Coated Sheep Erythrocytes Research Articles

Recombinant C1q variants modulate macrophage responses but do not activate the classical complement pathway

Complement protein C1q plays a dual role in a number of inflammatory diseases such as atherosclerosis. While in later stages classical complement pathway activation by C1q exacerbates disease progression, C1q also plays a beneficial role in early disease. Independent of its role in complement activation, we and others have identified a number of potentially beneficial interactions of C1q with phagocytes in vitro, including triggering phagocytosis of cellular and molecular debris and polarizing macrophages toward an anti-inflammatory phenotype. These interactions may also be important in preventing autoimmunity. Here, we characterize variants of recombinant human C1q (rC1q) which no longer initiate complement activation, through mutation of the C1r2C1s2 interaction site. For insight into the structural location of the site of C1q that is important for interaction with phagocytes, we investigated the effect of these mutations on phagocytosis and macrophage inflammatory polarization, as compared to wild-type C1q. Phagocytosis of antibody coated sheep erythrocytes and oxidized LDL was measured in human monocytes and monocyte-derived macrophages (HMDM) respectively that had interacted with rC1q wild-type or variants. Secreted levels of cytokines were also measured in C1q stimulated HMDM. All variants of C1q increased phagocytosis in HMDM compared to controls, similar to native or wild-type rC1q. In addition, levels of certain pro-inflammatory cytokines and chemokines secreted by HMDM were modulated in cells that interacted with C1q variants, similar to wild-type rC1q and native C1q. This includes downregulation of IL-1α, IL-1β, TNFα, MIP-1α, and IL-12p40 by native and rC1q in both resting and M1-polarized HMDM. This suggests that the site responsible for C1q interaction with phagocytes is independent of the C1r2C1s2 interaction site. Further studies with these classical pathway-null variants of C1q should provide greater understanding of the complement-independent role of C1q, and allow for potential therapeutic exploitation.

Functional Analysis of Recombinant Human Mannan‐binding Lectin and its Structural Variants expressed in Insect Cells

Mannan‐binding lectin alleles (MBL/B, C, and D) arise due to point mutations in the structural gene and are associated with increased susceptibility to recurrent infections in children and adults. Our data show that mutations in the collagen region have no effect on the ability of the recombinant forms of the structural variants (rMBL/B, C, and D) to form higher order oligomers. Purification of structural variants on mannose‐ and mannan‐Sepharose columns indicated that the structural variants bind to sugars. When binding to mannan was determined by ELISA using monoclonal antibody (mAb 131‐01), the structural variants bound poorly compared to wild type MBL purified from plasma (pMBL/A) or rMBL/A. The weak binding is attributed to the 16 to 60‐fold weaker avidity of the mAb for the variants than for pMBL/A or rMBL/A. Preliminary studies using surface plasmon resonance (Biacore) that does not require antibody, show pMBL/A and rMBL/A as well as rMBL/D bind to mannan with high affinity (KD range = 0.47 to 1.2 nM). The variants, rMBL/B and rMBL/C also bind to mannan but with a 35 to 54‐fold weaker affinity than pMBL/A. The effect of point mutations in MBL on complement activation was studied by hemolytic assay using MBL depleted serum and mannan coated sheep erythrocytes. The results show that rMBL/C was ineffective and rMBL/B activated complement weakly where as rMBL/D was fully functional like pMBL/A and rMBL/A.Research supported by NIH grant HL073804

Production and characterisation of two monoclonal antibodies recognising equine IgG Fc receptors

Despite the importance of IgG Fc receptors in the regulation of various immunological mechanisms, these receptors have not been well characterised in the domesticated animals including equines. This paper describes the production of two monoclonal antibodies (CVS 59 and CVS 61) that recognise equine IgG Fc receptors. Fusions were conducted using BALB/c mice hyperimmunised with equine peripheral blood mononuclear cells. Hybridoma supernatants were screened on the basis of their ability to inhibit the rosetting of equine antibody coated sheep erythrocytes with equine peripheral blood mononuclear cells. The mAbs were then characterised for cellular distribution of the antigen by flow cytometry. Using immunoprecipitation, it was shown that both under reducing and non-reducing conditions, CVS 59 and CVS 61 precipitated a 80 and 40 kD protein, respectively. From the functional study coupled with cellular distribution and molecular weight of the antigen recognised by these mAbs, it would appear that CVS 59 and CVS 61 recognise an epitope on equine equivalent of human and murine FcγRIII and FcγRII, respectively.

