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PubMed1987

Tetanus toxoid-anti-tetanus toxoid complexes: a potential model to study the complement transport system for immune complex in humans.

Complement and its receptor on erythrocytes appears to play a physiological role in the elimination of large immune complexes (IC) in monkeys, and a similar system is likely to work in humans. Here we define a safe IC model which is suitable for clinical investigations. Soluble tetanus toxoid (TT)-human anti-TT (IgG) antibody complexes were prepared in large antibody excess. The size of the complexes was approximately 45 S. When incubated in normal human serum, 50% of the IC increased further in size, but remained soluble, and bound rapidly to human erythrocytes in vitro. This binding was shown to require intact classical pathway function. When injected into normal guinea-pigs a comparable proportion of IC bound immediately to blood cells (mainly to platelets). No platelet binding of IC occurred in C4-deficient guinea-pigs, but this binding was restored when C4 was supplied. Initial immune complex elimination was faster in C4 deficient than in C4-supplemented and normal guinea pigs. Thus classical pathway function appeared to be necessary for the normal processing, transport and elimination of TT-anti-TT complexes.

Animals↗
PubMed1987

Comparison of blocking effects of monoclonal antibodies anti-MO1-alpha and anti-LFA1-alpha on human neutrophil functions.

In order to analyse the role of LFA1 and MO1 on neutrophil functions, the blocking effects of two monoclonal antibodies (MAb), one (anti-MO1) recognizing an epitope of the MO1-alpha chain and the other (25.31) an epitope of the LFA1-alpha chain, were measured. Adherence of 51Cr-labelled control neutrophils was 66 + 8% (mean +/- 1 SD) on plastic nuclon plates; this figure decreased to 33 +/- 5% and 23 +/- 6% of control adherence when the neutrophils had been pretreated with anti-LFA1-alpha (anti-alpha L) and anti-MO1-alpha (anti-alpha M), respectively. On another support (plastic culture chambers), 84 +/- 6% of control neutrophils adhered and the adherence of neutrophils pretreated with anti-alpha L or anti-alpha M was 10% and 43% of the control figure, respectively. These results show that adherence of neutrophils is dependent upon the plastic used. Moreover, inhibition of adhesion by the two MAbs was also dependent upon the support used for the assay, suggesting that MO1 and LFA1 may be surface proteins with different specificities. Both antigens capped upon adhesion, while they were randomly distributed in resting neutrophils. Anti-alpha L inhibited (congruent to 50%) locomotion more than did anti-alpha M (congruent to 25%), without altering chemoattractant-induced shape changes. These results suggest that the two MAbs inhibit chemokinesis but not chemotaxis. Many other adherence-associated functions, such as ingestion of opsonized Klebsiella pneumoniae, and cytotoxicity towards K/562 cells were decreased more by anti-alpha L than by anti-alpha M. In contrast, chemiluminescence and iodination induced by opsonized zymosan were inhibited more by anti-alpha M than by anti-alpha L. Degranulation induced by zymosan or opsonized zymosan was altered by anti-alpha M only, and this alteration involved azurophilic and not specific granules. Chemiluminescence induced by phorbol myristate acetate was inhibited to a greater extent by anti-alpha M than by anti-alpha L, while degranulation induced by phorbol myristate acetate was not altered by either of the two Mabs.

Adult↗
PubMed1987

Erythrocytes transfused into patients with SLE and haemolytic anaemia lose complement receptor type 1 from their cell surface.

Erythrocyte complement receptor type 1 (CR1) shows a numerical deficiency in patients with SLE and with haemolytic anaemias. This receptor is a cofactor for the enzymatic degradation of C3b and is believed to play a role in the transport of immune complexes from the circulation to the reticulo-endothelial system. Erythrocyte CR1 was enumerated on cells which had been transfused into patients with SLE and haemolytic anaemias. In three patients with active disease up to 60% of CR1 was lost from erythrocytes during 5 days after blood transfusion and up to 410 molecules of C3 were deposited on these cells. These are the first in vivo data showing that CR1 may be lost from circulating erythrocytes. This acquired deficiency of CR1 on erythrocytes may have pathological consequences in patients with SLE.

