Non-complement-dependent adsorption of soluble immune complexes to human red cells

Heat aggregated IgG and soluble immune complexes (IC) prepared by combining human serum albumin with rabbit anti-serum albumin and tetanus toxoid with rabbit antiserum to tetanus toxoid were shown to bind to human O+ RBC. The binding was greater for soluble IC prepared at antigen excess, and although it was usually maximal when IC were pre-opsonized, it could also be demonstrated using non-opsonized heat aggregated IgG or soluble IC prepared in the absence of complement. These observations suggest that two types of receptors may be involved in binding of soluble IC to human RBC: the classical C3b receptor, and a non-complement-dependent receptor, perhaps recognizing the Fc region of the immunoglobulin molecule exposed after heat aggregation or antigen-antibody reaction.     

BSA-anti-BSA immune complexes formed in the presence of human complement do not bind to autologous red blood cells.

The binding of 125I-BSA-anti-BSA immune complexes (IC) formed in the presence of complement (nascent IC) and those that reacted with complement after their formation (preformed IC) to human erythrocytes was studied. The effect of antigen-antibody ratios and serum dilutions on IC binding to red blood cells (RBC) were also investigated. A dramatic difference was found between the behaviour of the two types of complexes: while preformed IC bound effectively to human RBC after their interaction with complement, no efficient RBC-binding was observed with nascent BSA-anti-BSA complexes formed in the presence of complement. Changing the antigen-antibody ratio or dilution of serum did not affect markedly the extent of difference between the binding of preformed and nascent IC. Our findings do not support the essential role of red blood cells in the elimination of each type of IC.     

Kinetics of interaction of immune complexes with complement receptors on human blood cells: modification of complexes during interaction with red cells.

Antigen-antibody complexes, composed of 125I-BSA and guinea-pig or rabbit antibody, were incubated at 37 degrees C with human blood cells suspended in autologous serum and kinetics of binding analysed. When purified polymorphonuclear (PMN) or mononuclear cells (MNC) were studied, maximum binding was observed within 8 min, and immune complexes (IC) remained associated with cells even after 1 hr. When cells were studied unseparated (in the same amount of serum), maximum binding was observed slightly earlier (within 4 min), but within 15 min most of the IC were found in the serum. Separation of cell types at the time of maximal binding and studies with cell preparations depleted of different elements revealed that binding was principally to red blood cells (RBC). IC recovered in the serum 16 min after addition to unseparated cells bound very slowly to purified PMN or MNC; binding after 30 min was 10-15% of that observed with fresh IC at 8 min. Ultracentrifugal analysis revealed that reduction in binding efficiency correlated with decrease in the size of IC. RBC isolated after binding and release of IC bound newly-formed IC was identical rapidity and capacity as fresh RBC, indicating that receptors were not altered by IC. Kinetics studies with serum in the absence of cells suggested that interaction with RBC accelerated the rate of change in binding properties of IC. Rates of binding and release were independent of antigen/antibody ratio but were slowed and binding to RBC sustained when diluted or hypocomplementaemic (SLE) serum was substituted for neat serum. Our results suggest that competition for IC by RBC is associated with loss of ability of IC to bind to other blood cell types and reduction in size of IC, and that abnormalities of complement can lead to prolonged association of IC with RBC.     

Mechanism of transfer of immune complexes from red blood cell CR1 to monocytes.