Dynamics of actin filaments in microglia during Fc receptor-mediated phagocytosis.

The phagocytic ability of mouse microglia during their differentiation in culture and after stimulation with bacterial wall lipopolysaccharide (LPS) has been investigated using Fc receptor-mediated phagocytosis of immunoglobulin (IgG)-coated sheep erythrocytes (SRBCs). We observed that in 10-14 day-confluent neopallial cell cultures some immature microglia are not phagocytic but, on further culturing, they do become phagocytic. LPS-stimulated microglia are able to phagocytose larger numbers of IgG-coated SRBCs and at a faster rate than non-stimulated microglia. Within 5-10 min of phagocytosis the actin filaments of the LPS-stimulated microglia become depolymerized, leaving only bundles of actin filaments around the phagocytosed SRBCs (phagosome cups). At 30 min after the start of phagocytosis the actin filaments of the LPS-stimulated microglia begin to polymerize, and within 2 h the original pre-phagocytosis pattern of the actin filament network is re-established. The non-LPS-stimulated microglia exhibit actin filament depolymerization in only a few lamellipodia and polymerization of actin filaments around engulfed particles, but much later during phagocytosis.

The mechanism of activation of the alternative pathway of complement by cell-bound C4b

Investigations into the mechanism of alternative pathway-dependent lysis of C4b-coated cells are reported. Test cells (EAC1q4b) were formed by reaction of sheep erythrocytes with antibody, C1 and C4. In C5-deficient serum, more C3b was deposited onto EAC1qC4b than onto control cells (EAC1q). The possibility that the C4bBb enzyme could form was considered, but no C3 convertase activity was generated when magnesium, properdin and factors B and D were added to EAC1qC4b. Binding studies employing radiolabeled components provided evidence that C4b bound the C3 convertase, C3bBbP, through a weak interaction with C3b. These data implied C3 conversion would be localized to the cell surface, thereby amplifying C3b deposition. This could be demonstrated in vitro. C3b, properdin, factor B and factor D were all required and the amplified C3b deposition was not due to deposition onto C4b itself. In serum, C5 convertase activity would be consequently expressed and cell lysis would result. This could be the mechanism by which the sera of C2-deficient patients mediate lysis of antibody coated sheep erythrocytes.

Different effects of ethanol on particle phagocytosis via different receptors in human monocytes

Endocytosis of test particles by human blood monocytes (Mo) was tested in the presence of ethanol (80 mM). Phagocytosis via the Fc (IgG)- or C3b receptors (R) was assessed by an assay in which IgG- or C3b coated sheep erythrocytes (E) were used as test particles. Latex particles were tested in parallel with opsonized E. Phagocytosis of IgG-E was reduced to 67 +/- 5% of control (= without ethanol), while the corresponding value for C3b-E was 164 +/- 26% controls. Phagocytosis of latex particles was not affected by ethanol exposure (91 +/- 8% of control). The receptor functions were also tested without ethanol present during the assays. In this part of the study, Mo were incubated with or without ethanol in autologous serum for 15 min at 37 degrees C. After washing the cells free of ethanol, binding properties of the Fc-R or C3b-R were assessed by a rosette assay. Preincubation with ethanol reduced Fc-R binding, while attachment to C3b-R seemed to be more effective. The experiments thus indicate different effects of ethanol treatment in vitro on phagocytic receptors in human Mo. Control experiments revealed no direct effect of ethanol on the test particles.

Microbiology: An Evaluation of Two Hemagglutination Tests as Alternatives to F TA-ABS in Otologic Syphilis

The microhemagglutination Treponema pallidum test (MHA-TP, Ames Co, El-kart, Ind) and hemagglutination test for syphilis (HATTS, Difco Lab, Detroit, Mich) were evaluated as alternative procedures to the fluorescent treponemal antibody absorption (FTA-ABS) test for screening an older population with suspected otologic syphilis. Both hemagglutination tests are performed in microtiter plates; MHA-TP uses four hours for reaction of serum with Treponema pallidum (TP)-coated sheep erythrocytes, while HATTS takes only one hour using TP-sensitized turkey erythrocytes. Of 267 patients tested using MHA-TP and FTA-ABS, there were 78 presumptively positive for syphilis and 189 presumptively negative. The sensitivity and specificity were93.6%(73/78)and99%(187/189), respectively, for MHA-TP and 100% (78/78) and97.4% (184/189), respectively, for FTA-ABS. Of 103 patients tested with HATTS and FTA-ABS, 13 were positive and 90 were negative. The sensitivities of both HATTS and FT A-ABS were 100% (13/13); the specificities were 97.8% (88/90) and 93.3% (84/90), respectively. These results suggest that either MHA-TP or HATTS can be used to screen for otologic syphilis.

