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PubMed1987

The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation.

Flow cytofluorimetry identifies and quantifies cell markers of different leukocyte subpopulations by combining cytofluorimetry with the differences in the light scattering properties of the leukocytes in mixed populations. In the phagocytic assay, reported in this paper, the experimental conditions were selected in such a way that it was possible to analyse the phagocytic function of granulocytes in peripheral blood without time-consuming cell separation. The percentage of phagocytosing granulocytes was not dependent on the concentration of granulocytes at the selected incubation time and particle (yeast-C3b) concentration. Furthermore, it was possible to adapt a previously described fluorescence quenching technique (FQ method) to differentiate between attachment and ingestion. Crystal violet, originally used in the FQ method, could not be used in this assay due to its lysomotropic effect. Trypan blue at a concentration of 0.25 mg/ml or higher at pH 4.5 showed a plateau effect in fluorescence quenching indicating an effect on attached but not ingested particles. This assay offers a simple technique to screen the functional properties of phagocytic cells in peripheral blood.

Cell Membrane↗
PubMed1986

Altered erythrocyte C3b receptor expression, immune complexes, and complement activation in homosexual men in varying risk groups for acquired immune deficiency syndrome.

We studied levels of erythrocyte C3b receptors (E-CR1) and correlated them to the level of circulating immune complexes (CIC) and complement activation in patients with or at risk for acquired immunodeficiency syndrome (AIDS). A significant reduction was found in patients with AIDS (185 +/- 93 CR1/cell), AIDS-related complex, and generalized lymphadenopathy, whereas healthy male homosexuals or normal controls had 434 +/- 193 and 509 +/- 140 CR1/cell, respectively (P less than 0.001). Family studies indicate that this defect is acquired. Reduction in E-CR1 was associated with increased levels of CIC when assayed by binding to Raji cells, but not when tested by C1q binding. Complement activation was assessed by levels of C3bi/C3d-g in plasma, measured with a monoclonal antibody specific for a neoantigen in C3d. AIDS patients had increased C3 activation (2.68 +/- 1.67%) when compared with normal controls (0.9 +/- 0.22%) (P less than 0.01). The decreased E-CR1, the presence of CIC, and C3 activation suggest that complement activation by immune complexes may play a role in the clinical expression of the disease.

AIDS-Related Complex↗
PubMed2000

Molecular mechanisms of target recognition in an innate immune system: interactions among factor H, C3b, and target in the alternative pathway of human complement.

In the alternative pathway of complement (APC) factor H is the primary control factor involved in discrimination between potential pathogens. The APC deposits C3b on possible Ags, and the interaction with factor H determines whether the initial C3b activates the APC. Factor H is composed of a linear array of 20 homologous short consensus repeats (SCR) domains with many functional sites. Three of these sites are involved in binding C3b and regulating complement activation; others bind to sialic acid and/or heparin and are responsible for host recognition. Using site-directed mutations we have examined the contributions of each of these sites to target discrimination and to functional activities of factor H. Decay acceleration by SCR1-4 of C3/C5 convertases bound to nonactivators was strongly dependent on SCR domains 11-15 and 16-20. Loss of these regions caused a 97% loss of activity, with SCR16-20 being the most critical (>90% loss). On APC activators the pattern of site usage was different and unique on each. On yeast, deletion of the 10 C-terminal domains (SCR11-20) had no effect on specific activity. On rabbit erythrocytes, this deletion caused loss of 75% of the specific activity. An examination of binding affinity to C3b on the four cell types demonstrated that factor H exhibits a unique pattern of SCR involvement on each cell. The results reveal a complex molecular mechanism of discrimination between microbes and host in this ancient innate defense system and help explain the different rates and intensities of APC activation on different biological particles.

Animals↗
PubMed1997

Functional characterization of an isogenic meningococcal alpha-2,3-sialyltransferase mutant: the role of lipooligosaccharide sialylation for serum resistance in serogroup B meningococci.

