Effect of plasmapheresis on ligand binding capacity and expression of erythrocyte complement receptor type 1 (CR1) of patients with systemic lupus erythematosus (SLE).

The functional activity and the expression of CR1 on the erythrocytes (E) of patients with SLE were, respectively, determined by measuring the binding to E of either complement-opsonized bovine serum albumin (BSA)-anti-BSA immune complexes (ICC) or specific anti-ECR1 MoAbs. We found that both the functional activity and levels of ECR1 in SLE patients homozygous for ECR1 high density allele were significantly lowered compared with healthy controls having the same allele. Soon after plasmapheresis there was a significant increase in E ICC binding activity, and this increased functional activity was stable. Moreover, plasmapheresis reduced the level of immune complexes demonstrable in the circulation of the patients. The expression of ECR1 determined with several different anti-CR1 MoAbs was also elevated as a consequence of plasmapheresis. This elevation was observed for both MoAb 1B4, which competes for the ICC binding site of ECR1, and for MoAb HB8592, which does not, but the time course for the increase in binding of the two MoAbs was different, in that the epitope recognized by MoAb 1B4 increased more rapidly. The present results, considered in the context of previous findings, suggest that more than one mechanism may be operative with respect to the effects of the plasmapheresis in increasing ECR1 levels defined by different epitopes on the molecule.

Correlation of Fc gamma receptor expression of monocytes with clearance function by macrophages in systemic lupus erythematosus.

The expression of Fc gamma receptor II (Fc gammaRII) and Fc gammaRIII on monocytes in peripheral blood and the clearance of immunoglobulin (Ig)G-sensitized erythrocytes (EA) by tissue macrophages were investigated in parallel in patients with systemic lupus erythematosus (SLE). The relationship between receptor expression and the rate of clearance of EA (half-time) was analyzed. The detected decrease in mean fluorescence intensity of both FcR gammaII and Fc gammaRIII of patients' monocytes stained with specific monoclonal antibodies (IV.3 and 3G8) was inversely correlated with the prolonged clearance half-time of 51Cr-labelled and anti-D IgG-sensitized autologous erythrocytes in these patients. A correlation was found between the impaired clearance function and the severity of the disease manifestation expressed by either clinical activity or renal involvement in our SLE patients. From these results it can be concluded that the in-vitro determination of monocyte Fc gammaRII and Fc gammaRIII expression may predict the in-vivo macrophage function via the same Fc receptors.

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.

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.

Changes in superoxide anion production and phagocytosis by circulating neutrophils during tumor progression in a rat model.

The functional state of circulating neutrophils was monitored in a rat model of mesoblastic nephroma during tumor progression. Superoxide anion (O2.-) production in response to PMA and phagocytosis of yeast particles (Saccharomyces cerevisiae) were measured every second day after tumor cell implantation. Both phagocytosis and PMA-induced 02.- generation were found to be enhanced in the first period (on Days 6, 8, and 10), while they became significantly reduced in the advanced stage of cancer (on Days 12, 14, 16, and 18). The suppression of PMNL functions was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. Studies were also carried out on PMNLs isolated from normal rats and the cells were treated with plasma samples obtained from tumor-bearing animals at different stages of nephroma. Incubation of the normal cells with plasmas separated on the 2nd and 8th days of tumor growth influenced neither the 02.- generation nor the phagocytosis. In contrast, plasma preparations obtained on the 14th day significantly inhibited both 02.- production and phagocytosis by normal neutrophils. The alterations in 02'- generation and phagocytosis by PMNLs were observed in close association with tumor growth, thus they could be considered as indicators of tumor progression. However, further studies are required to see whether the granulocyte dysfunctions observed in our animal model could provide additional prognostic information in the case of human malignancies as well as to clarify the origin of inhibitory factor(s) present in the blood of tumorous animals.

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.

Correlation of IgG Fc receptors on granulocytes with serum immune complex level in systemic lupus erythematosus.

The expression of Fc gamma RI, Fc gamma RII and Fc gamma RIII on granulocytes in the blood of patients with systemic lupus erythematosus was investigated. The relationship between the receptor expression and serum immune complex (IC) concentration was analysed. The decrease in mean fluorescence intensity of both Fc gamma RII and Fc gamma RIII of patients' granulocytes stained by specific monoclonal antibodies (using MoAb IV.3 and 3G8) was significant. The detected decrease of Fc gamma RII was inversely correlated with the high circulating IC level in patients' sera.

