantitative CR1 phenotype and suggest that enhanced peripheral catabolism is not the sole mechanism of the acquired loss of CR1 (CD35) on E in patients with SLE.

Alveolar macrophages (AM phi) were examined for CR1 (C3b receptor, CD35), CR3 (iC3b receptor; CD11b/CD18), and CR4 (iC3b receptor; CD11c/CD18) by assays for binding of C3-opsonized sheep erythrocytes (EC3b or EC3bi) and uptake of specific monoclonal antibodies (mAbs). In AM phi isolates from nine normal volunteers, 49% of cells bound EC3b and 71% bound EC3bi. Quantitation of receptors per cell with [125I]mAbs showed 8.5 x 10(4) CR4, 5.1 x 10(4) CR3, and 2.6 x 10(4) CR1. With most AM phi preparations, CR3 was the major receptor mediating attachment of EC3bi, despite the predominance of CR4 antigens. Anti-CR3 inhibited EC3bi rosettes by > or = 50%, whereas anti-CR4 blocked rosettes by < or = 18%. U937 cells differentiated with phorbol myristate acetate resembled AM phi in receptor expression but exhibited almost no CR4-dependent rosetting. Despite the relative inability of CR4 to mediate EC3bi attachment, AM phi ingestion of [51Cr]EC3bi was blocked by either anti-CR3 or anti-CR4. Two lines of evidence indicated that CR3 were more mobile within the membrane than were CR4. Immunofluorescence staining demonstrated patching and occasional capping of CR3, whereas CR4 remained uniformly distributed. This patching and capping of CR3 required the actin cytoskeleton, as it was inhibited by cytochalasin D. Modulation experiments using surfaces coated with anti-CR3 or anti-CR4 also showed that CR3 was more mobile than was CR4. However, there was some variation among AM phi isolates from different donors. In seven isolates, no CR4 modulation was produced with anti-CR4, whereas in six other isolates, CR4 was modulated by 66%. Incubation of cells in cytochalasin D increased modulation of both CR3 and CR4 on mAb-coated surfaces. Cells exhibiting increased mobility of CR4 showed an increased ability to form CR4-dependent EC3bi rosettes. The data are consistent with the hypothesis that CR3 and CR4 exhibit a variable association with the cytoskeleton that regulates their mobility and function. A relatively mobile subset of CR3 and/or CR4 mediates EC3bi attachment, whereas a relatively immobile subset of CR3 and/or CR4 fails to mediate EC3bi attachment but functions to promote ingestion of EC3bi.

Herpes simplex virus type 1-infected cells bind C3b and iC3b, but not C3d, at the cell surface. Herpes simplex virus type 2 (HSV-2)-infected cells bind none of these C3 fragments. A transfection assay was used to demonstrate that binding of iC3b was to gC1. Although iC3b did not bind to HSV-2-infected cells, it did bind to mammalian cells transfected with the gC2 gene. Using linker insertion mutants, three domains on gC2 that are important for binding iC3b were mapped; these regions were similar to previously defined regions involved in binding C3b. These results suggest that some of the functions served by gC may be similar to those of CR3, the mammalian receptor for iC3b.

C3b-coated immune complexes adhere to the complement receptor 1 (CR1, CD35) on human erythrocytes. This multi-valent binding might be favoured by the known clustering of CR1 and by the multiple C3b-binding sites on each CR1. The size of the CR1 clusters correlates directly with the number of CR1/erythrocytes, and the different structural CR1 alleles bear between two and five C3b-binding sites. Using radiolabelled hepatitis B surface antigen-antibody complexes, we investigated whether CR1 numbers and structural alleles modulate the ability of erythrocytes to bind immune complexes, and assessed if any reorganization of immune complexes takes place at the erythrocyte surface after the initial binding reaction. The binding efficiency (immune complexes/CR1) correlated with CR1 number as determined by the maximal binding at 4 degrees C, the kinetics of binding at 37 degrees C, and the binding in the presence of excess immune complexes and of immune complexes of small size. Binding efficiencies were similar for erythrocytes with low CR1 from normal subjects and patients with AIDS or SLE. A monoclonal antibody blocking the C3b-binding sites (3D9) of CR1 interfered with binding efficiency at a lower concentration on cells bearing low CR1 numbers, suggesting that CR1 clustering is essential. The larger alleles of CR1 (DD and BB) were more efficient than AA alleles. The distribution of immune complexes, visualized by immunofluorescence, was heterogeneous on erythrocytes: about two out of three cells bore between one and 12 immune complexes. No visible immune complex reorganization took place after initial binding, as prefixed erythrocytes displayed the same immune complex distribution and number/erythrocytes as unfixed erythrocytes. The contribution of CR1 alleles in immune complex binding efficiency was confirmed by morphological analysis. These results demonstrate that immune adherence efficiency is the resultant of the CR1 clustering, as well as the particular alleles carried by erythrocytes. Moreover, there is little or no immune complexes surface reorganization after the initial binding reaction.