The neisserial alpha-2,3-sialyltransferase, which is encoded by the lst gene, terminally links sialic acid to the lacto-N-neotetraose residue of neisserial lipooligosaccharide (LOS). We used the recently published nucleotide sequence of the neisserial lst gene to construct an isogenic serogroup B meningococcal lst mutant by insertion of a kanamycin resistance gene. The resulting lst mutant expressed the unsialylated lacto-N-neotetraose structure. Using bactericidal assays and an infant rat model of meningococcal infection, we were able to demonstrate that lst mutation, in contrast to galE mutation, which results in a truncated LOS, or to siaD mutation, which results in loss of the capsule, neither had an effect on resistance to normal human serum, nor did it impair the ability of meningococci to spread systemically in the non-immune host. The lst mutant was serum resistant despite of the fact that the central factor of complement activation, C3b, was deposited on the lst mutant as efficiently as it was on the galE mutant. Thus, the terminal sialic acid residue linked to the wild-type LOS inhibited C3b deposition on the meningocuccus. However, in contrast to the galE mutant, where C3b deposition is promoted by IgM binding, the lst mutant's surface is not a target for IgM molecules. Thus, the lacto-N-neotetraose residue of neisserial LOS alone, without the presence of terminal sialic acid, is sufficient to block IgM epitopes either on the LOS itself, or on other surface molecules. Our data provide further insight into the complex interplay of capsular and LOS sialic acids in serogroup B meningococci with host effector mechanisms, and suggest that LOS sialylation in meningococci is of a less central importance as it is in gonococci.

Animals↗
PubMed2000

Mapping of the region of complement receptor (CR) 1 required for Plasmodium falciparum rosetting and demonstration of the importance of CR1 in rosetting in field isolates.

The malaria parasite Plasmodium falciparum induces a number of novel adhesion properties in the erythrocytes that it infects. One of these properties, the ability of infected erythrocytes to bind uninfected erythrocytes to form rosettes, is associated with severe malaria and may play a direct role in the pathogenesis of disease. Previous work has shown that erythrocytes deficient in complement receptor (CR) 1 (CR1, CD35; C3b/C4b receptor) have greatly reduced rosetting capacity, indicating an essential role for CR1 in rosette formation. Using deletion mutants and mAbs, we have localized the region of CR1 required for the formation of P. falciparum rosettes to the area of long homologous repeat regions B and C that also acts as the binding site for the activated complement component C3b. This result raises the possibility that C3b could be an intermediary in rosetting, bridging between the infected erythrocyte and CR1. We were able to exclude this hypothesis, however, as parasites grown in C3-deficient human serum formed rosettes normally. We have also shown in this report that rosettes can be reversed by mAb J3B11 that recognizes the C3b binding site of CR1. This rosette-reversing activity was demonstrated in a range of laboratory-adapted parasite strains and field isolates from Kenya and Malawi. Thus, we have mapped the region of CR1 required for rosetting and demonstrated that the CR1-dependent rosetting mechanism occurs commonly in P. falciparum isolates, and could therefore be a potential target for future therapeutic interventions to treat severe malaria.

Animals↗
PubMed1985

Genetic polymorphism of human factor I (C3b inactivator).

Genetic polymorphism of human factor I (C3b inactivator) has been described using polyacrylamide gel isoelectric focusing electrophoresis of neuraminidase-treated EDTA plasma samples followed by electrophoretic blotting technique. In 435 individuals three different common patterns were observed, and these were controlled by two common alleles at a single locus. The results of typing family material confirmed autosomal codominant Mendelian inheritance. Two common alleles were designated FI*B and FI*A, and gene frequencies were estimated to be 0.8931 and 0.1069 for FI*B and FI*A, respectively. The distribution of phenotypes fitted the Hardy-Weinberg equilibrium. Linkage studies failed to show close linkage between factor I and the major histocompatibility complex.

Complement C3b Inactivator Proteins↗
PubMed1986

[Serum immunoconglutinin in patients with a respiratory allergy syndrome, before and after specific immunotherapy].

Immunoconglutinins (Ic) are a group of predominantly IgM antibodies formed towards antigenic determinants exposed in fixed complement components (C3b and C4). Ic production is stimulated by bacterial and viral infections; elevated titers were also found in a number of other diseases involving completement-fixing reactions in vivo. High titers have been associated with the infectious morbidity within a particular population. The object of this investigation was to determine the correlations of autostimulated Ic titre with several parameters in 100 atopic nondesensitized subjects with respiratory allergies and in 140 non atopic, healthy, individuals. Eighty of them had rhinitis and/or conjunctivitis, 9 asthma and 11 rhinitis and asthma. All the atopics were studied also after at least 6 months of specific immunotherapy. None of the subjects received any other therapy at the time of study. Atopic subjects with high titers of Ic are significantly more numerous than controls, without significant correlation with the kind and beginning of atopic syndrome, and with immunotherapy. These results might be attributed to a higher incidence of infections in the atopic population. The Ic might play an important role in the immunoregulation involving complement system.