Complement receptor 1 (CR1) on primate red blood cells (RBC) binds most complement-fixing immune complexes in the circulation. It has been postulated that by binding them, RBC keep immune complexes in the intravascular space and deliver them to the tissue macrophages of the mononuclear phagocyte system. We have developed an in vitro model to study the transfer of RBC-bound immune complexes (heat-aggregated IgG and DNA-anti-DNA) to phagocytic cells (human monocytes). Transfer of immune complexes from RBC to monocytes occurred significantly more rapidly than monocyte uptake of the same immune complexes from solution. In the transfer process, complex-bearing RBC were not bound or sequestered by the monocytes. To define the monocyte receptors involved in binding immune complexes from the RBC surface, monocyte receptors were blocked with MoAbs (anti-CR1, anti-FcRII) or EDTA (to block CR3). Monocyte binding of immune complexes primarily used CR1 with a small contribution from FcRII, and with little or no contribution from CR3 and FcRI. Uptake of immune complexes from solution employed the same monocyte receptors as binding of complexes from the RBC surface. Immune complexes in solution bound to RBC and to monocytes with equally high avidity (approximately 1 x 10(11) l/M), but monocytes expressed a 15-20-fold greater number of immune complex binding sites. We propose that immune complexes distribute between RBC and monocytes according to the binding capacity of these cells, such that at equal or high RBC/monocyte ratios as would be seen in the circulation immune complexes bind to RBC, but at low RBC/monocyte ratios (as would be seen in the sinusoidal circulation of the liver and spleen), most immune complexes bind to monocytes. To define the pathway by which immune complexes move from RBC to monocytes, their release from RBC CR1 was examined. Under various conditions, the dissociation rate was extremely slow, and did not increase with the addition of monocyte supernatants. To examine whether factor I-mediated processing of immune complexes enhances binding of immune complexes to monocytes, RBC-bound complexes were released with factor I, and binding of these 'processed' immune complexes to monocytes was examined. Monocyte binding of these processed immune complexes was slower than of control ones; furthermore, performance of transfer experiments at 4 degrees C, which significantly shows enzymatic processes, did not decrease the rate of immune complex transfer from RBC to monocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

The binding of immune complexes to human red cells: complement requirements and fate of the RBC-bound IC after interaction with human phagocytic cells.

Soluble immune complexes (IC) are known to bind to human red blood cells (HRBC). Most authors have attributed this binding to the interaction between IC-bound C3b and a red cell CR1 receptor, but contradictory data has been published concerning the ability of IC to bind to HRBC in the absence of complement. Using soluble tetanus toxoid-rabbit anti-tetanus toxoid (TT-ATT) IC, we have shown that binding through the CR1 receptor takes place when IC are formed at antibody excess, while IC formed at antigen excess do not require complement for erythrocyte binding. Once absorbed to HRBC, IC are recognized by CR1 and/or Fc receptors on phagocytic cells. This interaction is not associated with red cell engulfment, but using radiolabelled S. aureus protein A as a probe, we have demonstrated the transfer of IC from HRBC to phagocytic cells. Such transfer without red blood cell (RBC) damage agrees with the postulated role of RBC in the elimination of soluble IC from circulation. However, we have also demonstrated that the interaction between HRBC-IC and phagocytic cells is associated with the release of mediators of inflammation. It is, therefore, not absolutely clear whether the interaction of RBC-adsorbed IC and phagocytic cells will always have beneficial consequences.     

Rosetting of human red blood cells to thymocytes and thymus-derived cells

Human thymocytes bind with human red blood cells (HRBC) and neuraminidase-treated HRBC (nHRBC) forming rosettes. Peripheral blood lymphocytes and tonsillar lymphocytes bind to nHRBC but not HRBC. The conditions which lead to nHRBC rosette formation are similar to those required for rosette formation with sheep red blood cells (SRBC). The rosetting of human lymphocytes to nHRBC does not require serum but is prevented by pretreatment of the lymphocytes with sodium azide, proteolytic enzymes, 50°C for 7 min, or anti-T cell (but not anti-B cell) serum. Peripheral lymphocytes from patients with chronic lymphatic leukaemia do not rosette with either HRBC or nHRBC. Therefore, the nHRBC rosetting lymphocyte is a living T cell. It is proposed that this in vitro test may simulate an in vivo mechanism for immune surveillance.     

DMO-transport in human red blood cells

The DMO (5,5-dimethyloxazolidine-2,4-dione) transport was studied in human red blood cells by measuring the¹⁴C-DMO back exchange under self exchange conditions from¹⁴C-DMO labeled cells at 0°C. The unidirectional DMO-flux was linearly related to the DMO concentration up to approx. 100 mM. The unidirectional DMO-flux increases as pH is reduced. The DMO transport is not inhibited by anion transport inhibitors like DIDS, SITS, dipyridamole, phlorhizin, salicylate or butanol. A close correlation between the unidirectional DMO-flux and DMOH, the unionized form of DMO, has been observed suggesting that DMO is transported predominantly by nonionic diffusion. The permeability of DMOH is 9.5·10^–6 cm/s (0°C) while the permeability of DMO^– cannot be assessed from our data.     

Antibodies for trypsinized human red blood cells in human sera.