Anemia, Hemolytic, Autoimmune↗
PubMed1987

Chemotactic factor-induced low density neutrophils express enhanced complement (CR1 and CR3) receptors and increased complement-dependent cytotoxicity.

We have studied chemotactic factor-induced 'complement receptor enhancement' to determine whether changes in receptor expression and complement-dependent cytotoxicity were associated with alterations in cell density. Ficoll-Paque separated normal human neutrophils (greater than 90%), when further fractionated on discontinuous metrizamide (MTZ) gradients (18, 19, 20, 21, 22, 23% MTZ), consistently gave two major bands at the 20/21% and 21/22% interfaces. Incubation with the synthetic chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP (10(-8) M)), converted virtually all neutrophils to low density cells sedimenting on MTZ at the 18/19% and 19/20% interfaces. There was a time-dependent change of density after fMLP-stimulation which was maximal at 30 min, with cells reverting towards normal density by 60 min. Control unstimulated cells did not alter their density at any of the time points examined. Activated, low density neutrophils had increased expression of CR1 and CR3 (as shown by flow cytometry and the uptake of 125I-F(ab')2 monoclonal anti-CR1 antibody (E11)). These cells also showed enhanced cytotoxic capacity in vitro for helminthic targets (schistosomula of Schistosoma mansoni) opsonized with autologous complement. There were highly significant correlations between cell density and anti-CR1 uptake (P less than 0.001), and between schistosomular killing and change in density (P less than 0.001). Increased CR1 expression also correlated with enhanced helminthicidal capacity of neutrophils (P less than 0.001). Complement dependent cytotoxicity was partially reduced after treatment of cells with anti-human CR1 and/or CR3 antibodies, but only in the presence of a second antibody. These findings indicate that chemotactic factor-induced complement receptor enhancement of human neutrophils is associated with a decrease in cell density and increased complement-dependent cytotoxicity (CTX).

Animals↗
PubMed1987

Quantitative analyses of the binding of soluble complement-fixing antibody/dsDNA immune complexes to CR1 on human red blood cells.

We have used direct binding isotherm analyses to measure the association constant (Ka) and number of binding sites for the binding of prepared complement-fixing antibody (Ab)/dsDNA immune complexes (IC) to human red blood cells (RBC). In order to generalize this study we have examined the binding reaction for a number of different anti-dsDNA Ab (from systemic lupus erythematosus plasmas), complement sources, RBC donors, and dsDNA sizes. The affinity of the IC for the RBC is quite high, and the Ka values fall within a narrow range (5 to 14 X 10(10) liter/mol). Similarly, the limiting stoichiometries for the number of IC bound per RBC were between 40 and 91. The very high affinity and limiting stoichiometries both suggest that the IC bind to the RBC via multiple contacts with clusters of complement receptor type 1 (CR1). Furthermore, we have used three specific monoclonal AB (mAb) to quantitate CR1 on human RBC in the presence and absence of bound IC. One of these Ab, mAb 1B4, is blocked from binding to the RBC if IC are previously bound, and we have used this observation to verify the multivalent nature of the interaction of complement-fixing IC with CR1 on human RBC.

Antibodies, Antinuclear↗
PubMed1988

Tumor necrosis factor is the major monocyte product that increases complement receptor expression on mature human neutrophils.

Interleukin-1 (IL-1) and other monocyte products have several important effects on the systemic response to infection in addition to their roles in lymphocyte stimulation. The present studies were carried out to determine whether products of stimulated monocytes activated circulating neutrophils (PMN) to increase expression of receptors for C3b (CR1) and C3bi (CR3), which are necessary for optimal margination, migration, and phagocytosis. Supernatants of human mononuclear cells that had been stimulated with lipopolysaccharide (LPS) or purified protein derivative (PPD) contained both tumor necrosis factor (TNF) and IL-1 and increased CR1 and CR3 expression on isolated PMNs. Supernatants of unstimulated cultures, media alone, or LPS or PPD alone had little or no effect. Supernatant effects were detectable at 1:3,000 final dilution and appeared to have a characteristic slow time course. These supernatants also caused dose- and time-dependent secretion of PMN granular constituents, but maximal receptor expression was accompanied by secretion of less than 10% of the cells' content of lysozyme and less than 16% of the B12 binding protein. Immunoadsorption studies showed that the supernatant's activity could be removed by anti-TNF but not by anti-IL-1. Recombinant IL-1 had no effect on receptor expression, but recombinant TNF increased CR1 and CR3 expression with kinetics similar to the supernatants. These results thus indicate that TNF is the major monocyte product that increases CR1 and CR3 expression on mature blood neutrophils. This would result in increased margination and phagocytic activity and may be an important systemic effect that would help the host eradicate infection.

