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PubMed1994

Immunohistochemical staining of retrobulbar adipose tissue in Graves' ophthalmopathy.

An increase of retrobulbar adipose tissue has been shown by imaging techniques in patients with Graves' ophthalmopathy (GO). Immunohistochemical staining was applied to investigate the involvement of different retrobulbar (especially adipose) tissue components in the autoimmune process of the disease. Cryostat sections from retrobulbar tissues of 15 GO patients and 11 controls were analyzed with a battery of monoclonal antibodies against CD2, CD4, CD8, CD11a, CD19/22, CD25, CD54, CD57, CD68, C3b, HLA-A, B, C, and HLA-DR. In contrast to controls, the retrobulbar adipose tissue showed an increase of HLA-DR expression, an activation of intercellular adhesion molecule I (ICAM-1, CD54), as well as a marked infiltration of activated T-cells (especially CD4) and macrophages (CD68), whereas a positive staining for interleukin-2 receptors (CD25), T-cells (CD2), B-cells (CD19/22), complement factors (C3b), and LFA-1 (CD11a) was present only in a few patients' specimens. Staining for natural killer cells (CD57) was negative in fat tissue of patients and controls. The perimuscular connective tissue sections of GO patients showed an accumulation of T-cells (CD4/CD8), an enhanced display of ICAM-1, LFA-1, HLA-DR-positive cells, and B-cells. Retrobulbar muscle tissue of GO patients exhibited an increase of CD4, CD8, CD54, CD68, and HLA-DR-positive cells, located in the intramuscular connective tissue, exclusively. In conclusion, the present study underlines the involvement of retrobulbar adipose tissue in the immunopathogenesis of Graves' ophthalmopathy.

Adipose Tissue↗
PubMed1989

The effect of tumor necrosis factor-alpha and interferon-gamma on neutrophil function.

Tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN-gamma) have been shown to regulate cell-mediated immunity and act as effective modifiers of immune function; however, their influence on neutrophil (PMN) function is not well defined. This study investigated the effect of these cytokines on PMN phagocytosis, respiratory burst, and complement receptor (C3b) expression. Human citrated whole blood was incubated with either phosphate-buffered saline (control), 20 micrograms Escherichia coli lipopolysaccharide (LPS), TNF (1, 10, or 100 units), or IFN-gamma (1, 10, or 100 units). Synergy was also assessed between TNF and IFN-gamma. Phagocytosis and respiratory burst were assayed by sequential incubation of blood with dichlorofluorescein diacetate followed by labeled Staphylococcus aureus. C3b receptor expression was assayed by labeled anti-CR3 monoclonal antibody. Measurements were expressed as the mean channel fluorescence of 2000 PMNs counted by flow cytometry. IFN-gamma alone at all doses had no effect on any of the parameters measured. TNF 1 unit/ml increased phagocytosis (666 +/- 47 vs 542 +/- 19), respiratory burst (326 +/- 33 vs 258 +/- 17), and C3b (374 +/- 42 vs 157 +/- 14; all P less than 0.05) over those of control. TNF also demonstrated dose-dependent PMN activation. The combination of TNF + IFN-gamma increased both respiratory burst and C3b compared to either agent alone. These data indicate that TNF enhances PMN function and cytokine interaction may be important in PMN activation.

Humans↗
PubMed2006

[The immune enhancement of the C3 to the Escherichia coli vaccine].

C3b was separated and purified from the SPF chicken serum. It was linked with E. coli antigen by the glutaral. 11 days aged SPF chicken were immunized by the complex antigen and the chickens of control group were immunised by the FCA- E. coli antigen . They were boosted at the age of 18 day. The immune response was monitored by an enzyme-linked immunosorbent assay(ELISA) for anti-E. coli anitibody. The ELISA results indicated that during the early several weeks, IgG titers elicited by FCA (FCA-E. coli) were higher than those elicited by C3 (C3b-E. coli), but decreased rapidly after a peak around the end of 4th week from being immunized. Chickens immunized with C3b always gave increased response, and the IgG titers were equal to that of FCA at the end of 5th week from being immunized and then higher and higher than that of FCA. Thus the adjuvant effect of C3b is different from that of FCA, it could induce production of memory cell and make the antigens stimulate immune cells consistently and stably.

Adjuvants, Immunologic↗
PubMed1991

Phagocytosis of autologous platelets by human neutrophil granulocytes.