The co-expression of C5a and formyl-methionine-leucine-phenylalanine-lysine (FMLPL) receptors with CR1, CR3, and Fc receptors on human neutrophils (PMN) was studied. Fluorescein-conjugated C5a (FL-C5a) and FMLPL (FL-FMLPL) were used to identify C5a and formyl peptide receptors. CR1, CR3, and Fc receptors were identified with monoclonal antibodies and a Texas red-labeled goat anti-mouse immunoglobulin second step reagent. The co-expression of chemotactic receptors with CR1, CR3, or Fc receptors was evaluated using two-color flow cytometry. A direct correlation between the degree of expression of receptors for FL-FMLPL and the expression of CR3, CR1, and Fc receptors on individual PMN was observed. In contrast, no correlation between the degree of C5a receptor expression and CR1, CR3, or Fc receptor expression was found. Similar results were obtained with PMN after up regulation of CR1, CR3, Fc, and FMLPL receptors by incubation at 37 degrees C for 10 min with or without phorbol myristate acetate. These data suggest that the expression of FMLPL, CR1, CR3, and Fc receptors are regulated in a similar manner, whereas C5a receptor expression is regulated independently. Furthermore, these data indicate that within a given population of PMN, a parallel exists between the degree of CR1, CR3, FMLPL, and Fc receptor expression on individual cells.

We investigated whether a decreased complement receptor expression or function of monocytes isolated from peripheral blood of 52 patients with rheumatoid arthritis (RA) and rheumatoid vasculitis (RV) could account for the previously observed diminished degradation of immune complexes by monocytes of patients with RA and RV. On average, monocytes from all patients expressed significantly less CR1, and degraded significantly less AC3b when compared with monocytes of healthy controls. In addition, monocytes from RV patients degraded significantly less AC3b when compared with monocytes from patients with RA. The expression of both CR1 and CR3 on monocytes of RV patients was lower compared with RA patients but this difference was only significant for CR3. No differences were found between AC3b degradation and the expression of CR1 and CR3 between patients with active and inactive RA. Using linear discriminant analysis on the variables AC3b, CR1 and CR3, 94% of the patients could be classified correctly as healthy controls, RA or RV, suggesting a true multi-variate relationship between these parameters and patients groups. Our results suggest that the diminished capacity of monocytes from RA patients to degrade AC3b is due partly to a decreased expression of CR1 and CR3.

Patient isolates of Candida albicans from blood, urine, or mucosal sites express a surface receptor for C3 fragment iC3b that is recognized by monoclonal antibodies (MAb) directed against alpha-chain epitopes of the neutrophil iC3b receptor, also known as the type 3 complement receptor (CR3) or CD11b/CD18. Because 60% of these patients were hyperglycemic, the effect of glucose on receptor expression was investigated. As assessed by flow cytometry, yeasts grown in 20 mM D-glucose exhibited a four- to six-fold increase in receptor expression compared with yeasts grown in 20 mM L-glutamate. Receptor expression increased as glucose concentration rose from 0 to 20 mM (equivalent to plasma glucose concentrations of 0-360 mg/dl). Augmentation of receptor expression by growth in glucose led to significant inhibition of phagocytosis compared with that of organisms grown in equimolar L-glutamate. SDS-PAGE, Western blotting, and immunodetection of extracts of yeast cell wall, membrane, and cytosol disclosed a protein of 165 kDa in membrane and cytosolic extracts, consistent with the published Mr of the alpha-chain of neutrophil CR3. These studies provide a mechanism to explain the predilection of C. albicans to infect the hyperglycemic host.

