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PubMed1975

Function of the classical and alternate pathways of human complement in serum treated with ethylene glycol tetraacetic acid and MgCl2-ethylene glycol tetraacetic acid.

An immunochemical and functional analysis of the classical and alternate complement pathways in human serum was performed in the presence of 10 mM ethylene glycol tetraacetic acid (EGTA) and MgCl(2)-EGTA (MgEGTA), chelating agents which have been recently utilized as a means of distinguishing between these two complement pathways. Total hemolytic activity, integrity of the C1 complex, hemolytic activity of C2, conversion of factor B (C3 proactivator), and complement-dependent bactericidal activity were studied. The effect of these chelators on activation of complement pathways by Escherichia coli, by sensitized erythrocytes as a prototype of activators of the classical pathway, and by zymosan as a prototype of alternate (properdin) pathway activators was studied. Human serum containing 10 mM EGTA, which provides almost no ionized calcium and considerably less ionized magnesium than unchelated serum, allowed consumption of complement via the alternate (properdin) pathway, but blocked the classical pathway as judged by disintegration of the C1 complex and lack of utilization of C2. However, activity of the alternate complement pathway in EGTA serum, as judged by conversion of factor B and bactericidal activity against gram-negative bacteria, was distinctly suboptimal. Addition of magnesium ion in a concentration equimolar to EGTA (MgEGTA serum), while still providing conditions in which the C1 complex dissociated, significantly enhanced alternate complement pathway-mediated bactericidal activity. However, in MgEGTA serum considerable fluid-phase activation of the alternate pathway, as indicated by decrease in 50% hemolytic complement (CH(5 0)) titers and conversion of factor B to its active form in the absence of any activating challenge, was observed. Moreover, some fluid-phase consumption of C2 was observed in MgEGTA serum, even though, as mentioned, the C1 complex was shown to be dissociated under these conditions. MgEGTA-related activation of C2 and of the alternate (properdin) pathway of complement was significantly enhanced by the presence of zymosan and E. coli. These results indicate that use of the chelating agents EGTA and MgEGTA to differentiate between classical and alternate pathway activation of human complement is more complex than has hitherto been suggested. In EGTA serum, spontaneous activation of either pathway does not occur but bactericidal activity, as a measure of biologic function of complement, is suboptimal. In MgEGTA serum, bactericidal activity is fully expressed, but there is considerable instability, in terms of fluid-phase activation, in Mg(2+)-dependent components of both pathways. Thus, caution is indicated in the use and interpretation of the effects of these chelating agents on biologic functions mediated by either pathway of human complement.

Blood Bactericidal Activity↗
PubMed2001

Complement and apoptosis.

Apoptosis and necrosis are two forms of cell death characterized by distinct morphologies. Until recently, complement-mediated cell lysis has been presented as a classical example of necrotic cell death. However, recent reports on apoptogenic effects of complement have shaken this dogma. The field has become even more confusing with descriptions of anti-apoptotic effects of complement. Necrosis has been associated traditionally with inflammation, whereas apoptosis has been regarded as noninflammatory. Therefore, first descriptions of the capacity of the complement system to identify apoptotic cells and to be activated by them, led to the development of the concept that complement opsonizes apoptotic cells for fast clearance by phagocytic cells. In the absence of such opsonization, (eg in C1q or C4 deficiency), apoptotic cells may remain longer in the body and may stimulate autoantibody production or undergo pro-inflammatory secondary necrosis. This has been associated in man and mouse with the development of an autoimmune disease like systemic lupus erythematosus. However, complement may also interfere with the programmed intention of apoptosis to avoid triggering of inflammation. Our recent results show that, under specific conditions, early apoptotic cells are not only opsonized by complement but may also be lysed, raising the possibility that under certain conditions apoptosis will be associated with an inflammatory reaction. The review describes and discusses the reports covering the various aspects of the interface between complement and apoptosis and its possible relevance to autoimmune diseases and inflammation and raises the following questions: 1. Can activated complement proteins induce apoptotic cell death? 2. Can complement protect cells from apoptosis? 3. Do apoptotic cells activate complement? and 4. What role is complement playing in clearance of apoptotic cells and which complement receptors are involved? Clearly, this research field is highly complex and still at its formation stage, yet interesting and important mechanisms are bound to emerge from these studies.

