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PubMed1990

Neutrophil elastase cleaves C3bi on opsonized pseudomonas as well as CR1 on neutrophils to create a functionally important opsonin receptor mismatch.

Neutrophil elastase has been implicated as a factor that impairs local host defenses in chronic Pseudomonas aeruginosa (Pa) lung infection in cystic fibrosis (CF). We recently showed that this enzyme cleaves the C3b receptor, CR1, from neutrophils (PMN) in the lungs of infected CF patients. The C3bi receptor on these cells, CR3, is resistant to elastase. We now show that purified neutrophil elastase markedly impairs complement-mediated PMN-Pa interactions including phagocytosis of opsonized Pa, stimulation by opsonized Pa of PMN superoxide production, and killing of opsonized Pa by PMN. When PMN and opsonized Pa were treated separately with elastase, additive levels of inhibition were observed in each of the above assays. The effects on the bacteria were due to cleavage of the bound C3bi from the surface of opsonized Pa by neutrophil elastase. C3bi was also cleaved by pseudomonas elastase, or bronchoalveolar lavage fluid from CF patients with chronic Pa lung infection. Inhibitors of neutrophil elastase eliminated C3bi cleavage by BAL fluid, while inhibitors of pseudomonas elastase had no effect. Blocking CR1 and CR3 on PMN with specific monoclonal antibodies reduced phagocytosis of opsonized Pa to an extent similar to that caused by elastase cleavage of CR1 on PMN and C3bi on Pa. We conclude that neutrophil elastase in the lungs of chronically infected CF patients cleaves C3bi from opsonized Pa as well as CR1 from PMN, creating an "opsonin-receptor mismatch" that severely impairs complement-mediated phagocytic host defenses against these bacteria.

Bacterial Adhesion↗
PubMed1988

Use of antisera to the isolated alpha and beta subunits of C3 as probes to study functional sites present on particle-bound C3b but absent on native soluble forms of C3.

The effect of antisera to the isolated alpha and beta chains of C3 on certain C3b-dependent reactions has been studied. C5-mediated haemolysis of EAC1423b was inhibited preferentially by antiserum to the alpha chain, whereas antiserum to the beta chain inhibited the formation of C3bBb. The anti-beta chain antiserum also stabilised C3bBbP, and rendered the enzyme relatively resistant to accelerated decay in the presence of factor H. These and previous findings that anti-alpha and anti-beta IgG bind to restricted subsets of antigenic determinants on C3/C3b suggest that these antisera affect C3b function through the binding of antibodies to active binding sites exclusively exposed by bound C3b. The anti-alpha and anti-beta antibody probes are currently being further developed to verify this interpretation.

Animals↗
PubMed1992

Identical point mutation leading to low levels of mannose binding protein and poor C3b mediated opsonisation in Chinese and Caucasian populations.

A common opsonic defect occurring in 7% of the Caucasian population is associated with low serum levels of the lectin mannose binding protein (MBP). This study sought to determine whether the deficiency was also present in a Chinese population using sera obtained from 100 healthy Chinese children (age range 6 weeks-16 years). The distribution profiles of MBP levels and C3b/C3bi fragments binding to mannan coated plates were both bimodal and similar to the corresponding Caucasian profiles. Serum MBP levels were low in 9% of the Chinese children and all of these sera generated low levels of C3b/C3bi fragments. Overall there was a high significant correlation between MBP levels and C3b opsonin generation (r = 0.77; P less than 0.001). By analogy with similar findings in a Caucasian population we believe this correlation to be a reflection of antibody independent complement activation by MBP. In a pilot study of DNA obtained from three adult Chinese with low MBP levels the point mutation causing MBP deficiency in Caucasians was identified in all three cases.

Adolescent↗
PubMedDate not supplied

Novel mechanisms of immune evasion by Schistosoma mansoni.