In vivo effect of fly ash on surface receptors of mice peritoneal macrophages

Abstract Functional activity of macrophages was studied in mice upto 15 days after intraperitoneal injection of 2.5 and 5.0 mg of fly ash using in vitro parameters. Fly ash did not cause any variation in the type of cellular response. The total cell number decreased significantly by 4 days after fly ash treatment but recovered subsequently. The decrease was dose dependent. Fly ash also caused a 50% depression in the FC receptor mediated phagocytosis of IgG coated sheep erythrocytes (SRBC) by macrophages at 2 days of dust treatment. However, the recovery began earlier with 2.5 mg fly ash than with 5.0 mg fly ash. These changes were not associated with any marked changes in esterase activity of macrophages following phagocytosis of fly ash.

In vitro function of indium-111 oxine-labeled human monocytes

Mononuclear phagocytes (monocytes and macrophages) are widely disttibuted throughout the body. Indium-111 ( 111In) oxine has been a useful radioactive label in studies of in vivo cellular kinetics and distribution. In preparation for in vivo monocyte investigations in humans, this study was designed to determine the effects of labeling human blood monocytes with 111In on their function in vitro. The monocytes labeled with an efficiency of 57.9 ± 14.2% (range of 47–95%). The 111In eluted from the cells at a rate of 0.25%/h over 48 h in vitro. The labeled monocytes had a slight, but statistically significant, reduction in their ability to phagocytize or mediate antibody-dependent cellular cytotoxicity of antibody- coated sheep erythrocytes. The labeled cells were not different than control monocytes with respect to viability (trypan blue exclusion and lactate dehydrogenase release), adherence to plastic, hydrogen peroxide production, chemotaxis to N-formyl methionylleucylphenylalanine, and antibody-dependent lysis of the tumor cells U937 and HeLa. 111In-labeled human monocytes should be useful for in vivo studies of mononuclear phagocyte kinetics and distribution in normal and diseased subjects.

Activation of neutrophil phagocytosis of complement coated and IgG coated sheep erythrocytes by platelet release products.

Phagocytosis by human neutrophils of opsonized sheep erythrocytes with rabbit immunoglobulin M and human complement (IgM-EACh) and those with rabbit immunoglobulin G (IgG-EA) increased 2-3 times of control neutrophils after treatment with released products from activated platelets (PRPr). The factors in PRPr which augmented IgM-EACh phagocytosis were suggested to be adenosine diphosphate (ADP) and triphosphate (ATP) by experiments using apyrase treated PRPr and by direct exposures of neutrophils with these nucleotides. The presence of two other stimulators for phagocytosis of IgG-EA were suggested. One was a macromolecular substance of molecular weight greater than 100 000 and the other was a substance of molecular weight less than 500. The latter may be a prostaglandin because the activity was easily removed by acid-extraction with ethylacetate from the water phase, and because prior incubation of platelets with 10 micrograms per ml indomethacin deprived PRPr of its low molecular weight activity. Exposure of neutrophils to thromboxane B2, prostaglandin F2 alpha and prostaglandin E2 at the concentration of 10(-6)-10(-7) mg/ml actually increased the phagocytosis of IgG-EA. The activity in the macromolecular fraction was not affected by prior incubation of platelets with indomethacin.

In vitro inhibition of the activation of the human complement and coagulation systems by chloroquine

The ability of chloroquine to inhibit the activation of the complement and coagulation systems was investigated. The activation of the classical pathway of complement by antibody coated sheep erythrocytes and aggregated IgG was blocked by chloroquine. Similarly, it prevented normal rabbit erythrocytes and zymosan from activating the alternative pathway of complement. The activation of C3 by immune complexes of different solubilities also was inhibited by this drug. In addition, it abrogated the clotting of plasma by calcium chloride and thrombin. This effect could be reversed by the addition of excess thrombin but not of calcium chloride. It is suggested that the inhibition of these two important phlogistic mediator systems of inflammation may contribute to the anti-inflammatory property of chloroquine.

Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram‐Positive Bacteria

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol-induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol-water and saline extracts of various gram-positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.

Peripheral blood leucocytes with receptors for extracts of dental plaque. I. Frequency in patients with periodontal disease and in normal subjects.