Apoptosis↗
PubMed2003

Complement resistance of human carcinoma cells depends on membrane regulatory proteins, protein kinases and sialic acid.

Nucleated cells employ several strategies to evade killing by homologous complement. We studied complement resistance in the human carcinoma cell lines (CA) T47D (mammary), SKOV3 (ovarian), and PC-3 (prostate) with emphasis on the following mechanisms of defense: 1. Expression and shedding of the membrane complement regulatory proteins (mCRP) CD46, CD55 and CD59; 2. Resistance based on protein phosphorylation; 3. Cell surface expression of sialic acid residues; 4. Desensitization to complement upon exposure to sublytic complement doses. Anti-mCRP antibody blocking experiments demonstrated that CD59 is the main mCRP protecting these CA from complement. Soluble CD59 was also found in supernates of PC-3> SKOV3 > T47D cells. Second, inhibitors of PKC, PKA and MEK sensitized the CA to lysis, thus implicating these protein kinases in CA complement resistance. Third, removal of sialic acid residues with neuraminidase also sensitized CA to lysis. Finally, exposure of CA to sublytic doses of complement conferred on them enhanced resistance to lytic complement doses in a PKC-dependent process. Combined treatment of CA with anti-CD59 antibodies, PD98059 (a MEK inhibitor) and neuraminidase produced a large enhancement in CA sensitivity to complement. Our results show that CD59 and sialic acid residues present on the cell surface, and intracellular processes involving protein phosphorylation act additively to secure CA resistance to complement-mediated lysis. Therefore, the effectiveness of antibody- and complement-based cancer immunotherapy will markedly improve by suppression of the various complement resistance mechanisms.

CD55 Antigens↗
PubMed2000

Human and rodent decay-accelerating factors (CD55) are not species restricted in their complement-inhibiting activities.

Homologous complement activation is restricted on cells by the complement regulators, decay-accelerating factor (DAF), membrane cofactor protein (MCP) and CD59. These proteins act in concert with other membrane structures to protect cells from homologous complement attack. In contrast, cells are usually sensitive to heterologous complement attack. It has been suggested that species-specific restriction of complement activation can be attributed to the inability of regulators to inhibit across species. We have investigated the capacities of human, rat and mouse analogues of DAF to regulate homologous and heterologous complement. Cells transfected with cDNA encoding these analogues were protected from heterologous complement attack. C3b-deposition experiments indicated that whilst cells were best protected by DAF from the same species, all three analogues inhibited human, rat and mouse complement. Comparable results were obtained in haemolysis assays using soluble, recombinant forms of the proteins. Inhibition of the classical pathway (CP) was best achieved with homologous DAF, although human DAF also inhibited rat complement, rat DAF also inhibited human complement and mouse DAF inhibited complement from all species. Human DAF was the best inhibitor of alternative pathway (AP)-mediated attack, inhibiting complement from all species. Mouse DAF inhibited mouse and rat AP, whilst rat DAF inhibited only rat AP. These data indicate that human and rodent analogues of DAF are not species restricted and highlights interesting differences in the capacity to regulate AP and CP. This has implications in broader fields of research, such as xenotransplantation, where cross-species regulation of complement is of paramount importance.

Animals↗
PubMed2002

Calcium-dependent protection from complement lysis in Naegleria fowleri amebae.

