Roles of plasma proteins and surface negative charge of erythrocytes in erythrocyte aggregation

The effect of plasma proteins (and IgG fragments) and sialic acid content of erythrocytes on the aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyser and a computer. (1) The velocity of erythrocyte aggregation by plasma proteins was increased with increasing in their molecular weight, i.e., IgG less than IgA less than fibrinogen less than IgM. F(ab')2. Fab and Fc could not induce the aggregation. (2) The aggregation induced by fibrinogen was accelerated by IgG and its peptic fragment, F(ab')2, but was unaffected by the plasmic fragments, Fab and Fc. The accelerating effect by IgG and F(ab')2 was inhibited by Fab and Fc. (3) The aggregation of erythrocytes was accelerated by decreasing the sialic acid content (due to the reduction of the electrostatic repulsive force among erythrocytes), and the effect of desialylation on the IgG-induced aggregation was greater than that of desialylation on the fibrinogen-induced aggregation. (4) The roles of plasma proteins and of sialic acid content of erythrocytes on the aggregation of erythrocytes were discussed.     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

Comparative study of conformation properties of Fc-fragments of human immunoglobulin G subclasses using 1H-NMR

Conformational properties of the Fc- and pFc'-fragments of human myeloma immunoglobulins G of the first and third subclasses were studied by 1H-NMR method (270 and 400 MHz). It was found that the globular structures (domains) of the Fc-fragments of IgG1 and IgG3 in solution are characterized by high segmental mobility, and have no significant differences in their spatial arrangement. Comparative analysis of the spectra obtained at different temperatures (30-70 degrees C) revealed that the Fc-fragment of IgG3 has a more heat-stable conformation than the Fc of IgG1. The intramolecular mobility of the Fc-fragment increased upon lowering the pH. The partial assignment of the signals in the NMR spectra of the Fc-fragments of immunoglobulins G1 and G3 was carried out, and the pKa values for histidines of the pFc'-fragment of IgG1 were determined.     

Idiotypic analysis of myeloma proteins: anti-DNA activity of monoclonal immunoglobulins bearing an SLE idiotype is more common in IgG than IgM antibodies

The anti-idiotype 3I which recognizes a determinant on kappa-chains of anti-DNA antibodies in SLE patients also recognizes a determinant on kappa-chains of 82/706 myeloma proteins tested. Twenty-nine of these 82 proteins bind to double-stranded DNA, including two monoclonal IgM, one monoclonal IgA, and 26 monoclonal IgG proteins. DNA binding is much more frequent in the IgG than in the IgM myeloma proteins (p less than 0.001), and is also associated with cationic antibody charge. Two-dimensional gel electrophoresis reveals markedly increased charge heterogeneity of both heavy and light chains of the monoclonal IgG as compared with the monoclonal IgM proteins. We postulate that the increased charge heterogeneity of the IgG-associated 3I-reactive kappa-light chains may reflect somatic mutation, and that DNA specificity within the 3I idiotype system arises by somatic mutation of germ-line genes found in normal individuals. DNA binding may be associated with those mutations that give rise to a cationic immunoglobulin charge.     

Separate Fc-receptors for immunoglogulins IgG2a and IgG2b on an established cell line of mouse macrophages.

The specificity of Fe-receptors on IC-21 cells, an established line of mouse peritoneal macrophages with antibody-dependent effector cell activity has been examined. Only IgG2a and IgG2b myeloma proteins bound readily to IC-21 Fc-receptors, the former in nonaggregated as well as aggregated form, the latter only as aggregated complexes. Thus, IgG2a bound in a manner characteristic of classically defined cytophilic antibody, whereas the binding of IgG2b appeared to be mediated by Fc-receptors for antigen-antibody complexes. Evidence is presented in support of the view that IC-21 macrophages possess separate and distinct Fc-receptor sites for these two immunoglobulins.     

Erythrocyte aggregation: bridging by macromolecules and electrostatic repulsion by sialic acid.

Relation between aggregating force (of fibrinogen and IgG) and disaggregating force (due to electrostatic repulsion among erythrocytes) in erythrocyte aggregation was investigated with a rheoscope combining a video camera, an image analyzer and a computer. (i) Erythrocyte aggregation was augmented with the increase of molecular weight of bridging macromolecules as far as examined for fibrinogen and the degradation products and IgG and the related macromolecules, and the augmentation seemed to be dependent on the molecular length of macromolecules. In accelerating the erythrocyte aggregation, fibrinogen was more effective than IgG, and some interaction between fibrinogen and IgG in their coexistence was suggested. (ii) The decrease of sialic acid content on the erythrocyte surface accelerated IgG-induced erythrocyte aggregation much greater than fibrinogen-induced one. (iii) Counteraction between aggregating force and disaggregating force in leading to erythrocyte aggregation was discussed relating to molecular length of bridging macromolecule and electrostatic repulsive force by sialic acid.     