The phenomenon that the autologous human platelets can be phagocytosed in vitro by neutrophils and monocytes of healthy human donors (being free of antiplatelet autoantibodies) was demonstrated earlier. In this study we have confirmed electron microscopically the phenomenon of phagocytosis. Besides, using flow cytometric analysis, we have shown that a possible way for the bridge-formation between the washed platelets and neutrophils can be the linkage via IgG + C3b complexes bound to the Fc receptors of platelets. This mechanism supposedly may play a role also in the clearance of platelets by peripheral phagocytes in vivo.

Blood Platelets↗
PubMedDate not supplied

Studies on the immunopathology of the nephrotic syndrome associated with Plasmodium malariae. 1. Serum levels of an immune adherence inhibitor.

A comparative study of the serum levels of an immune-adherence inhibiting factor was carried out on serum samples from eighty-seven nephrotic syndrome children, twenty-eight nephrotic adults, 202 normal school children, 116 blood donors, twenty-five falciparum malaria children and 172 patients with miscellaneous diseases. Low titres (1/32 and below) of the factor were present in sera from 63.2% of the nephrotic children 60.7% of nephrotic adults and 60.0% of children malaria, as compared with 30.7% of the normal children, 25.5% of the patients with miscellaneous diseases and 41.4% of the blood donors. There is a significant difference between nephrotic children and normal children with low titres (P less than 0.05). Furthermore, 36.8% of the nephrotic children had serum titres of 1/4 or less, as compared with 6.4% of normal children. The serum factor is tentatively referred to as 'C3b-inase'. Its similarity to conglutinogen-activating factor (KAF) and its possible role in the pathogenesis of the immune-complex nephropathy of childhood nephrotic syndrome associated with malaria are discussed.

Adolescent↗
PubMed1980

Establishment and characterization of a human acute monocytic leukemia cell line (THP-1).

A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia is described. This cell line had Fc and C3b receptors, but no surface or cytoplasmic immunoglobulins. HLA haplotypes of THP-1 were HLA-A2, -A9, -B5, -DRW1 and -DRW2. The monocytic nature of the cell line was characterized by: (1) the presence of alpha-naphthyl butyrate esterase activities which could be inhibited by NaF; (2) lysozyme production; (3) the phagocytosis of latex particles and sensitized sheep erythrocytes; and (4) the ability to restore T-lymphocyte response to Con A. The cells did not possess Epstein-Barr virus-associated nuclear antigen. These results indicate that THP-1 is a leukemia cell line with distinct monocytic markers. During culture, THP-1 maintained these monocytic characteristics for over 14 months.

Cell Line↗
PubMed1985

Leukocyte membrane receptors in meningitis and other bacterial infections.

Blood leukocytes from 37 patients with acute bacterial infections, and cerebrospinal fluid (CSF) granulocytes from 12 patients with bacterial meningitis, were examined for the distribution of membrane receptors (R) for (1) untreated sheep erythrocytes (E), (2) the Fc portion of IgG (Fc gamma), and (3) complement component C3b. We found a decreased percentage of granulocytes bearing Fc gamma-R in the CSF from patients with meningitis, and in blood from patients with respiratory tract infections. This group also had a decreased percentage of C3b-R bearing granulocytes on admission, whereas meningitis patients had lower levels of C3b-R and Fc gamma-R bearing granulocytes in the 2nd and 3rd week and even later. Several patients with meningitis and gastroenteritis had granulocytes bearing the E-R, previously considered specific for T lymphocytes. Such cells were also found in the CSF. Meningitis and respiratory tract infections were associated with a decreased percentage of 'active' T lymphocytes. The total percentage of T lymphocytes was also decreased in meningitis. Conversely the proportion of Fc gamma-R bearing lymphocytes (consisting mostly of B lymphocytes) was increased in most infections. During the first 3 weeks of bacterial meningitis, the percentages of Fc gamma- and C3b-R bearing granulocytes, and of Fc gamma-R bearing lymphocytes, gradually decreased, while the T lymphocyte percentage increased from the initial low values.

Antigens, Surface↗
PubMed1998

Nonopsonic and opsonic association of Mycobacterium tuberculosis with resident alveolar macrophages is inefficient.