The percentage of peripheral blood leucocytes with receptors for dental plaque has been determined with a rosetting assay. Patients with periodontal disease had a significantly greater mean fraction of leucocytes with receptors for a plaque extract than could be detected in leucocytes from healthy subjects. The percentage of plaque binding leucocytes was dependent upon the plaque concentrations used to coat the indicator sheep erythrocytes.Attempts to demonstrate the receptors directly by pre‐incubation of the leucocytes with high concentrations of plaque were unsuccessful in that subsequent rosette formation with plaque coated sheep erythrocytes was not inhibited.It is suggested that both antigen binding cells and mitogen binding cells are detected by the present method in proportions dependent upon the plaque concentration employed.If tolerance occurs to plaque in advanced periodontal disease, it is not due to a reduction in the number of specific antigen binding cells.

Lymphocyte Subpopulations in the Ferret

Several different surface markers were sought on ferret lymphocytes. Using a technique which attaches complement to zymosan particles, 16.7% of peripheral blood lymphocytes and 15% of splenic lymphocytes exhibited receptors for complement. Receptors for the Fc portion of IgG, measured by an assay using antibody coated sheep erythrocytes, were found on 11.7% of peripheral blood lymphocytes and 34.8% of splenic lymphocytes. Using an indirect fluorescent antibody technique, surface immunoglobulin was detected on 13.4% of peripheral blood lymphocytes and 10.3% of splenic lymphocytes. Spontaneous “T” lymphocyte rosettes were sought unsuccessfully using erythrocytes from 16 different animals. These studies increase our understanding of the ferret immune system and provide methods for the in vitro separation of lymphocyte subpopulations in ferrets.

Receptor sites for complement and for immune complexes on human nonhemopoietic tumor cells

Tumor cells in primary cultures derived from 11 untreated nonhemopoietic cancer patients were reacted with specifically coated sheep erythrocytes. Rosette formation between tumor and indicator cells was assessed, Eight of the primary cultures reacted positively with both IgG-coated (EAIgG) and with IgM-human complement coated (EAIgMC or EAIgMC 4,3) sheep erythrocytes. EAIgG rosette formation in positive cultures ranged from 25 to 85%, and for EAIgMC/EAIgMC 4,3 reactivity ranged between 22--95%. Rosette formation with E (uncoated) and EAIgM was negligible. These findings suggest that human nonhemopoietic tumor cells may carry on their surface receptor sites for an IgG component of immune complexes and for human complement. These receptor sites may be important in the host-tumor relationship.

Properties of a cell-bound homogeneous murine IgM anti-dextran-V: Effects of density of cell-bound IgM on immunological activities

O-stearoyl dextran coated sheep erythrocytes (OSD-E) were sensitized with increasing amounts of MOPC 104E IgM to give IgM-OSD-E in antigen excess, in equivalence and in antibody excess. These zones were defined operationally by susceptibility of the cells to complement-mediated lysis, and by the number of free Fab regions on the cell-bound IgM. The free Hab regions were measured by ability to (a) bind soluble dextran and (b) to react with and participate in complement-mediated lysis of bystander OSD-E. In antigen excess, complement-mediated lysis is proportional to cell-bound IgM and there are few free Fab regions. In the equivalence zone, complement-mediated lysis reaches a plateau, while the number of free Fab regions becomes significant. Throughout the zone of antihody excess, complement-mediated lysis diminishes while the number of free Fab region increase exponentially. Blocking these free Fab regions with soluble dextran prevents reactions with bystander OSD-E. he rate of reaction of IgM-OSD-E, in antibody excess, with bystander OSD-E is proportional to reaction temperature and the extent of reaction is proportional to cell concentration.

Complement receptors in pathological human renal glomeruli.

Glomerular complement receptors (GCR) were demonstrated by the adsorption of C3 coated sheep erythrocytes to cryostat sections of human kidneys. An epithelial localization of the GCR is implied by the binding pattern of the indicator cells. The GCR activity was reduced in all glomeruli where in vivo deposition of immunoglobulin and C3 along the capillary wall was observed. In fourteen out of nineteen biopsies where deposits in the mesangium were seen, reduced GCR activity was also observed, whereas GCR activity was normal in the other five. Immunofluorescence and haemadsorption tests performed on the same section showed that C3 deposits corresponded to areas showing reduced complement receptor activity. Areas outside the C3 deposits adsorbed indicator cells coated with various amounts of C3 similarly to the adsorption to glomeruli seen in normal adult and fetal kidneys. Sclerotic glomeruli showed no GCR activity. The results indicate that the complement receptors play a role for the binding of complement-containing complexes in glomeruli.