Adolescent↗
PubMed1984

Effects of interferon-alpha/beta and interferon-gamma preparations on phagocytosis by mouse peritoneal macrophages.

The influence of murine alpha/beta-interferon (Mu IFN-alpha/beta) and murine gamma-interferon (Mu IFN-gamma) preparations on the attachment and ingestion phase of phagocytosis by mouse peritoneal macrophages (MPM) was studied. A non-opsonized strain of Escherichia coli, IgG-opsonized E. coli, and sheep erythrocytes opsonized with IgG (E-IgG) and IgM plus complement factor C3b (E-IgMC) were used as test particles. Pretreatment of MPM with 10(2)-10(3) U/ml of Mu IFN-alpha/beta for 24 h enhanced both attachment and ingestion of bacteria or erythrocytes mediated by the non-specific receptor, the Fc receptor, or the C3b receptor. Higher concentrations had no such effects. In contrast, treatment of MPM with 10(1)-10(2)U/ml of Mu IFN-gamma suppressed attachment and ingestion of non-opsonized and IgG-opsonized E. coli and of E-IgG by 10-40%. Mu IFN-gamma did not influence attachment and ingestion of E-IgMC. The effects were neutralized by specific anti-IFN antiserum. The data indicate that the IFN effect on phagocytic activity is, at least to a large extent, due to modifications of the surface receptors.

Animals↗
PubMed2000

Host recognition and target differentiation by factor H, a regulator of the alternative pathway of complement.

Factor H is responsible for recognition of host cells and tissues and mediates discrimination among microbial pathogens during activation of the alternative pathway of complement (AP). Its unique structure of 20 SCR domains arranged in a flexible chain permits a variety of functional sites to interact with complement proteins and surface markers in a biological example of single-molecule combinatorial chemistry. In addition to the complement regulatory site located in the N-terminal four SCR domains, two other sites bind complement protein C3b and three sites appear to recognize a variety of polyanions that serve as host markers. Recent studies indicate that cooperativity among several C3b- and polyanion-binding sites influences the biological functions of factor H and that the degree of influence of each site varies on different cells. The engagement of one or more of the host marker recognition sites enables factor H to control activation of the AP. The absence of host-like markers allows AP activation, but many common pathogens have developed receptors for factor H or mimics of host markers of varying degrees of authenticity allowing them to escape detection by this innate defense system. Organisms using one or more of these evasive techniques include Neisseria gonorrhoeae, Streptococcus pyogenes, Yersinia enterocolitica, Trypanosoma cruzi, and the HIV virus.

Animals↗
PubMed1986

In situ characterization of the inflammatory cell infiltrates of hyperplastic denture stomatitis.

Cryostat sections from 14 surgical specimens were examined to determine whether selected factors of the immune response related to histopathological reactions are present in the palatal mucosa affected by hyperplastic denture stomatitis. By means of various immunological techniques the presence of IgG, IgA, IgM, complement factor C3c, receptors for the Fc region of IgG (FcR) and for complement factor C3b (C3bR), T lymphocytes, and macrophages were studied. The inflammatory infiltrate was mainly located in the papillary part of the lamina propria. IgG, IgA, and IgM appeared both in plasma cells and intercellularly. FcR, C3bR, and T lymphocytes were present in the areas with inflammatory cell infiltrate. Macrophages were found in the papillary part of the lamina propria and within the epithelium. The immunological response in the mucosa affected by denture stomatitis was in many respects similar to that of marginal and apical periodontitis. We conclude that hyperplastic denture stomatitis is a complex inflammatory lesion showing elements of both humoral and cellular immune responses.

Antibody Formation↗
PubMed1990

Studies on molecular regulation of phagocytosis and activation of the NADPH oxidase in neutrophils. IgG- and C3b-mediated ingestion and associated respiratory burst independent of phospholipid turnover and Ca2+ transients.