Calcium↗
PubMed1987

The relative roles of genetic and environmental factors in the regulation of erythrocyte C3b receptor (ECR1) numbers in normal individuals.

Erythrocyte C3b receptors (ECR1) have been measured in 122 pairs of twins (60 monozygotic, 62 dizygotic) using an [125I]-labelled monoclonal antibody (E11). The range of ECR1 numbers was wide, 99-4179 sites/cell, with a log-normal distribution around a geometric mean of 837 sites/cell. The intra-pair variance in dizygotic twins was no greater than that in monozygotic twins. These data indicate that ECR1 numerical expression is governed by environmental rather than genetic factors.

Adolescent↗
PubMed1987

Activated neutrophils exhibit enhanced phagocytosis of Cryptococcus neoformans opsonized with normal human serum.

We studied the effect of agents that activate neutrophils on phagocytosis of C. neoformans. The amount of CR3 on the surface of neutrophils was used as a marker for neutrophil activation. Surface CR3 was estimated by flow cytometry using phycoerythrin-labelled anti-CR3 (anti-Leu-15) monoclonal antibody. Phagocytosis was determined by incubation of neutrophils with cryptococci that had been preincubated with normal human serum. We found that treatment of neutrophils with (i) the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, (ii) zymosan activated serum, (iii) supernatant fluid from a mixed leucocyte culture, or (iv) supernatant fluid from human leucocytes cultured with phytohaemagglutinin produced a dose-dependent increase in CR3 density. These agents also markedly enhanced phagocytosis of opsonized cryptococci in a parallel dose-dependent fashion. These results indicate that phagocytosis of cryptococci opsonized with normal human serum is markedly enhanced by treatment of neutrophils with reagents that stimulate neutrophils. Our results demonstrate that neutrophils activated in an appropriate manner are capable of efficient phagocytosis of encapsulated cryptococci. This potential phagocyte activity may account in part for the high natural resistance to cryptococcosis.

Cryptococcus↗
PubMed1987

Abnormal podocyte CR-1 expression in glomerular diseases: association with glomerular cell proliferation and monocyte infiltration.

The expression of CR-1 complement receptors on glomerular epithelial cells, was studied in 77 renal biopsies from patients with (74) or without (3) glomerular diseases, employing an anti-CR-1 monoclonal antibody, and an indirect immunoperoxidase technique. Four patterns of CR-1 expression were recognised: normal (18); generally decreased (6); focal/segmental partial loss (44); and complete loss (9). Normal expression was detected in all three biopsies with non-glomerular diseases, and in glomerular diseases with normal glomeruli on light microscopy, but also in several glomerulonephritic biopsies (13), including diffuse proliferative lupus nephritis (1 of 7) and idiopathic membranous nephritis (5 of 14). However, the majority of biopsies from patients with glomerular diseases showed abnormal CR-1 expression (59 of 74), most evident in proliferative biopsies (43 of 49), with or without crescent formation (respectively, 18 of 20 and 25 of 29). Complete loss of CR-1 expression was almost restricted to crescentic biopsies (8 of 9). The abnormal CR-1 expression was unrelated to the presence of capillary immune deposits of Ig or C. More intraglomerular monocytes, assessed by monoclonal antibodies, were encountered in glomerulonephritic biopsies with partial CR-1 loss (median 6.2, P less than 0.05) or complete loss (median 14, P less than 0.03), than in biopsies with normal receptor expression (median 1.4). Thus, changes in glomerular CR-1 expression are frequently seen in many glomerular diseases and are associated with glomerular proliferative changes and monocyte infiltration, but not with the presence of capillary immune deposits.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗
PubMed1988

Sodium fluoride reveals multiple pathways for regulation of surface expression of the C3b/C4b receptor (CR1) on human polymorphonuclear leukocytes.