Pathogenic Naegleria fowleri amebae are resistant to the lytic effects of serum complement. The presence of surface glycoproteins or removal of the membrane attack complex (MAC) of complement from the cell surface by vesiculation serve to protect the amebae from complement lysis. The specific mediators important in stimulating complement resistance are not defined. These studies were undertaken to examine the effect of Ca(2+) ions in initiating complement resistance of N. fowleri in contrast to non-pathogenic complement-sensitive N. gruberi. Chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) or chelation of intracellular calcium with 1,2-bis-(O-Aminophenoxy) ethane-N,N,N,N tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM) increased complement lysis of N. fowleri. Chelation of calcium ions did not affect complement sensitivity of N. gruberi. Increased lysis of ionomycin-treated N. fowleri was detected after exposure to serum complement, suggesting that a threshold level of Ca(2+) mediates complement resistance before survival mechanisms are overwhelmed and lysis occurs. A differential influx of Ca(2+) ions occurred in fura-2 labeled N. fowleri after deposition of complement component C9 to form the MAC complex on the cell surface in comparison to N. gruberi. These studies suggest that Ca(2+) ions influence complement resistance in N. fowleri but do not play a role in altering the sensitivity of N. gruberi to complement.

Animals↗
PubMed2005

Controlling the complement system for prevention of red cell destruction.

PURPOSE OF REVIEW: Complement sensitization of red blood cells (RBCs) can lead to both intravascular and extravascular red cell destruction. Altered levels of naturally occurring complement regulatory proteins on red cells can result in hemolysis, while defective expression of these proteins on immune cells can cause breakdown of tolerance to self antigens and is associated with autoimmune disease. RECENT FINDINGS: To date several complement inhibitors, including recombinant forms of complement regulatory proteins, humanized antibodies, and synthetic molecules have been described that limit complement activation by interfering with different steps in the complement cascade. However, few have been evaluated for prevention of complement-mediated RBC destruction. In this review, possible applications of these complement inhibitors for treatment of complement-mediated hemolysis in specific disease states are described. Furthermore, the implication of the regulatory role of complement in the development of autoimmune hemolytic anemia is discussed. SUMMARY: Complement therapeutics has potential for effective and safe prophylactic use and treatment of hemolytic transfusion reactions and complement-mediated hemolytic diseases. Furthermore, the regulatory function of complement may be exploited to prevent and treat autoimmune hemolytic anemia.

Anemia, Hemolytic↗
PubMed2001

The complement system in renal diseases.

The complement system has long been recognized as having a role in immune glomerular disease. This review provides an update on this association, some strategies for the clinical testing of complement in disease, and a brief commentary on current research directions. Evidence of complement activation in glomerulonephritis comes from characteristic patterns of a decrease in the serum concentrations of specific components, some of which are virtually diagnostic of certain nephritides. These patterns are often accompanied by the presence of complement components in the glomeruli and the detection of complement breakdown products in the circulation. In certain diseases, circulating complement-activating substances can be detected. Although there are over 20 complement proteins, clinical analysis is most often directed at C3 and C4, with occasional measurement of B and C5. Recently, a variety of mechanisms for complement-induced injury has been recognized. These mechanisms go far beyond simple passive lysis of erythrocytes, the earliest functional effect of complement studied. The role of such mechanisms in renal disease is just beginning to be studied. Local synthesis of complement components in the kidney may play a role both in host defense and in the promotion of interstitial inflammation and scarring. Such mechanisms will likely be defined more precisely with the availability of animals with specific complement deficiencies. Ultimately, an understanding of the role of complement in renal disease may permit specific targeted inhibition of one or more complement functions as a form of therapy.

Animals↗
PubMed1980

Influence of the Escherichia coli capsule on complement fixation and on phagocytosis and killing by human phagocytes.