C3b/C4b receptor (CR1) and membrane cofactor protein (MCP) are integral membrane glycoproteins with factor I-dependent cofactor activity. They bind to C3b, allowing factor I to cleave C3b at two sites (first and second cleavage), which results in the generation of C3bi, a hemolytically inactive form which is a ligand for complement receptor type three (CR3). C3bi is further degraded by factor I and CR1 (third cleavage) to C3dg (a ligand for complement receptor type two, CR2) and C3c. Using two different substrates, fluid-phase C3b and cell-bound C3b, the cleavage of C3b by MCP and factor I was compared to that by CR1 and factor I under various conditions. The optimal pH for the first and second cleavage of either substrate was 6.0 for MCP and 7.5 for CR1. The third cleavage was mediated only by CR1 and factor I, the optimal pH being 8.0. Low ionic conditions enhanced the C3b binding and cofactor activity of both CR1 and MCP. The efficiency of binding C3b to CR1 or MCP was maximal at pH 6.2. The isoelectric point (pI) of MCP was acidic (approximately 4.0), while that of CR1 was 6.8. Therefore, compared to CR1, MCP possesses distinct functional profiles relative to C3b-binding and factor I-cofactor activity.

When sera diluted to 5% in a buffer containing calcium and magnesium were incubated with mannan-coated ELISA plates, C4 fragments, properdin and factor B were bound to the plates as well as the expected opsonic C3 fragments, C3b and C3bi. The calcium-dependent lectin mannan-binding protein, which is structurally similar to C1q, was also shown to bind in this assay and analysis of sera from 179 healthy blood donors revealed that the binding levels of all these proteins were highly significantly correlated. Results obtained with a previously described C3b opsonic assay using zymosan also correlated with the mannan-binding levels. When the sera were diluted to 5% in the presence of Mg-EGTA there was no detectable binding of complement proteins to the mannan surface, confirming that no alternative pathway activation occurred at this serum concentration. When sera were diluted to 5% in a buffer containing EDTA in order to study immunoglobulin binding in the absence of complement activation, the levels of bound IgG1, IgG2, IgG3, IgA and IgM antibodies were found to be completely unrelated to the C3bi binding levels previously observed. The results suggest that in this experimental system using low concentrations of serum, mannan-binding protein initiates an antibody-independent mechanism of cleavage of the classical pathway component C4, which subsequently regulates the degree of cleavage of C3 and recruitment of alternative pathway proteins.

Using a correction assay a factor essential for normal deposition of C3 fragments on zymosan was identified in fractions of serum obtained by Sephacryl S-300 gel filtration (eluting between IgG and albumin), preparative Pevikon block electrophoresis (eluting in the beta-region) and DEAE-ion-exchange chromatography (eluting immediately post IgG). Active material was also identified in a commercial preparation of transferrin. A combination of DEAE-ion-exchange chromatography and gel filtration was used to partially purify the co-factor. The factor has an approximate relative molecular mass of 70,000-80,000.

Limited proteolysis of C3b by C3b inactivator (factor I) consists of a two-step reaction; rapid cleavage of C3b to yield a nicked C3b derivative, iC3b, and followed by slow cleavage of iC3b to yield two antigenically distinct fragments, C3c and C3d,g. Using a fluorescence-labeled C3b as a substrate for I, we have investigated in detail the optimal conditions for the sequential cleavages of C3b by I. The pH optimum for the first cleavage was markedly affected by the ionic strength of buffers. The cleavage was maximum at pH 6.0 under physiological ionic strength but at pH 8.5 under low ionic strength (such as 1.7 mS). The second cleavage was a slow reaction and occurred only under low ionic strength and within a narrow pH range around pH 6.0. One of the products of the second cleavage, C3d,g, was isolated and shown to be a single polypeptide chain of 41,000 daltons with pI 5.0. C3d,g had leucocytosis-inducing activity, like C3d-k, which is a C3d fragment released by the action of plasma kallikrein. Trypsin digestion of C3d,g produced two fragments of 30,000 and 10,000 daltons and the 10,000-dalton fragment retained the leucocytosis inducing activity.