We have examined the effects of NaF on C3b receptor (CR1) expression and function in human polymorphonuclear leukocytes (PMN). Plasma membrane expression of CR1 was determined with a monoclonal antibody (3D9); CR1 function was assessed with erythrocytes bearing C3b (EC3b) or C3b oligomers prepared with avidin and biotin. NaF inhibited in a dose-dependent manner CR1-mediated phagocytosis and NaF inhibited f-met-leu-phe or phorbol dibutyrate-induced increases in CR1 expression, with 50% inhibition at 5 mM NaF. Increased plasma membrane expression of CR3 induced by f-met-leu-phe also was inhibited by NaF. However, increased CR1 and CR3 expression due to incubation at 37 degrees C were unaffected by 10 mM NaF. Incubation of PMN with 10 mM NaF depleted 80% of intracellular adenosine triphosphate (ATP) after 10 min. However, inhibition of CR1 function was unrelated to ATP level, inasmuch as normal increases in CR1 expression and in phagocytosis occurred 20 min after removal of NaF, whereas ATP levels remained below 25% of normal. Strikingly, internalization of soluble oligomeric C3b ligands was unaffected by 10 mM NaF, which completely inhibited phorbol dibutyrate-induced CR1 internalization and EC3b phagocytosis. We conclude that there are two different mechanisms for increasing plasma membrane expression of CR1, one of which is inhibitable by NaF. Moreover, there are two distinct pathways of CR1 internalization which can also be distinguished by their sensitivity to NaF.

Adenosine Triphosphate↗
PubMed1988

The clearance of tetanus toxoid/anti-tetanus toxoid immune complexes from the circulation of humans. Complement- and erythrocyte complement receptor 1-dependent mechanisms.

The role of complement and its receptor on erythrocytes (CR1) in the physiologic elimination of large immune complexes from the circulation of humans was assessed. Large radiolabeled soluble tetanus toxoid- anti-tetanus toxoid complexes were injected i.v. into three normal individuals and three patients with SLE. These complexes were prepared in antibody excess and were 45S in size, fixed C and bound to E CR1 in vitro. The percentage of complexes bound in vitro was directly proportional to CR1 number/E in four normal subjects and three SLE patients. After i.v. injection into normal subjects, complexes were cleared rapidly, with a monoexponential rate constant (10.3 to 11% complexes cleared/min). In the SLE patients, clearance was best explained by two phases: the first occurred within the first minute indicating immediate trapping of a fraction of the complexes (19.5 to 25.3% of injected complexes trapped), the second was monoexponential and was similar to the normal range. A large fraction of complexes bound within the first minute to E in vivo; the percentage of binding was variable, ranging from 16.3% to 71.5% and was related to E CR1 number. In a second study complexes were injected that had been attached to autologous E by opsonization with C in vitro. Their elimination was similarly monoexponential, except in one SLE patient in whom there was significant initial trapping (30.9%). A fraction of these complexes were released from E within the first minute, the percentage release being greatest in the patient with the lowest CR1 number (81.4%). E bearing immune complexes remained in the circulation and were not transiently sequestered in the liver or spleen. This is the first study of the clearance of soluble immune complexes in vivo in humans and shows that C and CR1 on E participate in immune complex clearance reactions, and that abnormal clearance can be detected in the form of rapid removal of immune complexes from the circulation.

Adult↗
PubMed1988

Identification of a C3b/iC3 binding protein of rabbit platelets and leukocytes. A CR1-like candidate for the immune adherence receptor.

Immune adherence is the attachment of C-bearing immune complexes via the major activation fragment of the third component of C(C3b) to C3b binding membrane proteins. On primate E, the C3b-R, termed CR1, mediates immune adherence. In nonprimates, immune adherence involves platelets instead of E. However, these functional data have not been corroborated by the identification of the binding protein. In this work, we have identified a C3b/iC3 binding protein of rabbit platelets and characterized it as a single chain structure with a Mr of 150 kDa (nonreducing) or 175 kDa (reducing). This protein binds to rabbit iC3 or C3b but not C3d. This specificity of binding and the ability to rebind to a second column of iC3- or C3b-thiol-Sepharose are comparable to human CR1. Also, a molecule with the identical Mr as well as other structural and binding characteristics is present on rabbit PBMC. No such protein was isolated from rabbit E. Our data strongly suggest that the C3b/iC3 binding protein of rabbit platelets is the homologue of human CR1. If so, this represents an interesting evolutionary switch in the tissue specific expression of the immune adherence R from platelets in the nonprimate to E in the primate.