To define mechanisms by which polysaccharide capsules confer enhanced virulence on gram-negative bacteria, we examined the effect of the Escherichia coli capsule on complement fixation to the bacterial surface and on phagocytosis and killing of these bacteria by mouse macrophages and human polymorphonuclear leukocytes (PMN) and monocytes. When E. coli were attached to mouse macrophages with concanavalin A, the macrophages readily phagocytosed unencapsulated but not encapsulated bacteria even in the presence of fresh mouse serum; macrophages did not phagocytose encapsulated E. coli unless antibacterial or anti-Con A antibody was added. Similarly, when these bacteria were attached to human PMN with Con A, PMN ingested unencapsulated but not encapsulated E. coli. PMN phagocytosed and killed encapsulated serum-resistant E. coli only in the presence of both complement and antibacterial antibody; PMN phagocytosed and killed unencapsulated E. coli of the same strain in the presence of complement alone. Fluorescence microscopy showed that antibody had to be present for encapsulated but not unencapsulated E. coli to fix complement to its surface. To examine the role of the complement receptors of human PMN and monocytes in phagocytosis and killing of encapsulated E. coli, we used human and rabbit antibacterial immunoglobulin (Ig)M to fix complement to the bacteria. PMN and monocytes phagocytosed and killed encapsulated E. coli in the presence of both IgM and complement, but not in the presence of either serum opsonin alone. In the presence of antibacterial IgG, PMN and monocytes required complement to effectively phagocytose and kill the E. coli. We conclude that (a) attachment by itself results in ingestion of unencapsulated but not encapsulated E. coli; (b) under physiologic conditions, E. coli are not phagocytosed or killed the absence of antibody, the E. coli capsule blocks complement fixation to the bacterial surface probably by masking surface components, such as lipopolysaccharide, capable of activating the complement pathway; (d) the E. coli capsule imposes a requirement for specific antibacterial antibody for complement fixation; and (e) the complement receptor of human PMN and monocytes mediates phagocytoses of complement-coated encapsulated bacteria and is the primary mediator of phagocytosis and killing of these bacteria.

Animals↗
PubMed1998

Complement can neutralize HIV-1 plasma virus by a C5-independent mechanism.

A previous study showed a portion of HIV-1 plasma virus was lysed by the addition of exogenous human AB+ seronegative complement. The current study was performed to determine whether infectious plasma virus was inactivated by complement. Incubation of plasma virus with AB+-seronegative serum resulted in substantial decreases in infectious titers, demonstrating that infectious plasma virus is susceptible to complement-mediated inactivation. Although complement also induced some lysis of plasma virus samples, virus was neutralized to a significantly higher degree, suggesting neutralization did not occur solely by lysis. Additionally, C5-deficient complement substantially neutralized virus, indicating coating of virus by early complement components was an important mechanism of neutralization. A portion of some freshly isolated plasma virus samples bound to complement receptor 2 in the absence of exogenous complement, indicating that early complement components bound virus in vivo. Furthermore, plasma virus samples that had less C3 deposited on their surface in vivo had higher infectious titers than samples with a larger fraction with surface C3. These findings suggest that complement can neutralize HIV-1 plasma virus in vivo by coating with complement proteins. This is the first study to provide evidence that coating by complement leads to functional inactivation of a virus in vivo.

Antiviral Agents↗
PubMed1985

Complement effector mechanisms in health and disease.

Complement is an effector system able to mediate a number of biological activities in vitro and in vivo. Most familiar is the ability of the system to mediate the lytic destruction of numerous kinds of cells and pathogenic organisms including bacteria, viruses, and virus-infected cells. In addition, the complement system also activates neutrophils, monocytes, basophils, mast cells, and lymphocytes to perform specialized functions. While generally considered to be confined to the effector side of immune reactions, recent evidence indicates that the complement system also directly recognizes and is triggered by a number of bacteria and viruses as well as virus-infected cells in the absence of antibody. In such reactions, complement fulfills the recognition role normally associated with the antibody molecule or immune lymphocyte. The complement system may thus also function as a natural surveillance system operative prior to the induction of specific immunity. Involvement of the complement system in biological reactions has been ascertained by several techniques over the years. These include quantitation of individual complement components in human sera and demonstration of complement deposition in diseased tissues in human diseases and in experimental diseases in animals. Such techniques, however, have limitations in specificity and sensitivity. Assays which detect specific features of the complement activation process have become available in recent years. These tests detect the physical, chemical, or antigenic changes characteristic of the complement activation process. These assays are extremely specific and quantitative; furthermore, most are usable with samples from patients. Three general approaches have been utilized to develop such specific quantitative assays for complement activation. The first includes assays which quantitate activation-specific limited proteolysis of the complement components. The second type of assay includes tests which detect and quantitate new antigens or other activation-specific antigenic changes. The third category is represented by assays which detect and quantitate the protein-protein complexes characteristic of the activation process. Examples of tests presenting each of these approaches are given.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Genetic analysis of temperature-sensitive mutants of HSV-1: the combined use of complementation and physical mapping for cistron assignment.