The interaction of Schistosoma mansoni with its host's immune system is largely affected by multiple specific and non-specific evasion mechanisms employed by the parasite to reduce the host's immune reactivity. Only little is known about these mechanisms on the molecular level. The four molecules described below are intrinsic parasitic proteins recently identified and studied in our laboratory. 1. m28--A 28kDa membrane serine protease. m28 cleaves iC3b and can thus restrict attack by effector cells utilizing complement receptors (especially CR3). Treatment with protease inhibitors potentiates killing of schistosomula by complement plus neutrophils. 2. Smpi56--A 56kDa serine protease inhibitor. Smpi56 binds covalently to m28 and to neutrophil's elastase and blocks their proteolytic activity. 3. P70--A 70kDa C3b binding protein. The postulated activity of P70 includes binding to C3b and blocking of complement activation of the C3 step. 4. SCIP-1--A 94kDa schistosome complement inhibitor. SCIP-1 shows antigenic and functional similarities to the human 18kDa complement inhibitor CD59. Like CD59, SCIP-1 binds to C8 and C9 and blocks formation of the complement membrane attack complex. Antibodies directed to human CD59 bind to schistosomula and potentiate their killing by complement. The structure and function of these four proteins as well as their capacity to induce protection from infection with S. mansoni are under investigation.

Animals↗
PubMed1981

C3b receptor in normal human skin.

Receptors for C3b in normal skin were studied. C3b was produced by treating normal human serum with cobra venom factor and by partial digestion of purified C3 with trypsin. Cryostat sections of normal human skin were incubated with C3b, followed by a direct immunofluorescent technique using monospecific goat antihuman C3. The histologic localization of C3b fluorescence was ascertained by fixing cryostat sections with glutaraldehyde and staining with hematoxylin and eosin. The following structures showed staining with anti-C3: (1) endothelial cells in capillaries, larger vessels, and arteries, (2) smooth muscle in arrector pilori muscles and artery walls, and (3) myoepithelial cells in the secretory portion of sweat glands. C3b did not bind to the intercellular substance nor to the basement membrane zone in normal human skin. Normal human sera treated with EDTA, EGTA, and heat (56 degrees C for 30 min) were negative, as was purified C3 by itself, thus indicating that native C3 did not bind to these receptors. Specificity for C3/C3b was shown by blocking with both unconjugated rabbit antihuman C3 and purified C3. The endothelial C3b receptor may have an important role in the localization of immune complexes in cutaneous vasculitis.

Antigen-Antibody Complex↗
PubMed1980

Effects of soluble immune complexes on Fc receptor- and C3b receptor-mediated phagocytosis by macrophages.

The effects of ingestion of soluble immune complexes upon macrophage phagocytic function was studied. Ingestion of immune complexes severely impaired the macrophage's ability to ingest IgG-coated particles but did not alter its ability to interact with particles by means other than its Fc receptors. Treatment of macrophages that had ingested immune complexes with supernates containing the previously described lymphokine that augments macrophage complement receptor function failed to enhance the cells' interaction with either IgG-coated erythrocytes or zymosan particles but markedly enhanced their ability to phagocytize via their complement receptors. The possible significance of these findings in immunologically mediated inflammation is discussed.

Animals↗
PubMed1986

Ligand binding by the p150,95 antigen of U937 monocytic cells: properties in common with complement receptor type 3 (CR3).

U937 cells (a monocytic cell line) grown in the presence of phorbol myristate acetate were surface labeled with 125I and the iC3b-binding proteins isolated by affinity chromatography on iC3b-Sepharose in the presence of divalent cations. Three polypeptides of 170, 150 and 95 kDa were found to bind specifically to iC3b-Sepharose. The polypeptides of 170 and 95 kDa were identified as the alpha and the beta subunits of CR3 by immunoprecipitation with OKM1 monoclonal antibody. The 150-kDa polypeptide was not immunoprecipitated by antibodies to the alpha subunit of CR3 or LFA-1. However, the 150-kDa polypeptide, together with the 95-kDa polypeptide, was immunoprecipitated with an anti-beta subunit-specific antibody IB4, which immunoprecipitates LFA-1, CR3 and p150,95. These results indicated that the 150-kDa polypeptide is the alpha subunit of the p150,95 antigen. The binding of p150,95 and CR3 to iC3b-Sepharose is specific as neither binds to C3u-Sepharose. A monoclonal antibody, 3.9, which immunoprecipitated the 150 and a 95-kDa polypeptide from U937 cells was characterized as being directed against the alpha-subunit of the p150,95 antigen. Phorbol myristate acetate-stimulated U937 cells from rosettes with EAC3b and EAiC3b but not with EAC3d cells. Monoclonal antibody 3.9 does not inhibit either type of rosetting, but we were unable to exclude a role for p150,95 in adherence of iC3b-coated particles. Since there is no rosetting with C3d-bearing particles it is unlikely that p150,95 is a receptor for C3d, a role for p150,95 which has been suggested by others (Wright, S.D., Licht, M.R. and Silverstein, S.C., Fed. Proc. 1984. 43: 413).