Macrophage function in tumor-bearing mice: Dissociation of phagocytic and chemotactic responsiveness

Macrophage function in mice with tumors was examined by two in vitro assays: chemotaxis to lymphocyte- or serum-derived stimuli and phagocytosis of 51Cr-labeled IgG coated sheep erythrocytes ( 51Cr-EAIgG). Resident peritoneal macrophages from mice with intrafootpad syngeneic fibrosarcomas were less responsive to chemotactic stimuli than macrophages from control mice. Chemotactic responses were depressed to about half of normal within 3 days of tumor transplantation and remained depressed until death of the mouse by 6–8 weeks. In contrast to this persistent depression of macrophage chemotaxis, phagocytosis of 51Cr-EAIgG was markedly increased in tumorbearing mice. Macrophage uptake of 51Cr-EAIgG was 4–5 fold greater than normal within 1 week of tumor transplantation, progressively decreased to normal levels by 2 weeks and remained normal through 4 weeks. Increased phagocytic capacity of macrophages from mice with tumors reflected both increased numbers of phagocytic cells and increased numbers of erythrocytes phagocytized per cell. A similar increase in macrophage phagocytosis was observed with cells from mice bearing another tumor but not from mice treated with syngeneic spleen cells, medium or mineral oil. Thus, growing tumors induce significant alterations in mononuclear phagocyte function. Changes observed in vitro with macrophages from tumor-bearing mice may provide cellular mechanisms for the abnormal phagocytic and inflammatory responses of cancer patients.

Immunological cross-reactivity of antibodies to a synthetic undecapeptide analogous to the amino terminal segment of carcinoembryonic antigen, with the intact protein and with human sera.

A peptide corresponding to the 11 amino acid residues of the NH2-terminal portion in the sequence of carcinoembryonic antigen(CNTHETIC CEA(1-11) peptide was attached by means of a water-soluble carbodiimide reagent to multichain poly(DL-alanine( as well as to bovine serum albumin. Both macromolecular conjugates provoked in rabbit anti-CEA(1-11) peptide antibodies. The specificity of this immunological system and the crossreactivity between the peptide and intact CEA were investigated by two methods--passive hemagglutination and modified bacteriophage inactivation. Hemmagglutination experiments showed that not only anti-CEA(1-11) sera, but also anti-CEA sera, agglutinated CEA(1-11)-coated sheep erythrocytes, and both these reactions were inhibited with CEA(1-11) peptide. In experiments with the chemically modified bacteriophage technique CEA(1-11)-coated phase was efficiently inactivated with antisera against the CEA(1-11) conjugates, and the inactivation reaction could be totally inhibited with the free peptide. The semipure CEA, but not the pure protein, could also inhibit the phage inactivation, even though less efficiently. On the basis of the above results, sera of some cancer patients were tested for their capacity to inhibit the inactivation of CEA(1-11)-coated phage by means of anti-CEA(1-11) antiserum. The results indicate that sera from a large proportion of patients with adenocarcinomas of the digestive tract, pancreas, and breast are capable of inhibiting the above inactivation, whereas most normal sera do not inhibit.

Numbers of rosette forming cells in human peripheral blood

Thymus-derived (T-cell) and “bursal” derived (B-cell) lymphocytes in human peripheral blood were quantitated by assaying percentages of cells forming erythrocyte rosettes. T-cell rosettes were formed with neuraminidase treated sheep erythrocytes. B-cell rosettes were formed with complement coated sheep erythrocytes. Large differences in the percentages of T-rosette forming cells were noted depending on the method used to assay these cells. When rosette forming cells (RFC) and non-RFC were counted concurrently the percentage of T-cell rosettes were 53–75% whereas methods involving the separate counting of RFC and total cells gave T-cell RFC percentages of 23–40%. These differences were due to the “co-rosetting” of non-RFC into the T-cell rosette clusters. This occurred because of the gentleness required to resuspend the fragile T-cell rosettes. “Co-rosetting” was demonstrated by forming stable complement receptor rosettes with complement-coated human erythrocytes and resuspending them either gently or vigorously. Significantly higher percentages of rosettes were noted with gentle cell suspension than with vigorous resuspension. The percentages of rosette forming T-cells in human peripheral blood are therefore lower than previously estimated.