To date, mutations in mutants representing 19 of the 33 recognized HSV-1 complementation groups have been mapped. The physical map locations of mutations in 10 ts mutants of HSV-1 strain KOS representing 8 of the 19 complementation groups are reported herein. The mutations in three mutants were found to lie between coordinates 0.086 and 0.194--two of these were mapped finely to between coordinates 0.095 and 0.108--and in seven mutants, between 0.301 and 0.448. The mutation in 1 of the 10 mutants, tsQ26, was mapped finely to a sequence between 500 and 1000 base pairs to the left of the 3' end of the TK gene (0.301-0.304). The availability of physical mapping data has (1) confirmed the usefulness of the complementation test as a means of identifying viral gene functions, (2) facilitated the rapid assignment of mutants to new and recognized cistrons, and (3) prompted a reevaluation of previously ambiguous complementation for mutants in 2 complementation groups. Thus, the 10 mutants whose ts mutations were mapped in this study had been assigned previously to 8 complementation groups based on the assumption that complementation indices of 2 or greater signified that 2 mutants were in different genes. Combined with physical mapping data, however, the results of complementation tests now indicate that indices between 2 and 10 may reflect either inter- or intragenic complementation. Thus, the 10 mutants have now been assigned to 7 complementation groups. Although physical mapping data have confirmed the results of previous complementation tests for 6 of 8 groups analyzed, reevaluation of complementation data in the light of physical mapping data has resulted in a more precise genetic definition of the locus for viral DNA polymerase and of a locus (represented by mutants in complementation group 1-10) which maps in the left hand portion of UL.

DNA Restriction Enzymes↗
PubMed1986

The role of complement in inflammation during experimental pneumococcal meningitis.

The mechanism whereby an effective bactericidal inflammatory reaction develops in the subarachnoid space is not clearly defined. While normal cerebrospinal fluid is deficient in complement, immunoglobulin and leukocytes, these serum components appear in cerebrospinal fluid (CSF) during the course of bacterial meningitis. Using a rabbit model of pneumococcal meningitis we examined the role of the alternate complement pathway in three early events important to the defense of the subarachnoid space: leukocyte chemotaxis, phagocyte mediated bacterial killing, and clearance of bacterial components from the cerebrospinal fluid space. Rabbits treated with cobra venom factor to deplete complement were inoculated intracisternally with encapsulated (type II or XIX) pneumococci. Following complement depletion, there was a dramatic (at least 100-fold) decrease in the LD50 for these strains. Nevertheless, complement depletion did not affect the magnitude of CSF leucocytosis or the rate of clearance of bacterial particles from CSF. A short delay in the appearance of leukocytes in CSF was found in the absence of complement. The major effect of complement depletion, however, was to diminish the efficiency of leukocyte mediated killing of encapsulated bacteria in the CSF. Although the short delay in the onset of leukocytosis in the complement depleted animals is consistent with a chemotactic role of complement in the normal animal, the quantitatively normal leukocytosis in the complement depleted rabbits clearly indicates that important chemotaxins other than complement function in CSF. Inhibition of leukocytosis by indomethacin and diclofenac suggests that metabolite(s) of the arachidonic acid pathway may perform such a chemotactic role. A major role of complement in the defense of the subarachnoid space appears to be as an opsonin needed for the effective bactericidal activity of leukocytes. It is the lack of this function that best explains the greatly decreased LD50 value of encapsulated pneumococci in the complement depleted animal.

Animals↗
PubMedDate not supplied

Complement resistance of tumor cells: basal and induced mechanisms.