Antibodies, Monoclonal↗
PubMed1984

Complement C3 receptors: structure and function.

Significant progress has been made during the past few years toward elucidating the structure and function of various C3 receptors. Better understanding of the biochemical basis of the immune adherence phenomenon has been achieved. The availability of polyclonal and monoclonal antibodies to C3 receptors allowed more accurate studies of the cellular distribution of these receptors. Finally, advances in our knowledge of the functional properties of the C3 receptors and their relationship to specific cell surface molecules was facilitated by recognition of a patient deficient in various receptor-mediated functions. All together these developments will soon make it possible to describe in detail the molecular events involved in the modulation of complement receptor coupled cell functions.

Animals↗
PubMed1995

Altered immunoexpression of microglia and macrophages after mild head injury.

In this study we examined the temporal response of microglia and macrophages to mild head injury in the rat. Microglia and macrophages were identified by their distinct morphology and by immunophenotype. With regard to the latter, antibodies to OX42 and ED1 were used to define microglia and macrophages, respectively. Although there was no change in the morphology of brain macrophages after mild head injury, the morphology of microglia was dramatically altered. Microglial cell bodies appeared larger with a more elaborate arborization of cellular processes. After head injury certain populations of macrophages and microglia were more intensely immunostained. By 3 days postinjury these intensely stained cells exhibited a characteristic distribution in the brain. Prominently stained microglia were detected in the thalamus, hippocampus, lateral and medial geniculate body, and the substantia nigra. Intensely stained macrophages were located primarily in the cortex and subarachnoid space adjacent to the site of impact. By 7 days postinjury intensely immunostained macrophages and microglia were widespread throughout the injured cortex. These results demonstrate that microglia and macrophages are sensitive to mild head injury. Early changes in the macrophage population are more directly correlated with the most damaged tissue and may reflect migration of these cells from either the subarachnoid space or across the damaged blood-brain barrier. The early widespread microglial response in regions exhibiting no overt neuronal cell damage suggests that these cells are responding to more subtle factor(s) that are expressed in the mildly traumatized brain.

Animals↗
PubMed1991

GM-CSF primes and modulates neonatal PMN motility: up-regulation of C3bi (Mo1) expression with alteration in PMN adherence and aggregation.

Neonatal polymorphonuclear leukocytes (PMNs) are deficient in the expression of the adherence protein C3bi (Mo1), and are associated with reduced physiological inflammatory responses. We evaluated the priming and direct stimulating effect of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) on newborn PMN expression of C3bi (Mo1), PMN adherence and PMN aggregation. Cord PMNs were incubated with rhGM-CSF (Amgen 4 x 10(7) U/mg) for 0-15 min, and C3bi surface receptor expression measured by immunofluorescence with CD11b, adherence with nylon wool, and aggregation using a Payton aggregometer. RhGM-CSF (15 min) significantly induced Mo1 expression: 250 pM/L 129.4 +/- 5.3% of C (p less than 0.001) 500 pM/L 141.5 +/- 4.1% (p less than 0.001), 1,000 pM/L 150.2 +/- 1.3% (p less than 0.0001). RhGM-CSF (1,000 pM/L x 5 min) followed by A23187 also primed newborn PMNs for increased Mo1 expression 122 +/- 4.5% of C (p less than 0.001). Additionally, rhGM-CSF (10 min) induced significant PMN adherence 50 pM/L 117.9 +/- 8.3% and 100 pM/L 131.5 +/- 5.7% of C (p less than 0.04). RhGM-CSF additionally primed newborn PMNs (100 pM/L) for increased adherence following A23187 (107.9 +/- 0.6% of C), p less than 0.02. Lastly, rhGM-CSF primed newborn PMNs for increased aggregation following FMLP: 100 pM/L, 15 min, 138.1 +/- 14.1%, p less than 0.0001. Co-incubating murine-antihuman GM-CSF AB 100 micrograms/ml neutralized 86.8 +/- 7.0% of newborn PMN Mo1 up-regulation. These studies demonstrate that rhGM-CSF primes and directly stimulates newborn PMNs for increased in vitro expression of Mo1, adherence, and aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1984

Natural release of covalently bound C3b from cell surfaces and the study of this phenomenon in the fluid-phase system.