Streptococcal Fc receptors. II. Comparison of the reactivity of a receptor from a group C streptococcus with staphylococcal protein A

The reactivity of a soluble Fc receptor from a group C streptococcus ( FcRc ) was compared antigenically and functionally with the staphylococcal Fc receptor, protein A. Protein A and FcRc were found to inhibit each others' binding to the Fc region of human IgG, indicating that they bind to sites that are in close proximity on the Fc region of human IgG. The two bacterial Fc receptors were antigenically unrelated. Differences were observed in the species and subclass reactivity of the two receptors. The patterns of binding of protein A and FcRc under various conditions suggested that these receptors reacted with distinct regions on the Fc region of immunoglobulins. FcRc bound more efficiently to goat, sheep, and cow IgG, protein A bound more efficiently to dog IgG, and neither receptor bound to rat IgG. Differences were also observed in the reactivity towards human IgG subclasses. The FcRc bound to all samples of the four human IgG subclass standards. Protein A bound to IgG1, IgG2, and IgG4, and to one of two IgG3 myeloma proteins tested. The reactivity of our soluble FcRc corresponds to a type III streptococcal Fc receptor classified by the reactivity of intact bacteria.     

Antigen-specificity of oligoclonal abnormal protein bands in multiple myeloma after allogeneic stem cell transplantation

Myeloma patients may develop oligoclonal immunoglobulins, so-called abnormal protein bands (APB), after stem cell transplantation. APB do not correspond to the patient's paraprotein and confer a good prognosis. We set out to investigate whether such APB represent a humoral anti-myeloma immune response by screening immunoglobulins of 15 myeloma patients after allogeneic stem cell transplantation and a control group of healthy donors for reactivity with myeloma protein extracts. While the immunoglobulins of healthy donors did not react with myeloma protein extracts, patient-derived immunoglobulins showed variable levels of interaction, depending on the presence of APB on immunofixation. Most commonly, we detected interactions with heat-shock proteins, followed by neutral alpha-glucosidase, alpha-enolase and vimentin, as well as proliferating cell nuclear antigen and MAGEA4. More than 80% of targets were upregulated in myeloma. Heat-shock protein 60 (HSP60) was subsequently evaluated as an exemplary antigen. We found that HSP60 was aberrantly displayed on the surface of primary myeloma cells. Indeed, patient-derived APB-containing immunoglobulins recognized surface HSP60 suggesting that this antigen becomes accessible to the immune system after aberrant membrane exposition. We conclude that immunoglobulin fractions with APB recognize recurrent myeloma antigens and that this humoral response may contribute to the more favorable prognosis in patients with APB.     

Analysis of paraprotein transport into the saliva by using anti-idiotype antibodies

To determine the extent of clonal involvement of the secretory immune system and the origin of salivary immunoglobulins (Ig) in monoclonal gammopathy patients, saliva and serum samples were collected from five affected individuals (two IgA myelomas, one IgG myeloma, one IgG benign monoclonal gammopathy, and one IgM lymphoma) and were assayed for the presence of monoclonal Ig. Purified polyclonal or monoclonal anti-idiotype (Id) antibodies were prepared against each of the isolated serum paraproteins. In all five individuals, the patient saliva samples inhibited the binding of 125I-labeled homologous Ig to the corresponding anti-Id antibodies, but normal saliva did not. The concentration of Id in patients' saliva varied from 1 to 400 micrograms/ml; i.e., 0.004 to 1.0% of the corresponding serum values. Saliva of a lymphoma patient whose IgM kappa protein exhibited rheumatoid factor (RF) activity also contained RF. The salivary Id-bearing molecules were found to have the same Ig isotype as the serum paraproteins. The myeloma IgA represented a minor component (0.4 and 3.9%) of the total salivary IgA. The salivary IgA myeloma proteins were associated at least in part with secretory component, but the salivary IgG paraproteins were not. In an IgA myeloma patient, a minority (17%) of the IgA+ plasma cells found in the lacrymal gland biopsy specimen were Id+, whereas the great majority (98%) of bone marrow IgA plasma cells were Id+. The results suggest active transport rather than passive transudation of myeloma IgA into the patients' saliva, and the integrity of the secretory immune system was not compromised by the neoplastic process.     

