Enzyme-linked immunosorbent assay for antibodies to native DNA in sera of patients with SLE.

A micro-ELISA technique has been developed to measure antibodies to native DNA and used in SLE patients. The distribution of antibody to native DNA in the main immunoglobulin classes was studied, using anti-human globulin conjugates labelled with peroxidase. the antigen (double-stranded DNA from calf thymus) used in the assay was adsorbed to the surface of polystyrene plates treated with methylated bovine serum albumin. The standardization of the method was carried out by use of globulin calibration curves.

Evaluation of different methods for detecting circulating immune complexes. An inter-laboratory study.

Forty serum samples of healthy blood donors and 60 sera of SLE patients were tested in parallel by 7 different assays for detecting immune complexes. Significantly higher titres and significant higher incidence of positive results were observed in the patient group than in the control group in 6 tests. No test discriminated between patients in an active stage of the disease and those whose disease was inactive. Significantly higher immune complex levels were found in lupus nephritis than in the non-nephritic patients by the complement consumption test. Significant positive correlation was obtained between the results of the macrophage aggregated IgG uptake inhibition and PEG-precipitation tests and between two tests based on the interaction of the complexes with the complement system. A characteristic 'profile' of the immune complex assay was observed in the course of the repeated testings of the same patients. The results indicate that the different methods detect different types of immune complexes present in the blood of the patients.

Evaluation of different methods for detecting circulating immune complexes. Studies in patients with lung cancer.

In a collaborative study involving 7 laboratories, sera from 53 patients with lung cancer, 37 primary and 16 secondary tumours, and sera of 40 healthy blood donors were tested by 19 different assays or assay modifications used for detecting immune complexes. In 12 out of 19 assays, significantly higher immune complex levels were found in the cancer patients than in the healthy subjects. Assays based on interactions between immune complexes and Fc receptors of different cells (lymphocytes, macrophages of platelets) discriminated between cancer patients and health subjects and a high percentage (47-87%) of positivity was observed in such assays in patients with lung cancer. In contrast, none of the tests based on immune complex-complement interactions discriminated between cancer patients and health subjects. Immunochemical analyses of the PEG precipitates obtained from the sera tested revealed that the concentrations of IgG, IgA and C3 were significantly higher in the precipitates obtained from patients sera than from control sera, but no significant differences were seen in IgM and C1q concentrations. A 100% correct classification of individuals tested was obtained on discriminant analysis of results with 3 assays: EA rosette inhibition, ADCC inhibition and C3 concentration in PEG precipitates. Correlation between results obtained with individual sera by the different assays was very poor: significant correlation coefficients were found in only 13% of all possible paired comparisons. Our results suggest that Fc receptor-dependent assays are more suitable for detection and measurement of circulating immune complexes in lung cancer than tests based on interactions with complement.

Monocytes of patients congenitally deficient in plasma factor XIII lack factor XIII subunit a antigen and transglutaminase activity.

Monocytes isolated from patients with severe deficiency in plasma Factor XIII of blood coagulation (FXIII) were tested for FXIII antigen and transglutaminase activity. By immunoperoxidase method the patients' monocytes, in contrast to normal controls, showed no reaction with a monospecific antibody against FXIII subunit a. This result was confirmed by immunoblotting technique, as well. In addition, tissue macrophages tested in one of the patients were also exempt of FXIII subunit a antigen. The transglutaminase activity in FXIII deficient monocytes was below the limit of the detection of the dansylcadaverine incorporation assay. The results suggest that FXIII subunit a of monocytes/macrophages and its plasma and platelet counterparts are closely related or identical proteins and demonstrate that the transglutaminase activity in monocytes is of FXIII origin and tissue transglutaminase is present, if at all, only in insignificant amount.

Autoantibodies specific to beta-2-microglobulin inhibit the Fc receptor-dependent phagocytosis of human monocytes.

Effect of anti-beta2-microglobulin (beta2m) antibodies was investigated on the in vitro Fc and C3 receptor dependent phagocytic capacity of human peripheral monocytes. Both rabbit and human anti-beta2m antibodies inhibit the Fc receptor-mediated phagocytosis. Anti-beta2m antibodies do not influence the spontaneous or C3b receptor-mediated ingestion. The inhibitory activity by human autoantibodies but not by rabbit anti-human beta2m antibodies was diminished when incubation was performed at 37 degrees C instead of 20 degrees C.

Acid phosphatase activity in monocytes and sera of patients with Hodgkin's disease.

Acid phosphatase (AP) levels have been found to be decreased in monocytes of 38 patients with Hodgkin's disease as compared to 16 healthy subjects. Low cellular AP activity was associated with the presence of active disease, stage IV and lymphocyte depletion type. Lactate dehydrogenase (LDH) was decreased in monocytes of patients but the difference did not reach statistical significance. In parallel, serum AP activities have been found to be increased in patients. There was an inverse correlation between the monocyte and serum AP levels which can be explained by assuming AP secretion from monocytes of patients in vivo.