Animals↗
PubMed1988

Intracellular degradation of the complement C3b/C4b receptor in the absence of ligand.

Human neutrophils (PMN) respond to various soluble stimuli by translocating intracellular complement C3b/C4b receptors (CR1) to the cell surface. Ligand-independent internalization of surface CR1 has been demonstrated previously, but the fate of total cellular CR1 during PMN stimulation has not been determined. In order to study the fate of CR1 during neutrophil activation, we have employed a unique approach for the quantitative analysis of intracellular antigens which allows simultaneous measurement of total cellular and surface membrane antigen pools. Stimulation of isolated PMN with N-formyl-Met-Leu-Phe or ionomycin resulted in a mean 7-fold increase in surface CR1 expression within 15 min. Total cellular CR1 decreased by as much as 45% within 15 min, with loss continuing for up to 1 h. Inclusion of NH4Cl during PMN stimulation inhibited the loss of total CR1 without affecting surface CR1 expression. Addition of phenylmethylsulfonyl fluoride inhibited loss of total CR1 and enhanced the stimulus-induced increases in surface CR1. These data suggest that intracellular degradation of CR1 occurs during stimulation of PMN and may involve proteolysis in an acidic intracellular compartment. Since our experiments were done with isolated PMN in the absence of serum and complement components, this degradation occurred in the absence of C3b, the ligand for CR1. To our knowledge, ligand-independent degradation of a cell surface receptor has not been previously detected.

Cell Separation↗
PubMed1988

Interaction of human monomeric C3b with its receptor (complement receptor type 1, CR1) on neutrophils. Evidence for negative cooperativity.

The binding of highly purified monomeric 125I-C3b to its receptor (CR1) on resting human polymorphonuclear neutrophils (PMN) was analyzed under equilibrium conditions, at 4 degrees C and low ionic strength. Scatchard analysis of specific binding data yielded curvilinear concave upward plots, which resulted from the presence of site-site interactions of the negative type among PMN C3b-receptors (negative cooperativity), as shown by dissociation kinetic experiments. Indeed, the dissociation rate of 125I-C3b from PMN was markedly increased in the presence of an excess of unlabeled C3b in the dilution medium and was directly dependent on the degree of initial receptor occupancy with the radioligand. These interactions occurred when 2% of the receptors were occupied with 125I-C3b and resulted in a 4-fold decrease in CR1 affinity when the receptor went from its "empty" to its "filled" conformation. In a disease associated with a continuous production of C3b (factor I deficiency), CR1 on in vivo circulating PMN was found to be in a "low affinity" and "high dissociating" state similar to that of normal CR1 at high occupancy. Finally, negative cooperativity among CR1 sites disappeared after PMN activation with chemotactic peptides.

Afibrinogenemia↗
PubMed1988

Monoclonal antibodies to mouse complement receptor type 1 (CR1). Their use in a distribution study showing that mouse erythrocytes and platelets are CR1-negative.