Correlations of monocyte phagocytic receptor expressions with serum immune complex level in systemic lupus erythematosus.

The expression of Fc gamma RI, Fc gamma RII, and Fc gamma RIII (the IgG receptors CD64, CD32, CD16) as well as CR3 (the C3bi receptor, CD11b) on monocytes in the blood of patients with systemic lupus erythematosus (SLE) was investigated. The relationship between the receptor expression and the serum immune complex (IC) concentration was analysed. The decrease in mean fluorescence intensity (FI) of the Fc gamma RII of patients' monocytes stained by specific monoclonal antibodies (MoAb IV3) was very close to statistical significance (P = 0.052). The expression (FI) of CR3 (using MoAb OKM1) on monocytes of patients was also decreased, but not significantly. The detected decrease of Fc gamma RII and CR3 was inversely correlated with the high circulating immune complex level in patients' sera. At the same time, Fc gamma RI expression on SLE monocytes (using MoAb 32) was significantly elevated and this change was in parallel with the serum IC concentration.

Triggering of respiratory burst by phagocytosis in monocytes of patients with systemic lupus erythematosus.

The triggering of the respiratory burst by phagocytosis via different receptors in monocytes of patients with systemic lupus erythematosus (SLE) was investigated. The superoxide anion synthesis was assayed by reduction of ferricytochrome C that was inhibited by superoxide dismutase. The mononuclear cell suspensions were triggered by IgG-coated latex, C3 complement fragment-coated and uncoated yeast (Saccharomyces cerevisiae). Superoxide generation induced by phagocytosis via Fc gamma R was decreased in monocytes of patients with SLE. On the other hand, MoAbs against Fc gamma RI, Fc gamma RII and especially CR3 could also induce superoxide anion synthesis. At the same time, superoxide generation induced by anti-CR3 could be inhibited with C3-coated yeast.

A sensitive simple ELISA for quantitation of sensitizing IgG from dissolved erythrocytes.

A simple enzyme-linked immunosorbent assay has been developed for quantitating the rabbit IgG present on the surface of sensitized red blood cells. A suitable cell lysis was carried out by an alkaline buffer, which dissolved the erythrocytes without forming any precipitate and without disruption of IgG, and facilitated the dissociation of the immune complexes, i.e. the erythrocyte-anti-erythrocyte rabbit IgG. In this alkaline buffer of pH 11.4 the IgG adsorbed directly into wells of microtitration plates unprecoated with anti-rabbit IgG. The detection was performed with alkaline phosphatase-conjugated goat anti-rabbit IgG and p-nitrophenyl phosphate as substrate. Constructing a calibration curve from rabbit IgG made possible the calculation of molecules of IgG bound per red cell. The method was sensitive for the detection of fewer than 500 bound IgG molecules per erythrocyte.

Marker profile, enzyme activity, and function of a human myelomonocytic leukemia cell line.

Morphological and functional characteristics of a permanent human leukemia cell line (DD) that possesses myelomonocytic features were investigated. The cells bear a second type Fc gamma receptor and form rosettes with sheep erythrocytes sensitized with rabbit IgG (EA). However, the surface-bound EA is not internalized. The cell line lacks the surface markers CD2, CD19, CD14, HLA-DR, Fc gamma receptor I, Fc gamma receptor III, and CR3. alpha 1-Antitrypsin, lysozyme, Factor XIII a subunit of blood coagulation, and acid phosphatase reactions were negative. A terminal differentiation of the DD cell line was observed when the expression of CD14, CR3, Fc gamma receptor I, and Fc gamma receptor III was induced. The DD cells induced with 12-O-tetradecanoylphorbol-13-acetate or Escherichia coli lipopolysaccharide can internalize EA via Fc gamma receptor II and complement-coated yeast in the function of the inducers. The phagocytic ability appears to be parallel with the appearance of enzymes which participate in phagocytosis.

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.

Ligand-specific cross-inhibition of monocyte phagocytosis.