Aberrant glycosylation of Igg heavy chain in multiple myeloma

Although the majority of eukaryotic proteins are glycosylated, there is a dearth of knowledge regarding protein sugar moieties and their changes in disease. Most multiple myeloma cases are characterized by production of monoclonal immunoglobulins (Ig). We studied galactosylation and sialylation of IgG heavy chains in 16 patients with IgG myeloma using lectin blotting and densitometry. In comparison to age and sex matched controls, galactosylation was reduced in multiple myeloma (median 317 vs. 362, range 153-410 vs. 309-447 relative units, p = 0.015, Student's t-test). Sialylation was stage dependent; samples from patients with stage IIA had lowest amounts of sialic acid, IIIA intermediate and IIIB highest (142.6 vs. 185.9 vs. 248.5 relative units, correlation coefficient r = 0.55). Both galactosylation and sialylation levels were independent of age, sex, treatment type, response to treatment, disease duration and IgG and b2 microglobulin concentration. These data indicate that multiple myeloma is characterized by aberrant immunoglobulin glycosylation.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

Determination of the subclasses of monoclonal human immunoglobulins G and of the light chain type with the aid of specific antisera]

Sera of 86 patients suffering from G-myeloma were studied for the purpose of determination of subclasses of monoclone IgG. Investigations were carried out by means of antisera to subclasses IgG by the double diffusion method in gel after Ouchterlony. The following distribution of myeloma Ig was revealed: G1--70%, G2--17%, G3--11%,and G4--2%. In typing of the light igG chains by the method of immunoelectrophoresis, using antisera to the light chains of immunoglobulins of the chi and lambda type it was found that IgG1 chi was encountered more frequently than IgG1 lambda (3:1 ratio). The amount of the sera with the IgG2, IgG3, and IgG4 was insufficient for the reliable conclusion of their distribution by the type of light chains.     

Immunoglobulins in uterine secretions of mares with differing resistance to endometritis

The immunoglobulins IgA, IgG, IgG(T) and IgM were measured in uterine secretions from mares with normal uterine defense capability against bacterial contamination, and in mares with lowered resistance. Samples were collected for analysis at two stages of estrus and two stages of diestrus. All mares were then challenged with a pathogenic culture of Streptococci inoculated into the uterus. The immunoglobulins were quantitated on a similar schedule post-inoculation. Generally higher amounts of IgA, IgG and IgG(T) were found in the uterine secretions of mares which had an imparied resistance to endometritis than in mares with an efficient defense mechanisms. IgM was not detected in enough samples to suggest any differences.     

Disturbances of platelet function in patients with multiple myeloma

Coagulation disturbances in patients with multiple myeloma are presented. Platelet disfunction was especially observed, but only in patients with marked hyperproteinemia and a high level of immunoglobulins in plasma, whereas normal adhesion and aggregation were noticed in 3 out of 17 patients without protein disturbances. Chemotherapy as well as plasma exchange caused not only a decrease of the paraprotein level in plasma, but also a normalization of the failure previously observed in platelet function. Our experiments confirm the opinion that protein M in plasma is responsible for the disturbances observed in multiple myeloma.     

Opposite effect of albumin on the erythrocyte aggregation induced by immunoglobulin G and fibrinogen

The effect of albumin on the immunoglobulin G (IgG)-induced and fibrinogen-induced aggregation of human erythrocytes was quantitatively examined by using a rheoscope combined with a television image analyzer and a computer. As albumin concentration in the medium was increased, the IgG-induced erythrocyte aggregation was inhibited, while the fibrinogen-induced erythrocyte aggregation was accelerated (albumin itself was not able to aggregate erythrocytes). These relations were empirically expressed by the equations, v = aG1.8/A and v = a'F1.5 (A + b'), respectively (v, the velocity of erythrocyte aggregation; A, G and F, the concentrations of albumin, IgG and fibrinogen, respectively; a, a' and b', constant). The IgG-induced erythrocyte aggregation was remarkably inhibited by the addition of poly(glutamic acid), but the fibrinogen-induced erythrocyte aggregation was not. A mechanism for the interaction of immunoglobulin G and fibrinogen with the surface of erythrocytes was proposed.     

Interaction of specific and innate factors of immunity: IgA enhances the antimicrobial effect of the lactoperoxidase system against Streptococcus mutans

The antimicrobial effect of the lactoperoxidase (LPO) system (enzyme with the thiocyanate ion and hydrogen peroxide) on Streptococcus mutans NCTC 10449 (serotype c) was significantly enhanced when the system was combined with secretory IgA. Similar enhancement was observed with LPO-myeloma IgA1 or IgA2 combinations. This enhancement of the antimicrobial efficiency was not dependent on the presence of specific antibodies to S. mutans in the IgA preparation, but seemed to require binding between LPO and immunoglobulin. However, neither human polyclonal nor myeloma IgG or IgM nor rabbit IgG enhanced the antibacterial activity of the LPO system. None of the immunoglobulins, when added alone, produced antimicrobial effects. LPO was shown to bind to colostral secretory IgA, myeloma IgA1, IgA2, and to a lesser degree to monoclonal and polyclonal IgG and monoclonal IgM. This binding had a stabilizing effect on the enzyme activity. Our results suggest that IgA significantly enhances the antibacterial efficiency of one of the innate immune factors--the LPO system.     