Clinical and experimental studies have suggested that complement may play a role in tumor cytotoxicity. However, the efficiency of complement-mediated tumor cell lysis is hampered by various protective mechanisms, which may be divided into two categories: basal and induced mechanisms. The basal mechanisms are spontaneously expressed in cells without a need for prior activation, whereas the induced mechanisms develop in cells subjected to stimulation with cytokines, hormones, drugs or with sublytic doses of complement and other pore-formers. Membrane-associated complement regulatory proteins, such as CD55 (DAF, Decay-Accelerating Factor), CD46 (MCP, Membrane Cofactor Protein), CD35 (CR1, Complement Receptor type 1) and CD59, which serve as an important mechanism of self protection and render autologous cells insensitive to the action of complement. appear to be over-expressed on certain tumors. Furthermore, tumor cells secrete several soluble complement inhibitors. Tumor cells may also express proteases that degrade complement proteins, such as C3, or ecto-protein kinases which can phosphorylate complement components, such as C9. Besides this basal resistance, nucleated cells resist, to some extent, complement damage by removing the membrane attack complexes (MAC) from their surface. Several biochemical pathways, including protein phosphorylation, activation of G-proteins and turnover of phosphoinositides have been implicated in resistance to complement. Calcium ion influx and activation of protein kinase C (PKC) and of mitogen-activated protein kinase (MAPK) have also been demonstrated to be associated with the complement-induced enhanced resistance to lysis. The complete elucidation of the molecular mechanisms involved in basal and induced tumor cell resistance will enable the development of strategies for interfering with these evasion mechanisms and the use of the cytotoxic complement system against tumor cells.

Animals↗
PubMed1996

Leishmania major-human macrophage interactions: cooperation between Mac-1 (CD11b/CD18) and complement receptor type 1 (CD35) in promastigote adhesion.

It has been suggested that the developmental maturation of Leishmania major promastigotes can affect their interaction with human complement receptors. To study this, we measured the adhesion of metacyclic and logarithmic-phase L. major promastigotes to complement receptors expressed on primary macrophages, to recombinant receptors expressed on transfected cells, or to purified complement receptors in a cell-free system. We demonstrate that complement-opsonized promastigotes can bind to both Mac-1 and complement receptor type 1 (CR1) and that the transition of promastigotes from the noninfectious logarithmic phase of growth to the infectious metacyclic stage does not affect this interaction. Furthermore, we show that Mac-1 and CR1 can cooperate to mediate the efficient adhesion of complement-opsonized metacyclic promastigotes to cells expressing both receptors. On human monocyte-derived macrophages, Mac-1 appears to make a quantitatively greater contribution to this adhesion than does CR1, since blocking macrophage Mac-1 diminishes metacyclic promastigote adhesion to a greater extent than does blocking CR1. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic decrease in complement-dependent promastigote adhesion, relative to normal monocytes. The predominance of Mac-1 in these interactions is due, at least in part, to the factor I cofactor activity of CR1, which facilitates the conversion of C3b to iC3b. The stable adhesion of complement-opsonized metacyclic promastigotes to Mac-1 is a prerequisite for phagocytosis by human monocyte-derived macrophages. Blocking Mac-1 on macrophages abrogates the majority of the complement-dependent phagocytosis of promastigotes, whereas blocking CR1 has no detectable effect on phagocytosis. In addition, bovine monocytes lacking Mac-1 exhibit a dramatic reduction in promastigote phagocytosis relative to normal bovine monocytes. We conclude, therefore, that the two complement receptors, Mac-1 and CR1, can cooperate to mediate the initial complement-dependent adhesion of metacyclic promastigotes to human monocyte-derived macrophages and that Mac-1 is the predominant complement receptor responsible for the phagocytosis of complement-opsonized metacyclic promastigotes.

Adhesiveness↗
PubMed2003

[In vitro effects of antiallergic eyedrops on complement activation induced by particulate matter].