Human monocytes exposed to particles reacting with receptors of one specific type demonstrate a markedly reduced phagocytosis of particles reacting with receptors of another type. The phagocytosis of yeast particles (Saccharomyces cerevisiae) coated with C3 fragments and of uncoated ones by monocytes can inhibit the subsequent endocytosis of sheep erythrocytes sensitized with IgG antibody and vice versa. The erythrocytes were labelled with 51Cr and the yeast particles with 125I. The dependence of the receptor-ligand reaction on temperature and time of preincubation, as well as on ratio of cell and particle, suggests that this cross-inhibition may be associated with plasma membrane modulations beginning with the attachment of ligands to a receptor and followed by their endocytosis. The differences between the functions of temperature, time, and concentration in inhibition caused by different receptor-ligand reactions suggest that these membrane modulations are probably ligand-specific processes.

Inefficient binding of IgM immune complexes to erythrocyte C3b-C4b receptors (CR1) and weak incorporation of C3b-iC3b into the complexes.

The binding of soluble complement-reacted IgM immune complexes (IC) to erythrocyte (E) C3b-C4b receptors (CR1) and the incorporation of C3b-iC3b into solid phase IgM-IC was investigated. The optimal binding of liquid phase IgM-IC to E-CR1 was obtained with IC formed at moderate antibody excess, but the binding was low (2-3%) when compared to the binding of the corresponding IgG-IC (50-60%). Solid phase IC were prepared by coating microwells with heat-aggregated bovine serum albumin (BSA) followed by incubation with rabbit IgM anti-BSA antibody. The IC were reacted with human serum at 37 degrees C. The binding of C3b-iC3b was determined by use of biotinylated F(ab')2 antibodies to C3b-C3c and avidin-coupled alkaline phosphatase. The incorporation of C3b-iC3b into solid-phase IgM-IC increased when increasing amounts of IgM antibody were reacted with the antigen. The binding reaction was slow, reaching a maximum after about 2 h at 37 degrees C. The binding of C3b-iC3b to the IgM-IC was remarkably inefficient when compared to the incorporation into IgG-IC reacted with the same amounts of BSA-precipitating antibody.

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.

Altered monocyte functions in patients with angioimmunoblastic lymphadenopathy.

Monocyte function was investigated in ten patients with angioimmunoblastic lymphadenopathy (AILD). Although spontaneous migration, phagocytosis and opsonisation of monocytes were unimpaired, the chemotactic response and erythrocyte-antibody-rosette (EA-rosette) formation were decreased significantly. The migratory response of normal monocytes was inhibited on preincubation with serum from AILD patients. It is suggested that the immune abnormality in AILD, previously thought to involve T-B and natural killer lymphocytes, extends to the monocyte-macrophage system.

Lymphokine effect upon the erythrocyte antibody uptake by human monocytes.

Preincubation of human monocytes with different amounts of human lymphokine at 37 degrees C dose dependently increased the uptake of EA cells at both 37 degrees C and 4 degrees C. Phenylmethanesulphonyl fluoride (PMSF), an inhibitor of serine esterases, inhibited the process. It seemed that the serine esterase and not the gamma interferon component of the lymphokine played the main role in the phenomenon.

Defective immune complex degradation by monocytes in patients with systemic lupus erythematosus.

The binding of 125I-labelled anti-bovine serum albumin (BSA)-BSA immune complexes (IC), giving a final molar antibody to antigen ratio of 1:1, to monocytes isolated from 18 patients with systemic lupus erythematosus (SLE) and from 10 normal healthy donors was quantitatively investigated. The degradation of the bound IC by the same monocytes was kinetically determined at the same time. The assays were performed on monocyte monolayers. Scatchard plots at 4 degrees C demonstrated that monocytes from patients with active SLE expressed a mean Fc gamma receptor (FcR) number that was 22% higher than that of the controls, although this did not reach statistical significance. The FcR number of normal monocytes and the degradation rate of anti-BSA-BSA complexes by the same cells showed a positive correlation. At the same time, the digestion of anti-BSA-BSA complexes by monocytes of SLE patients with active disease was prolonged, despite their enhanced FcR-ligand binding. The dissociation of FcR-ligand binding and FcR-mediated degradation suggests that the IC degradation is controlled by altered biochemical mechanisms in the monocytes of SLE patients.

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.