mAb to murine C receptor type 1 (CR1) were produced and three of them were characterized. One antibody, designated as 8C12, immunoprecipitated a protein of 190,000 Mr from a detergent extract of surface-labeled spleen cells and stained spleen B but not T lymphocytes in fluorescent flow cytometry. It inhibited both CR1-mediated rosette formation and the cofactor activity of CR1 for factor I-mediated cleavage of C3b, suggesting that it recognizes the ligand-binding site of CR1. The two other antibodies, designated as 7G6 and 7E9, recognized different epitopes from that recognized by 8C12, and they cross-reacted with a protein of 150,000 Mr that is present in a spleen extract. The distribution of CR1 in murine hemopoietic cells was studied by binding experiments with radiolabeled 8C12 and fluorescent flow cytometry. When CR1 was not detected by 8C12 alone, the two other antibodies were used in combination with 8C12 to confirm the negative results. Almost all B lymphocytes from the spleen, lymph nodes, and peripheral blood were CR1 positive. Most of the Thy-1-positive lymphocytes from these tissues were CR1 negative. Thymus lymphocytes were also CR1 negative. Peritoneal macrophages and chemotactic factor stimulated but not unstimulated peripheral blood granulocytes were CR1 positive. In contrast to human E, mouse E were CR1 negative. This pattern of distribution was consistent with previous results obtained by rosette assays. Although mouse platelets cause immune adherence hemagglutination with C3b-bearing SRBC, they are CR1 negative. Three other lines of evidence also indicated that platelets are CR1 negative. First, no band of CR1 was demonstrated by immunoprecipitation with 8C12 of an extract of surface-labeled platelets. Second, 8C12, which inhibited rosette formation by lymphocytes, alone or in combination with 7G6 and 7E9, did not inhibit immune adherence between platelets and C3b-bearing SRBC. Third, polyclonal rabbit IgG prepared from anti-mouse CR1 antiserum did not inhibit immune adherence by platelets. These results strongly suggest that the C3b-binding factor(s) on mouse platelets is different from CR1 and that processing of C3b-bearing immune complexes in mouse blood may be mediated by a new and as yet unidentified C3b-binding factor(s).

Animals↗
PubMed1987

Decreased expression of the C3b/C4b complement receptor (CR1) in AIDS and AIDS-related syndromes correlates with clinical subpopulations of patients with HIV infection.

Expression of the C3b/C4b receptor (CR1) was studied on erythrocytes of 153 individuals infected with HIV and 104 age-matched normal individuals by measuring the uptake of 125I-labelled monoclonal anti-CR1 antibody. The mean number of CR1 sites on erythrocytes of asymptomatic seropositive individuals (822 +/- 270; mean +/- s.d.) and of patients with persistent generalized lymphadenopathy (PGL; 775 +/- 320) did not differ significantly from that of normal subjects. The number was significantly lower in patients with AIDS-related complex (ARC; 543 +/- 233; P less than 5 x 10(-3)) and further decreased in patients with AIDS (442 +/- 271; P less than 1 x 10(-4)), whether they presented with Kaposi's sarcoma (KS) or opportunistic infections. An additional finding was that of decreased expression of antigenic and functional CR1 in neutrophils from patients with AIDS, as assessed by radioimmunoassay of CR1 in detergent-solubilized cells and the capacity of intact cells to form rosettes with C3b-coated erythrocytes. Low numbers of CR1 on cells from patients with AIDS were not due to occupation of the receptor by C3 fragments on immune complexes. The correlation that was observed between decreased numbers of CR1 on erythrocytes and clinical subpopulations of symptomatic HIV-infected patients suggests that CR1 expression on erythrocytes may represent a valuable marker of the severity and natural history of HIV-associated disease.

AIDS-Related Complex↗
PubMed1988

Immune complexes and erythrocyte CR1 (complement receptor type 1): effect of CR1 numbers on binding and release reactions.

We performed experiments to investigate whether immune complexes opsonized with C3b and iC3b transferred from CR1 on one erythrocyte to CR1 on others, and studied the effect of variation in erythrocyte CR1 number on the transfer reaction. We used populations of cells of different blood groups to study this phenomenon which were separated by differential agglutination with monoclonal anti-group antibodies. The rate of transfer of immune complexes between erythrocytes was related to CR1 concentration of both donor and recipient cells; fastest transfer occurred from donor cells of low CR1 numbers to recipient cells of high CR1. These results were not explained by a difference in the binding constant of immune complexes to erythrocytes bearing different numbers of CR1. In the absence of factor I, complexes partitioned between erythrocytes according to their relative concentrations of CR1 with no release of complexes into solution. In serum, the proportion of complexes bound to donor and recipient erythrocytes was similarly related to their respective CR1 numbers with progressive release of complexes into solution. Erythrocyte CR1 may act as a dynamic buffering system which prevents immune complexes that have bound complement from fixing to vascular endothelium.

Antigen-Antibody Complex↗