The association of Mycobacterium tuberculosis with alveolar macrophages (Mphi) in a serum-free environment is a crucial first step in the pathogenesis of this facultative intracellular pathogen. We present data demonstrating that freshly explanted alveolar Mphi do not efficiently bind M. tuberculosis in a serum-free system, although a small subpopulation of these Mphi (10-15%) can bind mycobacteria. In contrast, almost 100% of a peritoneal Mphi population bind mycobacteria under the same conditions. The poor binding of mycobacteria by alveolar Mphi does not reflect a general inability to associate with particles; binding and ingestion of latex beads and zymosan particles were comparable with that seen with peritoneal Mphi. Resident alveolar Mphi did not efficiently bind mycobacteria in the presence of serum and expressed poorly several Mphi surface receptors, including CR3. Furthermore, we demonstrate that bovine surfactant protein A does not enhance the association of M. tuberculosis with alveolar Mphi. Differentiation of alveolar Mphi in vitro resulted in increased expression of Mphi surface receptors and an increased capacity to bind mycobacteria in the presence and absence of serum. Evidence is presented that opsonic binding of M. tuberculosis by differentiated alveolar Mphi is mediated by complement and CR3, and that the poor binding by resident alveolar Mphi is due to their poor expression of CR3. The receptor mediating nonopsonic binding of M. tuberculosis to differentiated alveolar Mphi was not unequivocally identified in this study, but could also be CR3.

Animals↗
PubMed1983

The interaction of C3b bound to pneumococci with factor H (beta 1H globulin), factor I (C3b/C4b inactivator), and properdin factor B of the human complement system.

Pneumococcal cell walls are potent activators of the alternative complement (C) pathway; pneumococcal capsules are not. C3b that is deposited onto the cell walls of encapsulated organisms, however, functions inefficiently in host defense compared to C3b deposited onto capsular polysaccharides. Results of previous studies with guinea pig erythrocytes suggested that C3b deposited onto surfaces that do not activate the alternative pathway is rapidly inactivated. In the present study, we examined the interactions of C3b bound to pneumococcal capsules, to pneumococcal cell walls, and to the surface of sheep erythrocytes (E) with the serum control proteins, Factor H (beta 1H globulin) (H) and Factor I (C3b/4b inactivator) (I), and with Factor B (B) of the alternative C pathway. Conversion of bound C3b to C3bi was assayed by binding of radiolabeled conglutinin in a quantitative binding assay. Neither pneumococcal cell wall C3b nor capsular C3b was converted efficiently to a conglutinin-binding form by serum incubation. Experiments with purified C components showed that, after incubation with H and I, fewer conglutinin-binding sites were created on pneumococci than on E bearing equal numbers of C3b. Molecular analysis demonstrated that this did not result from cleavage of pneumococcal-bound C3b to an unusual, nonconglutinin-binding form of the molecule. Binding studies in which radiolabeled H was used demonstrated that the majority of C3b that is bound to both pneumococcal capsules and cell walls bound H with a lower affinity than did E-bound C3b. Studies of the binding of radiolabeled B demonstrated that C3b that was bound to pneumococcal cell walls and to E demonstrated equal affinity for B. In contrast, the majority of C3b that was fixed to pneumococcal capsules bound B with only 1/30 as high affinity. We conclude that pneumococcal capsules are not alternative pathway activators because the low affinity of capsular C3b for B leads to inefficient formation of an alternative pathway convertase, C3bBb. With regard to H binding, both cell wall- and capsular-bound C3b act as if they were in a "protected site" and resist degradation by the control proteins.

Binding Sites↗
PubMed1984

C3b receptor (CR1) on erythrocytes in various diseases.

Complement receptor for C3b (CR1) on erythrocytes was investigated in various diseases by immune adherence hemagglutination (IAHA) using aggregated human IgG. In normal controls, 21 out of 312 (6%) revealed defective CR1 reactivity, and there was no difference in the prevalence of defective CR1 reactivity between female (11/157, 7%) and male (10/155, 6%). Among diseases examined significantly high prevalence of defective reactivity of CR1 on erythrocytes was seen in systemic lupus erythematosus (SLE) (22/30, 73%) and malignancy of hematopoietic system, especially in acute myelogenous leukemia (AML)(6/11, 55%).

Connective Tissue Diseases↗
PubMed1980

Unique aspects of the modulation of human neutrophil function by 12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid.