BACKGROUND: Recent decades have been marked by an increasing number of patients suffering from ocular allergic-like symptoms without being associated with an increase in IgE levels. These symptoms include heaviness of the lid, foreign body sensation, burning, stinging and photophobia. Both epidemiological studies and controlled human exposure clinical studies have shown cause-effect relationships between allergic-like symptoms and environmental factors such as outdoor air pollutants or poor indoor air quality. An ocular surface subclinical inflammation is thought to be responsible for pseudoallergic, pollution-related conjunctivitis. The complement system is considered as one of the major effector mechanisms involved in initiation of the subclinical inflammation that leads to IgE-independent eye irritation. PURPOSE: To study the capability of nine antiallergic eyedrops commonly used in the treatment of allergic conjunctivitis to inhibit complement activation induced in vitro by pollutants. METHODS: Normal human serum obtained from healthy individuals was used as a source of complement. Activation of complement was assessed using the complement hemolytic 50% (CH50) assay, in the absence or the presence of antiallergic eyedrops and in the absence or the presence of various stimuli, including sand, common house dust, eye mascara, and Dactylis glomerata pollen extract. Zymosan was used as a standardized complement activator. The following eyedrops were studied: Naabak (4.9% N-acetyl aspartic acid-glutamic acid, NAAGA, sodium salt), Almide (lodoxamide 0.1%), Levophta (0.05% levocabastine), Emadine (0.05% emedastine), Tilavist (2% nedocromil), Allergodil (0.05% azelastine), Patanol (olopatadine), and Zaditen (0.025% ketotifen). Effects of preservative-free lodoxamide and ketotifen were also assessed and compared to those of the preserved formulations. A solution of 0.01% benzalkonium chloride (BAC), the most widely used preservative in topical eyedrops, was also tested. RESULTS: Zymosan-induced activation of complement (30+/-6%) was significantly lowered by preincubation of serum with unpreserved NAAGA (16.6+/-4%, p=0.0026) or benzalkonium-preserved nedocromil (20+/-2%, p=0.022). Preserved levocabastine, emedastine, olopatadine and ketotifen did not interfere with zymosan-induced complement activation, whereas preserved azelastine, lodoxamide and benzalkonium chloride significantly aggravated complement activation induced by zymosan. Similar results were obtained when complement activation was triggered by sand, common house dust, mascara, or by an allergenic extract of Dactylis glomerata pollen. In the absence of complement activator, none of the antiallergic eyedrops induced a significant change in CH50 titer, indicating that the deleterious pro-inflammatory effect of preserved azelastine and lodoxamide may occur only once complement activation has been initiated, i.e., on an inflamed ocular surface. CONCLUSION: Among the antiallergic eyedrops tested in this study, only Naabak and Tilavist were found to significantly inhibit complement activation triggered by particulate matters or pollen allergenic extract. Such an anticomplement activity confers these two molecules a potential in the therapeutic management of pollution-related pseudoallergic conjunctivitis.

Air Pollutants↗
PubMed1985

Unusual complement-mediated hemolytic kinetics at low ionic strength.

The dilution of human serum in relatively low ionic strength buffer (mu = 0.070) caused the spontaneous activation of C1 and a limited activation of C4 and C3 in the fluid phase. The unusual degree of activation of the complement system in the fluid phase suggested that the optimal functions of complement-regulatory systems such as C1 inhibitor might be reduced. As a function of the time of preincubation (PI) of diluted serum at 37 degrees C, under low ionic strength conditions, an unusual complement-mediated hemolytic kinetic pattern was observed upon adding sensitized erythrocytes (EA). For a 1:36 dilution of human serum, there was an initial progressive decrease in complement hemolytic activity (from 3 to 20 min PI, phase I), followed by an apparent functional reversal (increase) in hemolytic activity (20-50 min PI, phase II) and finally a gradual irreversible depletion of the hemolytic activity (after 50 min PI, phase III). This hemolytic pattern could only be adequately demonstrated using a kinetic assay which followed the course of lysis of EA in the presence of low dilutions of human serum as a complement source. Others might have missed this observation due to the use of end-point titration methods which required the use of relatively elevated serum dilutions at the time of EA addition. Mechanisms which governed the variations in hemolytic activity at low ionic strength were not clear. Speculatively, partial consumption of early complement components, generation of free C1q and generation of complement fragments might have accounted for the initial decrease in the hemolytic activity observed in phase I. The apparent functional reversal of hemolytic activity observed in phase II might have involved a critical depletion of C1 inhibitor which occurred secondary to C1 inhibitor binding to C1 (activated by low ionic strength effects) and to the C1 activated at the time of EA addition. Without sufficient regulation, a rapid unrestricted C1-mediated complement activation could have occurred, which resulted in a rapid deposition of complement on the EA. Finally, prolonged exposure of serum to low ionic strength effects appeared to induce a significant complement consumption, which caused a time-dependent irreversible depletion of complement hemolytic activity (phase III). Excess exogenous C1 inhibitor, when co-incubated with diluted serum at low ionic strength, reversed the time-dependent effects of low ionic strength and enhanced the subsequent specific complement-mediated hemolytic activity as compared to controls.(ABSTRACT TRUNCATED AT 400 WORDS)