CR1 density polymorphism and expression on erythrocytes of patients with systemic lupus erythematosus.

The present study investigated the expressed number of CR1 on erythrocytes (E) in relationship of the CR1 density genotype from 46 patients with systemic lupus erythematosus (SLE) and 47 healthy volunteers. The CR1 genotype was determined by a method based on polymerase chain reaction (PCR) amplification of the genomic DNA fragment of 1.8 kb separated by HindIII endonuclease digestion and agarose gel electrophoresis. Our data supported the earlier results that the number of binding sites/E for monoclonal anti-CR1 decreased among SLE patients compared with normal individuals having the same alleles for the CR1/E density. At the same time the novelty of our recent results was that the decreased expression of CR1 on E correlated significantly with kidney involvement in patients homozygous for the CR1/E high density allele (HH). These data suggest that the deficiency of the detectable number of CR1 on erythrocytes is acquired in this SLE population.

Serum complement activation of SLE patients during plasmapheresis.

The erythrocyte complement receptor 1 (ECR1)-immune complex binding assay is a sensitive method for the determination of complement fragments which can be activated by bovine serum albumin (BSA)-anti-BSA in vitro. When the C3b/C4b containing bovine serum albumin (BSA)-anti-BSA was formed in the presence of the serum of patients with systemic lupus erythematosus (SLE) its binding to ECR1 was found to be lower than that formed in sera of normal volunteers. The plasmapheresis of SLE patients homozygous for the CR1/E high density allele displays a beneficial effect on the formation of C3b/C4b containing BSA-anti-BSA and its binding to ECR1. There was no significant correlation between the serum C3/C4 level and the percentage of C3b/C4b containing BSA-anti-BSA binding to the ECR1 of SLE patients during plasmapheresis. At the same time, there was an inverse correlation between the serum immune complex level and the ECR1 binding, which was significant in 3 of 5 cases. These data suggest that, besides the determination of different components of complement activation, the functional assay of complement activation might be useful in monitoring the effect of plasmapheresis in SLE.

Factor XIII of blood coagulation in human monocytes.

The presence of Factor XIII subunit a was demonstrated in human monocytes by immunoperoxidase staining using specific antisera against Factor XIII and its subunits. This finding was verified by immunobiochemical techniques, as well. In an immunoblotting system after SDS polyacrylamide gel electrophoresis of denatured monocyte homogenate a protein band comigrating with Factor XIII subunit a showed positive reaction with antibodies against this subunit or whole Factor XIII. In contrast, no subunit b of Factor XIII could be detected by either of these methods in monocytes. Activity measurements were carried out by the dansylcadaverine incorporation assay in the absence and presence of anti-Factor XIII antibody with and without thrombin activation. The expression of transglutaminase activity required thrombin and was completely abolished in presence of anti- Factor XIII antibody, which clearly indicate that practically all the transglutaminase activity measured in monocytes comes from Factor XIII. Factor XIII of monocytes and macrophages might have a role in formation of focal fibrin thrombi as well as in organization of stable, fibrinolysis resistant fibrin clot at the site of inflammation or around tumor cells.

Recombinant human erythropoietin modulates erythrocyte complement receptor 1 functional activity in patients with lupus nephritis.

Deposition of immune complexes (IC) is an important step in the pathogenesis of lupus nephritis. Impairment of IC-clearance contributes to the accumulation of IC. It may be partly attributed to decreased complement containing immune complex (ICC) binding by erythrocytic complement receptor 1 (ECR1). Stimulating erythropoiesis with recombinant human erythropoietin (rHuEPO) may enhance the IC-clearance as increasing ECR1 expression and/or functional activity. Ten anemic patients with lupus nephritis were treated with 50 IU rHuEPO (Eprex) per kg body weight three times a week during a five week period. ICC-binding capacity of ECR1 was determined with 125I-labelled, C3ib containing BSA-anti-BSA complexes. In addition to effective correction of anemia, indicated by increased red blood cell count (RBC), hemoglobin concentration and reticulocyte ratio, rHuEPO significantly improved decreased ECR1 functional (ICC-binding) activity in patients with lupus nephritis. This improvement correlated with the increase in reticulocyte ratio. Although patients were kept on their previous therapy during Eprex administration, their clinical condition also improved. That was shown by a decrease in Westergreen ratio, serum creatinine concentration and anti-dsDNA level and also by an increase in creatinine clearance. Results suggest a beneficial immune modulatory effect of rHuEPO in lupus nephritis.

Characteristics of human monocyte subpopulations with the use of flow cytometer.