The role of messengers derived from hydrolysis of phosphoinositides and other phospholipids, of the basal level of [Ca2+]i and of the increase in [Ca2+]i in phagocytosis and respiratory burst was investigated, using normal neutrophils and neutrophils Ca2(+)-depleted by pretreatment with Quin2/AM and EGTA. 1) Phagocytosis and respiratory burst in control neutrophils challenged with yeast opsonized with IgG or C3b/bi were associated with a stimulation of the production of inositol phosphates, diacylglycerol, phosphatidic acid, arachidonic acid, and rise in [Ca2+]i. 2) In Ca2(+)-depleted neutrophils (basal [Ca2+]i 10 to 20 nM) the phagocytosis of yeast-IgG was similar to that in control neutrophils, the respiratory burst was slightly depressed (-30%), while the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, and phosphatidic and arachidonic acid did not occur. 3) In Ca2(+)-depleted neutrophils the phagocytosis of yeast-C3b/bi was slightly lower than that in control neutrophils, and the respiratory burst, related to the same number of particles ingested, was depressed by about 60%, whereas the increase in [Ca2+]i and production of inositol phosphates, diacylglycerol, phosphatidic acid, and arachidonic acid release did not occur. These findings demonstrate that transmembrane signaling pathways involving the hydrolysis of phosphoinositides by phospholipase C and D and of other phospholipids by phospholipase C and Az, and the rise in [Ca2+]i are not essential processes for triggering the ingestion of yeast particles opsonized with IgG and C3b/bi and the activation of the NADPH oxidase.

Arachidonic Acid↗
PubMed1988

Appearance of acceptor-bound C3b on HLA-DR positive macrophages and on stimulated U937 cells; inhibition of Fc gamma-receptors by the covalently fixed C3 fragments.

The appearance and the functional role of acceptor-bound C3b during differentiation of human monocytes into macrophages were studied. Acceptor-bound C3b could be detected by the immune adherence (IA) test parallel to the expression of antigenic determinants specific to mature cells--i.e. on days 4-5 of culture. Consequently, the capacity of these phagocytes to fix C3b covalently via C3b-acceptors (C3bAs) can be considered as one of the signs of their activation/differentiation. All the mature macrophages positive in the IA test were also found to express HLA-DR antigens on their membrane. Using solubilized extracts of stimulated, 35S-cysteine-labelled cells of the human monocytic cell line, U937, we demonstrate that C3 synthesized by these cells can bind to C3bAs of the same cells. Covalently fixed C3 fragments were found to inhibit Fc gamma-receptor-mediated ingestion of immune complexes and also antibody-dependent cellular cytotoxicity of monocyte-derived macrophages.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1991

[The characteristics of Staphylococcus aureus peptidoglycan in the spectrum of the reactivity of human lymphocytes to bacterial peptidoglycans].

The sensitivity of lymphocytes of healthy persons to S. aureus peptidoglycan as compared with that to the polyclonal stimulator zymosan C3b and peptidoglycans of other bacteria (Streptococcus faecalis, Escherichia coli, Bacterium bifidum) was analyzed with a test system permitting the determination of specific reactivity to peptidoglycans. The analysis showed that at the peak of luminol-dependent chemiluminescence (25-30 minutes) individual reactivity to S. aureus peptidoglycan varied within wide limits (the coefficient of lymphocyte stimulation was 1.4-9.6, 3.5 +/- 0.6), exceeding sensitivity to other bacteria, as well as the values obtained in the negative control. The conclusion of the wide spread of sensitization to S. aureus peptidoglycan and the possibility of using this preparation for the study of cell-mediated immunity reactions was made.

Bifidobacterium↗
PubMed1983

Modifications of granulocyte subpopulations and of their adherence property during filtration leukapheresis in man.

Polymorphonuclear leucocytes (PMN) isolated from the peripheral blood of 23 blood donors undergoing filtration leukapheresis were examined with regard to their number, expression of membrane receptors for rabbit IgG or human C3b and their ability to adhere to nylon fibres. All the donors showed an early granulocytopenia followed by a gradual return of PMN into the circulation, but differed in the number of PMN appearing in the rebound phase. Evaluation of rosette forming cells (RFC) and cells adhering to nylon fibres in this phase led to the recognition of two main groups of donors. In one of these groups the number of PMN, RFC and adhering cells remained unchanged compared to the pre-apheresis values. The other group showed marked leucocytosis accompanied by a relative increase in the number of non-RFC and non-adhering cells. Possible mechanisms involved in the changes of PMN subpopulations are discussed.

Cell Adhesion↗
PubMed1984

Myeloperoxidase modulates the phagocytic activity of polymorphonuclear neutrophil leukocytes. Studies with cells from a myeloperoxidase-deficient patient.