12-L-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-OOHETE), a labile intermediate generated by the lipoxygenation of arachidonic acid in platelets, and 12-L-hydroxy-5,8,10.14-eicosatetraenoic acid (12-OHETE), the reduction product of 12-OOHETE, were examined for their effects on human neutrophil function in vitro. 12-OOHETE elicited a maximal neutrophil chemotactic response at 4 microgram/ml, that exceeded by over 50% the maximal chemotactic response to 10-20 microgram/ml of 12-OHETE. Similarly 12-OOHETE was more potent than 12-OHETE in evoking neutrophil chemokinetic responses and in enhancing the expression of C3b receptors on neutrophils. The concentration of guanosine 3':5' cyclic monophosphate (cGMP) in neutrophils was increased to the same plateau level by 5 ng/ml of 12-OOHETE and by 50 ng/ml of 12-OHETE. Elevations in the concentration of cGMP were maintained for 30 min or longer by a single dose of 12-OOHETE, but fell between 10 and 20 min after the introduction of 12-OHETE. The release of neutrophil lysosomal enzymes by the chemotactic fragments of C5 was augmented substantially by 12-OOHETE, while 12-OHETE had only a marginal effect. The non-chemotactic methyl ester of 12-OHETE failed to inhibit the chemotactic responses to 12-OOHETE at molar ratios that suppressed comparable response to 12-OHETE by 42-86%. Thus 12-OOHETE is more potent than 12-OHETE in the stimulation of some human neutrophil functions and in the elevation of the cellular concentration of cGMP. Furthermore, 12-OOHETE may activate neutrophils by pathways not available to 12-OHETE.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1990

Functional consequences of the genetic polymorphism of the third component of complement.

The third component of complement, the central protein of the complement cascade, occurs in two principal allotypes, C3S and C3F. An excess frequency of the F allotype has been implicated in a number of disease states, including some forms of glomerulonephritis. These associations have been explained by functional differences between C3S and C3F. We examined several complement functions, using purified preparations of C3S or C3F. The C3S allotype was 1.3 times more efficient than C3F in a hemolytic assay employing sensitized sheep erythrocytes; this difference was shown to arise from a slightly more efficient deposition of C3F on the cell surface. These differences are trivial and of much less magnitude than the functional differences between C4A and C4B. There were no differences between allotypes in their ability to be converted to inactive C3b (C3bi) by complement factors H and I or by CR1 and factor I. No significant differences were seen between the allotypes and their ability to support solubilization of preformed immune complexes.

Animals↗
PubMed1996

Changes of expression of complement 3bi receptors on granulocytes after physical exercise in rats.

OBJECTIVE: The purpose of this study was to examine the effects of physical exercise on the expression of clusters of differentiation (CD)11b, CD18 and CD 54 on granulocytes and complement 3bi (C3bi)-dependent functions in rats. EXPERIMENTAL DESIGN: The rats were separated into two groups: the control group (n = 21) and the training group (n = 14). The rats in the training group were subjected to a program that consisted of swimming 6 days in a week for 7 weeks. MEASURES: The phagocytic and hydrogen peroxide (H2O2) generating activities and the expression of receptors on granulocytes were measured using flow cytometric techniques. RESULTS: The acute swimming and the 7 weeks of swim training caused high C3bi-dependent phagocytic activity of granulocytes. Swim training led to an increase of H2O2 generating activity together with C3bi-dependent phagocytosis both immediately after and 24 hrs after the 120 min swimming. The 24 hrs recovery from the 120 min swimming in trained and untrained rats caused an increase of H2O2 generating activity of granulocytes. In the training group, an increase in this activity could be observed immediately after the 120 min swimming exercise. The upregulation of the CI1b on granulocytes was induced by the 120 min of swimming regardless of swim training. CONCLUSIONS: These results suggest that the increase of C3bi receptor-dependent phagocytosis and H2O2 generating activities induced by the physical exercise might depend on the upregulation of the CD11b that is the component of C3bi receptor.

Animals↗
PubMed1991

Phagocytosis of fluorescent latex microbeads by peritoneal macrophages in different strains of mice: a flow cytometric study.

The phagocytosis of uniform fluorescent latex particles by resident and thioglycollate-elicited macrophages was analysed by flow cytometry. The percentage of phagocytosing macrophages and the number of internalized microspheres per cell was determined from cell size and fluorescence histograms. Results were corrected for the adherence of microbeads to the cells in the presence of sodium azide in the medium. Human C3b- or murine monoclonal IgG-coated microspheres were applied to assess receptor-mediated phagocytosis in different inbred strains of mice. Phagocytic activity of thioglycollate-elicited macrophages was consequently higher than that of resident macrophages. A decreasing gradient of C3b and Fc receptor-mediated phagocytosis was established in the following order: B10.BR, B10, C3H/Di and C3H.SW strains. Our results indicate that the phagocytic function of murine macrophages is under control of both the somatic (non-H-2) and H-2 genes.