Levels of the lysosomal enzyme N-acetyl-beta-glucosaminidase were measured in alveolar macrophage extracts and lung lavage fluid obtained from patients with cryptogenic fibrosing alveolitis and a control group. Evidence is presented which suggests that macrophages from patients with cryptogenic fibrosing alveolitis secrete this enzyme in vivo. To investigate one of the possible mechanisms of stimulating this secretion, an assessment was made of the C3b receptor sites on alveolar macrophages. While there were fewer receptor sites on the macrophages from patients with cryptogenic fibrosing alveolitis, which might indicate that they were occupied in vivo, this difference did not reach statistical significance. It is suggested that the alveolar macrophage is not merely a phagocyte but that it also has a more active role in the pathogenesis of cryptogenic fibrosing alveolitis.

Plasmid-cured variants of virulent strains of Yersinia enterocolitica and Y. pseudotuberculosis were obtained by selection after growth in calcium-deficient medium. To obtain antigen preparations consisting of whole bacteria the original plasmid-containing strains and the plasmid-cured variants were grown in conditions favouring expression of the temperature-inducible outer membrane proteins of Yersinia (YOP) (37 degrees C, calcium-deficient culture medium). The presence or absence of the YOP on the bacteria was verified by immunoblotting. Opsonophagocytosis of YOP-negative Yersinia preparations (YOP-) was compared to that of YOP-containing ones (YOP+) in human polymorphonuclear leukocyte (PMN) chemiluminescence (CL) assay. The attachment of complement C3b on the surface of the bacteria after opsonization with normal human serum was determined by using a fluorescent anti-C3c-antibody and flow cytometry. YOP+ bacteria resisted opsonization in the absence of specific antibodies, as indicated by diminished C3b-fixation on bacteria and weaker CL response. This implies that virulence-plasmid-coded structures provide Y. enterocolitica and Y. pseudotuberculosis with an ability to avoid complement-mediated opsonization and phagocytosis.

Recent studies employing sheep red cells, rabbit anti-sheep cell IgM, guinea-pig R3 reagent and purified human C3, factor I, factor H and human serum have led to the discovery of a new C3b breakdown product antigen, C3g, which is present on the alpha 2D fragment of C3 but is removed by trypsinization. The present studies were undertaken to confirm the above findings in entirely human red cell and serum systems, including studies on cells from nine patients with a variety of complement-mediated autoimmune haemolytic anemias. The results confirm the recently reported findings in every respect. The presence of both C3d and C3g on all patient's red cells so far tested supports the concept that red cells coated by C3d alone are likely to be a laboratory artefact and that the alpha 2D fragment (C3d,g) is the natural end product of cell bound C3b breakdown in vivo.

The number of receptors for complement component C3b per erythrocyte reportedly is decreased in over half of adults with systemic lupus erythematosus. We have devised an immunoradiometric assay for C3b receptor (CR1) on erythrocytes, with which one can assess CR1 saturation due to in vivo binding of immune complexes or activated complement fragments (C3b). Using this assay, we examined binding by CR1 in normal adults and newborns, in lupus and juvenile rheumatoid arthritis patients, and in a population of patients with various general medical problems, including other connective tissue diseases. Binding by CR1 was decreased in eight of 15 SLE patients, four of 25 juvenile rheumatoid arthritis patients, and one of 14 patients with other diseases. We found no significant correlation between CR1 binding and either C1q binding, antinuclear antibody titer, results for complement C3 and C4, or the presence of renal disease. Using this assay, we were also able to show that the observed reduction in CR1 binding was not ascribable to prior saturation of CR1 or to blocking antibody against CR1. The assay is precise and easy enough for routine application.