Orientation and kinetics of surface denaturation of normal human and myeloma IgA in monolayers at the interface of air-NaCl aqueous solutions

Normal serum immunoglobulin A (IgA) and abnormal IgA isolated from serum of patients with multiple A-myeloma have been studied by monolayer technique at air--NaCl solution interfaces. Normal IgA analogous to human normal IgG and secretory IgA was shown to have horizontal orientation at air--water interface. Only some abnormal IgA were similar to myeloma IgG and differed from the normal ones by their orientation at phase border. Majority of myeloma IgA under study could not be distinguished from the normal ones by orientation and denaturation kinetics at interface. B-lymphocytes of the first group of patients were assumed to carry IgA-receptors at their surface, but B-lymphocytes of the second group of patients carried Ig receptors of some other class of immunoglobulins.     

IgD Plasma Cell Neoplasia: Clinical Manifestations and Characteristic Features

Three patients, one with plasma cell leukemia and clinically asymptomatic hypernephroma and meningioma, and two others with multiple myeloma, had M-components of IgD/λ type. In the first case, IgD globulin was found in the serum, ascitic and pleural fluids. Including our patients, 50 cases of IgD myeloma have been reported in the literature. A review of this group showed some significant differences from the other classes of multiple myeloma. IgD myeloma seems to involve a larger proportion of younger people, 66% being less than 59 years of age. The involvement of internal organs and renal damage were more frequent in IgD myeloma than in other classes. Serum total protein was frequently not increased, the relative concentration of M-component was often low and in 12% there was no spike in electrophoresis. The diagnosis therefore was sometimes difficult. In a quarter of the cases Bence Jones proteinemia was found and in 15% there were multiple spikes, both these manifestations being rare in IgG or IgA classes of myeloma. In 89%, IgD globulin had lambda type light chain, clearly contrasting with the figure of approximately 30% in other classes. Bence Jones protein was found in the urine in 91%. The survival time seemed to be shorter than in other myelomas.     

In vitro binding of an IgE protein to human platelets

Bronchoconstriction in extrinsic asthma is initiated by mediators released from IgE-sensitized leukocytes after contact with polyvalent antigen. Because platelets also contain soluble mediators that can cause bronchoconstriction, platelet activation and release of the contents of platelet granules may play a role in IgE-mediated responses under some circumstances. We therefore sought to determine if platelets are capable of binding IgE and if cross-linking this cell-bound IgE initiates secretion of platelet granule contents. Platelets from 10 normal donors were studied by using automated fluorescence analysis and fluorescence microscopy. We detected binding of a purified myeloma IgE protein to 24.1 +/- 9.6% (mean +/- 2 SD) of the gel-filtered platelets from these normal individuals. Although we could detect the binding of IgE and anti-IgE to a minority of cells, every normal individual had a population of platelets that bound IgE. The amount of IgE that bound to normal platelets appeared to be distributed heterogeneously among the IgE-positive platelet population. Platelets from two individuals with type II Glanzmann's thrombasthenia bound normal amounts of heat-aggregated IgG, but less than 3% of the platelets bound detectable IgE. Moreover, a combination of monoclonal antibodies to glycoproteins IIb and IIIa inhibited the binding of the IgE protein to normal platelets but did not affect the binding of aggregated IgG. Thus, the binding of IgE to human platelets appeared to require the presence of the glycoprotein IIb-IIIa complex. Binding of monomeric IgE to platelets, by itself, did not initiate either platelet aggregation or release of 14C-serotonin. However, both aggregation and secretion of serotonin followed the addition of anti-IgE to IgE-sensitized platelets. These studies indicate that human platelets can bind an IgE myeloma protein in vitro and that cross-linking of surface-bound IgE with anti-IgE initiates aggregation and secretion. If platelets have a similar capacity to bind normal IgE in vivo, it is possible that platelets may participate directly in several atopic or inflammatory disorders in man mediated by this class of antibody.     

Relations between macro- and microstability of CH2 domains and human IgG2 and their biological activity: 1. Analysis of calorimetric and optical melting curves

In this study, we examined the human myeloma second-class immunoglobulins, LOM and SIN, and their Fc fragments, by a number of physical methods, such as scanning calorimetry, fluorescence spectroscopy and analytical centrifugation. In addition, we obtained and carried out a separate analysis of their hFc fragments, which contain not only the lower portion of the hinge region, but its complete core peptide, Cys-Cys-Val-Glu-Cys-Pro-Pro-Cys. Joint analysis of calorimetric and optical melting curves revealed that only the first low-temperature heat absorption peak in all of the melting curves corresponded to the melting of the two C_H2 domains. Thus, we demonstrate that the C_H2 domains of the intact IgG2 are present in a less compact conformation compared to their state within the hFc and Fc fragments.