Animals↗
PubMed1986

Murine protein H is comprised of 20 repeating units, 61 amino acids in length.

A cDNA library constructed from size-selected (greater than 28 S) poly(A)+ RNA isolated from the livers of C57B10. WR mice was screened by using a 249-base-pair (bp) cDNA fragment encoding 83 amino acid residues of human protein H as a probe. Of 120,000 transformants screened, 30 hybridized with this cDNA probe. Ten positives were colony-purified, and the largest plasmid cDNA insert, MH8 (4.4 kb), was sequenced by the dideoxy chain termination method. MH8 contained the complete coding sequence for the precursor of murine complement protein factor H (3702 bp), 100 bp of 5'-untranslated sequence, 448 bp of 3'-untranslated sequence, and a polyadenylylated tail of undetermined length. Murine pre-protein H was deduced to consist of an 18-amino acid signal peptide and 1216 residues of H-protein sequence. Murine H was composed of 20 repetitive units, each about 61 amino acid residues in length. Similar repetitive units are present in the C4b binding protein, the C3b-receptor (CR1), complement factor B and C2, and in beta 2-glycoprotein I and the interleukin 2 receptor. This finding suggests a common evolutionary origin for regions of these proteins.

Amino Acid Sequence↗
PubMed1986

Human complement factor H: isolation of cDNA clones and partial cDNA sequence of the 38-kDa tryptic fragment containing the binding site for C3b.

We isolated cDNA clones coding for the functionally important tryptic N-terminal 38-kDa fragment of human complement control protein factor H using polyclonal and monoclonal antibodies to screen a human liver cDNA library cloned in a bacterial expression vector, PEX-1. By testing the reactivity of antibodies specific for the recombinant proteins produced by individual clones with proteolytic fragments of serum H the exact position of these cDNA clones within H was mapped. One clone, H-19, coding for the 38-kDa fragment of H was sequenced and found to code for 289 amino acids derived from the 38-kDa N-terminal fragment as well as for the first 108 amino acids belonging to the complementary 142-kDa tryptic fragment. The derived protein sequence could be arranged in 6 highly homologous repeats of about 60 amino acids each, the homology between the repeats being determined by the characteristic position of cysteine, proline, glycine, tyrosine and tryptophane residues. The region coding for the epitope recognized by one of our monoclonal antibodies was localized by subcloning restriction fragments of H-19 into the expression plasmid and testing for the expression of this epitope.

Amino Acid Sequence↗
PubMed1993

[The diagnostic significance of neutrophil adhesive reactions in rheumatoid arthritis].

Literature and original data on 97 RA patients concerning the diagnosis of rheumatoid vasculitis (RV) are presented. Study was made of CIC, rheumatoid factor, migration profiles of skin fenestra, IgG- and C3b-dependent adherence of peripheral blood neutrophils. The latter was found when elevated to relate to RV symptoms which was confirmed by skin biopsy. Feasibility of using the results of the study for RV diagnosis is discussed.

5'-Nucleotidase↗
PubMed2005

Selective and efficient inhibition of the alternative pathway of complement by a mAb that recognizes C3b/iC3b.

The alternative pathway (AP) of the complement system plays an important role in tissue damage and inflammation associated with certain autoimmune diseases and with ischemia-reperfusion injury. Selective inhibition of the AP could prevent such pathologies while allowing the classical and lectin pathways of complement activation to continue to provide protection. Here we present data describing selective inhibition of the AP of complement by anti-C3b/iC3b monoclonal antibody (mAb) 3E7, and by a chimeric, "deimmunized" form of this mAb, H17, which contains the human IgG1 Fc region and was further modified by substitution of amino acids in order to remove T cell epitopes. Both mAbs block AP-mediated deposition of C3b onto zymosan or Sepharose 4B, and they also inhibit AP-promoted lysis of rabbit erythrocytes. MAbs 3E7 and H17 also successfully compete with both factors B and H for binding to C3b-opsonized substrates, and the ability of both mAbs to inhibit the AP is blocked by pre-incubation with two different sources of C3(H2O). Kinetic measurements demonstrate that mAb 3E7 effectively stops progression of C3b deposition after AP activation is initiated. Our results therefore suggest that these mAbs block activation of the AP by binding to both C3(H2O) and to C3b, and thus prevent binding and activation of factor B. Based on these and other observations, mAb H17 may find future use in therapeutic applications focused on selective inhibition of the AP.

Antibodies, Monoclonal↗