Human monocytes separated from peripheral blood by Ficoll-Hypaque and by adherence to serum-coated dishes showed a bimodal volume distribution measured with a fluorescence activated cell sorter. The rate of the fluorescein diacetate hydrolysis by small monocytes was lower than that of large ones. The majority of large monocytes reacted with sensitized SRBC while only a minority of small monocytes bound sensitized SRBC. Scatchard plots on the binding of FITC-labeled human monoclonal IgG1 to the two subpopulations indicated similar association constants, K = 1.2 +/- 0.3 x 10(5) M-1. The number of FcR was significantly different for the small (3.3 +/- 0.6 x 10(5)) and the large monocytes (10 +/- 1 x 10(5)).

Monocyte activation by immune complexes of patients with SLE.

1. IC precipitated by PEG from patients with SLE inhibit in vitro the FcR dependent reaction of normal monocytes with sSRBC, while the C3bR dependent reaction of the cells with sensitized yeast is reduced only by some of them. The monocytes were preincubated with the IC for 30 min at room temperature. 2. When the monocytes were incubated with the IC for 22 hours at 37 degrees C the reaction of FcR with sSRBC increased, while the C3bR dependent reaction did not altered. 3. Simultaneously with the increasing FcR dependent reaction, the secretion of lysosomal beta-glucuronidase of monocytes cultivated with IC is greater than those of the controls.

The lack of detectable immune complexes in rat anaphylaxis.

Attempts were made to detect soluble immune complexes (IC) in sera of rats submitted to anaphylactic shock 12 days following BPV plus antigen treatment, and their in vitro formation following the addition of specific antigens to sera or heparinized platelet rich plasma of sensitized rats. We failed to detect ICs or IC formation by studying Hageman factor activation and platelet aggregation or by using a very sensitive radio-assay of ICs. It is established that the anaphylactic activation of Hageman factor as well as the aggregation of platelets are not mediated by immune complexes, therefore, other explanations must be found.

Functional heterogeneity of macrophages.

The antibody-binding activities of rabbit peritoneal macrophages separated on discontinuous gradients of Ficoll were investigated. The antibody was rabbit anti-ovalbumin IgG labelled with 125I. Of the five fractions obtained, one macrophage fraction was found to bind substantially more antibody than the others. These macrophages possessed more Fc receptor sites than the others and the number of Fc receptors (n) and the association constant (K) of these cells was calculated. By electron microscopy, the phagocytic activity of the subpopulation with most Fc receptors was less than that of the others.

Characterization of human monocyte subpopulations by flow cytometry.

Human monocytes separated from peripheral blood by Ficoll-Hypaque and by adherence to serum-coated dishes show a bimodal volume distribution measured with a fluorescence-activated cell sorter. In the first peak of size distribution histogram of living mononuclear cells, lymphocytes and small monocytes were characterized by latex phagocytosis and non-specific esterase staining, whereas in the second peak the large monocytes dominated. The percentage of esterase stained small monocytes was lower than that of the large ones. Parallel to these data, the rate of the FDA hydrolysis of the small monocytes was lower than that of the large ones. The majority of the large monocytes reacted with sensitized sheep red blood cells (sSRBC) while only the minority of the small monocytes bound sSRBC. Scatchard plots on the binding of fluorescein isothiocyanate (FITC)-labelled human monoclonal IgG1 to the two subpopulations indicated similar association constants. K = 1 . 2 +/- 0 . 3 X 10(5) M-1. The number of Fc receptors was significantly different for the small (3 . 3 +/- 0 . 6 X 10(5)) and the large monocytes (10 +/- 1 X 10(5)).

Binding and endocytosis of erythrocytes sensitized with rabbit IgG via Fc gamma receptors of human monocytes.

The properties of the monocyte Fc gamma receptors (FcR) were investigated with monoclonal antibodies (mAb) against FcRI (10.1) and FcRII (IV3). mAb against FcRI inhibited partially the binding of sheep red blood cells (SRBC) sensitized with anti-SRBC rabbit IgG (EA) at 37 degrees to monocytes pretreated with N-ethyl maleimide, which inhibits the EA ingestion. The erythrocytes (E) were sensitized with varying concentrations of anti-E rabbit IgG. The EA binding to different FcR depends on the concentration of specific antibody used to sensitize the erythrocytes. At high levels of sensitization a high proportion of rosettes form via FcRII which can be inhibited with mAb IV3. As sensitization decreases it is more difficult for FcRII to form rosettes, so an increased percentage of them is mediated via FcRI. Sensitization of SRBC with 1-1.5 x 10(3) anti-SRBC rabbit IgG molecules per erythrocyte is the threshold to allow FcRII to mediate rosettes. At the lowest levels of sensitization the total number of rosettes is even lower and all rosettes are mediated via FcRI, hence mAb 10.1 is fully inhibitory. In addition, our data strongly support the view that the ingestion of EA takes place mainly via FcRII. We show in this study that while binding of slightly sensitized erythrocytes was blocked efficiently by mAb 10.1, the ingestion of the equivalent EA was hardly inhibited by it.