Patients lacking the primary granulae enzyme, myeloperoxidase (MPO), do not usually show any increased susceptibility to infection or altered inflammatory response, in contrast to several other biochemical defects in polymorphonuclear neutrophils. We have now evaluated the role of MPO on phagocyte function in a patient with complete MPO deficiency suffering from generalized pustular psoriasis. We found that the MPO-deficient neutrophils showed enhanced phagocytosis (greater than 200% of normal) of IgG- and C3b-opsonized yeast particles and prolonged N-formylmethionyl-leucyl-phenylaline-mediated stimulation of superoxide production. When purified human MPO was added to normal neutrophils during cell adhesion, their Fc- and C3b-mediated phagocytosis was reduced without affecting cell viability. 1 microgram/ml of MPO reduced the Fc and C3b phagocytosis to 47 and 65%, respectively, whereas 10 micrograms/ml reduced the activity to 20 and 54%. Both attachment and ingestion were reduced to a similar extent, indicating that MPO affected the receptor function per se. When MPO was added to the hyperactive MPO-deficient cells, phagocytosis was reduced more rapidly. Catalase, azide, and methionine eliminated the inhibitory effect, and catalase and methionine, in fact, enhanced the phagocytic activity of adherent neutrophils. These data indicate that, apart from being a potent antimicrobial system, the oxidizing activity of the MPO-H2O2-halide system may modulate the inflammatory response by impairing certain receptor-mediated recognition mechanisms of phagocytic cells, which otherwise could elicit inflammatory reactions and tissue injury.

Complement C3b↗
PubMed1976

A new activity of complement component C3: cell-bound C3b potentiates lysis of erythrocytes by C5b,6 and terminal components.

EAC4b,3b (sheep erythrocytes carrying rabbit antibody and guinea pig complement component fragments C4b and C3) adsorb human C5b,6 reversibly; the avidity of binding varies inversely with ionic strength. We believe that the receptor of C5b,6 is contributed by the cell-bound C3b because the binding capacity of EAC4b,3b varies with C3b multiplicity and can be blocked with rabbit antibody to guinea pig C3. The fixation of C5b,6 to the erythrocyte-bound C3b serves to concentrate C5b,6 on the cell surface; as a consequence, the hemolytic efficiency of C5b,6 is almost 100 times greater when assayed with EAC4b,3b than with plain erythrocytes. This potentiation represents a hitherto unrecognized function of cell-bound C3b.

Animals↗
PubMed1997

Monocyte adherence to Paracoccidioides brasiliensis, zymosan-C3b and erythrocyte-hemolysin in patients with paracoccidioidomycosis.

Independent and dependent (C3b/Fc receptors) opsonic adherence ability of monocytes from thirty-three patients with acute or chronic paracoccidioidomycosis and from 13 healthy individuals were studied in the presence of Paracoccidioides brasiliensis (Pb), Paracoccidioides brasiliensis opsonized by patient's serum (PbPS) or normal serum (PbNS), zymosan opsonized by fresh sera from healthy donors (ZyNS) and erythrocytes opsonized by hemolysin (EA). Statistically significant differences concerning the percentage of adhered monocytes to PbPS (number of adhered monocytes/total number of monocytes) were detected between control and chronic (active and inactive) groups. Significant differences in relationship to the mean number of PbPS (number of fungi in monocytes/total number of monocytes) were also observed between control and chronic active mycosis. Present data suggest that patients with chronic disease have more ability in the first step of phagocytic activity, considered as the main effector mechanism to control the dissemination and severity of paracoccidiodomycosis.

Acute Disease↗
PubMed1993

The influence of IL-3, IL-5, and GM-CSF on normal human eosinophil and neutrophil C3b-induced degranulation.

The priming effect of interleukin-3 (IL-3), interleukin-5 (IL-5), and granulocyte/macrophage-colony stimulating factor (GM-CSF) on eosinophil and neutrophil degranulation was studied. Granulocytes were obtained from normal donors, and degranulation was induced by incubation with serum-opsonized Sephadex particles. The released amounts of eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO), and lactoferrin (LF) were measured by radioimmunoassay (RIA). The effect of IL-5 was dose- and time-dependent, with a maximal enhancement of ECP and EPX release of 71% (P < 0.03) and 66% (P < 0.03), respectively. Neutrophil degranulation, however, was unaffected. IL-3 was marginally effective, whereas GM-CSF seemed to act as a secretagogue for both eosinophil and neutrophil degranulation. We conclude that IL-5 selectively primes eosinophil degranulation, whereas IL-3 and GM-CSF seem to act as secretagogues for eosinophils and neutrophils. The results indicate that IL-5 may be involved in the priming of eosinophils as observed in patients with asthma and hypereosinophilic syndrome (HES).

Blood Proteins↗