Complement Activation↗
PubMed1998

Complement regulatory proteins in glomerular diseases.

Complement activation plays a critical role in the pathogenesis of many forms of glomerulonephritis. Complement activation leads to tissue injury through various mechanisms including the generation of chemotactic factors and activation of the resident glomerular cells following C5b-9 insertion. Recent advances have disclosed the mechanisms of regulation of complement activation by discovery of a number of complement regulatory proteins. Decay accelerating factor (DAF), membrane cofactor protein (MCP), and complement receptor type 1 (CR1) act by inactivating C3/C5 convertase. They belong to the gene superfamily known as the regulators of complement activation (RCA), and share a common structural motif called a short consensus repeat (SCR). In contrast, CD59 works by inhibiting formation of C5b-9. The glomerulus is particularly well endowed with these membrane-bound complement regulatory proteins. DAF, MCP, and CD59 are ubiquitously expressed by all three resident glomerular cells, while CR1 is localized exclusively in podocytes. Expression of complement regulatory proteins can be changed by many factors including complement attack itself, and their expression levels are affected in various glomerular disorders. Studies utilizing cultured glomerular cells and animal models of glomerular diseases suggest important protective roles of complement regulatory proteins against immune-mediated renal injury. Recent progress in molecular biological techniques has made new therapeutic strategy feasible. Systemic administration of soluble recombinant complement regulatory proteins and local overexpression of complement regulatory proteins are promising therapeutic approaches.

Animals↗
PubMed2005

Mortalin/GRP75 promotes release of membrane vesicles from immune attacked cells and protection from complement-mediated lysis.

The membrane attack complex (MAC) of the complement system is causing membrane damage and cell death. For protection, cells have adopted several resistance mechanisms, including removal of the membrane-inserted MAC by vesiculation. To identify proteins involved in MAC vesiculation, extracellular proteins released from K562 cells in response to treatment with sub-lytic complement were separated by acrylamide gel electrophoresis and protein bands were extracted, digested into peptides and the peptides were analyzed by mass spectrometry. A 75-kDa protein that was abundant in the supernatant of complement-treated cells was identified as mortalin/GRP75. Analysis by western blotting demonstrated that as early as 5 min after exposure to sub-lytic doses of complement, mortalin was released from K562 cells. Mortalin was released after complete activation of the complement system and formation of C5b-8, and even more so when C5b-9 was formed. Other pore formers, such as streptolysin O and melittin, did not induce release of mortalin. As shown, mortalin can bind to complement C8 and C9 and is shed in vesicles containing C9 and complement MACs. Anti-mortalin antibodies reduced mortalin release from complement-treated cells and elevated the extent of cell death by complement. Inhibitors of protein kinase C and extracellular signal-regulated protein kinase also prevented mortalin release from complement-activated cells. These results suggest that mortalin/GRP75 promotes the shedding of membrane vesicles loaded with complement MAC and protects cells from complement-mediated lysis.

Complement C8↗