Covalently bound C3b is released from cell surfaces (EAC1423 and zymosan-C3b) on incubation under physiologic conditions. The release of C3b from cell surfaces occurs by the cleavage of the covalent bond. Sodium dodecyl sulfate (SDS) abolishes the release, thereby indicating the requirement of the native structure of C3b in this process. The phenomenon of release of C3b from cell surfaces has also been observed in the fluid-phase system by using C3b-[3H]glycerol. The kinetics of the release of [3H]glycerol from C3b-[3H]glycerol were studied at 37 degrees C in 0.15 M phosphate buffer, pH 7.4. The first-order rate constant was found to be 0.028 +/- 0.003 hr-1. The release does not take place in either 8 M urea or 6 M guanidine hydrochloride, at pH 7.4. Under alkaline conditions, the rate of release is unaffected in the presence of SDS, indicating that the release in this pH range is not dependent on the native structure of the protein. From the Arrhenius plot in the temperature range 18 to 37 degrees C, an apparent activation energy for the hydrolysis reaction of 21.2 kcal/mol was calculated. The release phenomenon is exclusive for ester-linked complexes, as inferred by the absence of release of [3H]threonine from C3b-[3H]threonine, wherein the linkage is of the amide type. The presence or absence of the C3a portion of the molecule has no effect on the rate of release. The modification of the -SH group of C3i-/C3b-[3H]glycerol alters the rate of hydrolysis of the ester bond between C3i/C3b and [3H]glycerol. Protease inhibitors (PMSF, benzamidine HCl, and DFP) do not alter the rate of release, indicating that the hydrolysis reaction is not due to trace amounts of contaminating proteases. Thus, it appears that some chemical group(s) of C3i/C3b is (are) involved in the intramolecular hydrolysis of the ester bond between C3i/C3b and small molecules. This phenomenon may play an important role in the release of C3b from receptive surfaces once the biologic functions that require covalently bound C3b have been mediated.

Alkylation↗
PubMed1976

Studies of the presence of membrane receptors for complement, IgG and the sheep erythrocyte rosetting capacity on the same human lymphocytes.

Normal peripheral blood lymphocytes were tested by a mixed rosette method, employing different sized erythrocytes as indicators to identify lymphocytes simultaneously possessing membrane markers found commonly on B and T cells. Only small populations of these lymphocytes were detected regularly in normal lymphocyte preparations. One type of lymphocyte (ranged from 0.5%-8%) was shown to possess the following markers: receptors for human and rabbit IgG, receptors for the third complement component C3b and C3b inactivator-cleaved C3b (C3d), and the capacity to rosette spontaneously with uncoated sheep erythrocytes (SRBC). Another lymphocyte cell type was shown to possess both SRBC and IgG receptors but lack membrane immunoglobulins and complement receptors. This population was detected in lymphocyte preparations depleted of complement receptor cells, and an increased number of these cells was found in rosette preparations incubated with human serum. The possible presence of some lymphocytes possessing both complement and SRBC receptors and lacking other markers was considered. The possibility that these small populations of human lymphocytes are sub-populations of T cells with certain cytotoxic function is postulated.

Adolescent↗
PubMed1984

Substances that can trigger activation of the alternative pathway of complement have anti-melanoma activity in mice.

Intraperitoneal (i.p.) injection of substances that can ignite the alternative pathway of complement, namely isolated human C3b or C3(H2O), guinea-pig C3(H2O) or cobra venom factor, or conventionally prepared zymosan, will reproducibly and very significantly increase the mean survival time of C57BL mice previously inoculated i.p. with melanoma cells. The effect is greater at higher doses and earlier post-inoculation (p.i.) administration, but the substances are active at low doses (30-100 micrograms/mouse) if given early enough. It is likely that C3b or C3(H2O) was the previously unidentified anti-tumour factor activated in serum by S. aureus treatment or serum fractionation and described elsewhere. Activation of the alternative pathway of complement appears to have potential interest for cancer therapy.

Animals↗
PubMed1986

Structure and functions of gp 140, the C3d/EBV receptor (CR2) of human B lymphocytes.

Analysis of the interaction of human C3 fragments with human B lymphoma cell line, led us to isolate gp 140, the C3 receptor of Raji cells. Rabbit anti-gp 140 was prepared against this highly purified receptor. Using these polyclonal antibodies, it was found that: gp 140 is the C3d receptor (CR2) which reacts with the C3d site expressed on C3d, C3dg, C3bi and at a less extent on C3b. Gp 140 is a specific marker of human B lymphocytes; gp 140 is also the Epstein-Barr virus receptor (EBVR); CR2 is a membrane site involved in B-cell regulation; and the C3d/C3dg receptor (CR2) of human B lymphocytes is distinct to the C3dg receptor (CR4) of human neutrophils.

B-Lymphocytes↗
PubMed1986

Identification of a nonproteolytically activated C3 with CR 1-binding properties in sera from subjects with primary biliary cirrhosis.

Complement factor C3 associated with immune complex-like material was precipitated by 2.7% (w/v) polyethylene glycol (PEG) in sera from patients with primary biliary cirrhosis. Radioimmunoassay analysis demonstrated that this C3 expressed antigens which are specific for bound and denatured C3 but absent in native C3. Gelfiltration of the PEG precipitate revealed a C3 peak which corresponded to a molecular weight of more than 2 X 10(7) daltons but sucrose-gradient centrifugation of the same material showed that the C3 had a sedimentation coefficient of 4-7 S. SDS-PAGE followed by immunoblotting presented intact C3 alpha and C3 beta chains, and immune electrophoresis showed that the PEG-precipitated C3 was slower than native C3. The biological properties of the PEG-precipitable C3 were demonstrated by the inhibitory effect on the binding of immune complexes containing C3b to complement receptor 1. These data are not compatible with coprecipitation of native C3, nor with immune complex-bound C3b.

Antigen-Antibody Complex↗
PubMed1994

Inhibition of human spermatozoon-oocyte interaction in vitro by monoclonal antibodies to CD46 (membrane cofactor protein).

CD46 (membrane cofactor protein) is a cell surface complement regulatory glycoprotein that facilitates enzymatic cleavage of complement component C3b; it is expressed by both human oocytes and acrosome-reacted spermatozoa. Murine anti-CD46 monoclonal antibody (mAb) has been reported to decrease significantly the ability of human spermatozoa to penetrate hamster oocytes. We have investigated the effect of purified anti-CD46 mAbs on spermatozoon-oocyte interaction in an autologous zona-free oocyte penetration test. Oocytes and/or spermatozoa were preincubated with either of two anti-CD46 murine mAbs, TRA.2.10 (a non-blocking mAb) and MH61 (a mAb that functionally blocks C3b-ligand binding activity), or a control isotype-matched mAb, in medium supplemented with human serum albumin. Preincubation of both spermatozoa and zona-free oocytes with TRA.2.10, but not MH61, caused a significant decrease in the number of oocytes showing sperm binding and pronuclear formation (9/23) compared with controls (21/26) in this complement component-depleted medium. This effect was not observed if oocytes or spermatozoa alone were preincubated. These data suggest that CD46 has a role in human spermatozoon-oocyte interaction at the level of the oocyte plasma membrane, and indicate that a novel function other than direct C3b binding could be involved.

Animals↗
PubMed1992

Phagocytosis by the murine testicular TM4 Sertoli cell line in culture.

To investigate phagocytosis, an assay enabling flow cytometric analysis of single cells having internalized fluorescent carboxyl microspheres was employed. Greater than 80% of murine testicular Sertoli line (TM4) cells were found to phagocytose one or more microspheres within six hours and electron microscopy confirmed carboxyl microsphere internalization. This level was equivalent to that of a macrophage-like cell line and much greater than the levels of testicular Leydig (TM3) cells. Reducing extracellular calcium or using a calcium channel blocker profoundly inhibited phagocytosis suggesting that phagocytosis by Sertoli cells requires extracellular Ca++. Although follicular stimulating hormone, luteinizing hormone, and testosterone had no significant effects on Sertoli cell phagocytosis, insulin, epidermal growth factor, and hydrocortisone enhanced activity. In contrast, beta-endorphin and 8-bromoadenosine-cyclic monophosphate had an inhibitory effect. In contrast to augmenting macrophage phagocytosis, 1,25-(OH)2D3, interferon-gamma, prostaglandin E2, and lipopolysaccharides, had no apparent effect on that by Sertoli cells. Additionally, neither C3bi receptors (Mac-1 antigen) nor FcRII could be detected on Sertoli cells. In total, the findings demonstrated that the murine Sertoli line exhibits potent phagocytic function and suggest the regulation of this activity may differ from that in "professional" phagocytic cells.

8-Bromo Cyclic Adenosine Monophosphate↗