During studies of agglutination of trypsinized human red blood cells (RBC) by anti-Rh sera, it was noticed that some human sera without Rh antibodies agglutinated these erythrocytes. Of 933 normal and pathological sera subsequently screened, 116 produced such agglutination. The agglutinins were of IgM nature and were directed against an antigen exposed or created by tryptic digestion of RBC. They reacted equally well with autologous as with allogeneic trypsinized RBC and were different from T agglutinins and from Paul-Bunnell antibodies. The mode of formation of these antibodies remains unknown.     

Assessment of immune phagocytosis of Plasmodium falciparum infected red blood cells by human monocytes and polymorphonuclear leukocytes. A method for visualizing infected red blood cells ingested by phagocytes

A method is described for the visualization of red blood cells infected with Plasmodium falciparum ingested by monocytes or polymorphonuclear leukocytes (PMN) after in vitro incubation. Smears were stained with peroxidase followed by 4,6-diamino-2-phenylindole (DAPI) staining specific for DNA. Monocytes or PMN were identified under normal illumination by the peroxidase stain and the nuclei of these cells as well as the parasites were identified by means of the DAPI stain with ultraviolet light. Using this method we found that monocytes and PMN from normal blood donors preferentially phagocytose plasmodium falciparum infected red blood cells in the presence of sera from subjects living in areas endemic for malaria.     

Suppression of the primary immune response in vivo to sheep red blood cells by B-cell mitogens.

Polyacrylic acid (PAAC), lipopolysaccharide (LPS), dextran sulphate (DS) and purified protein derivative of tubercle bacilli (PPD), compounds mitogenic for B lymphocytes in vitro, suppressed the immune response of mice to SRBC in vivo, when injected 4-2 days before immunization. The same compounds enhanced the immune response when injected half an hour before immunization of the animals with a suboptimal antigen dose. A subsequent injection of PAAC given shortly before immunization, abolished the immunosuppressive effect expected by pretreatment of the animals with either PAAC or LPS. A second injection of LPS abolished the immunosuppressive effect of LPS only. The results indicate that when B lymphocytes react with a mitogen in the absence of a particular antigen, they temporarily lose their capacity to respond to antigen.     

Alternating current electrophoresis of human red blood cells

An a.c. electrophoretic technique in the frequency range between 2–10 Hz, using a rectangular chamber design, has been developed for measuring the surface electric charge density of human red blood cells. With the a.c. approach, mobility can be determined rapidly, electrode polarization is minimized and the construction of the chamber simplified. For frequencies under 10 Hz and a chamber thickness of 150 μm, a theoretical analysis of the electroosmotic flow profile shows that it is almost identical to the d.c. case. With the a.c. method and using a 0.145 N NaCl solution buffered with NaHCO₃, the mobility of red cells measured at the lower stationary level is found to be −1.07±0.02 (S.D.) μmsec⁻¹ V⁻¹ cm. The zeta potential and charge density calculated from the mobility are −14.03±0.26 (S.D.) mV and −3603±69 (S.D.) esu cm⁻², respectively.     

L-asparaginase: suppression of the immune response of mice to sheep red blood cells

L-asparaginase was immunosuppressive in mice. A single injection of 1000 I.U. of L-asparaginase markedly suppressed the direct haemolytic plaque forming cell response to immunization with sheep red blood cells. The effect was not due to cytotoxicity but to delayed proliferation of plaque forming cells. Haemagglutinin production was delayed by a single injection and was suppressed when the enzyme was given daily from 3 days before immunization to the day before antiserum collection. L-asparaginase in this dosage form both reduced total antibody production and markedly prolonged 2-ME sensitive antibody production. L-asparaginase treatment was only weakly immunosuppressive and none of the animals studied showed complete suppression of the immune response. It was suggested that asparaginase would be most effective in the prevention of the development of primary immunity when combined with another agent with an independent mechanism of action.     

Calcium transport in human red blood cells under hypertonic conditions.

Calcium accumulation in intact human erythrocytes is enhanced by incubation in hypertonic solutions. Hypertonicity produces an increased permeability of the membrane to calcium that is time-dependent and occurs in the presence or absence of calcium. When hypertonically treated cells are incubated for more than 30 min in 300 mosmol/kg solutions the permeability of the membrane to calcium returns to basal values. Oligomycin inhibits the effect of hypertonicity on calcium uptake. The inhibitory action of oligomycin diminishes as the external sodium increases and can only be observed when the external concentration of potassium is at or below 3 mM. Low intracellular sodium and high intracellular potassium concentrations increase the uptake of calcium. It is concluded for human erythrocytes that 1) the increased permeability of the membrane to calcium produced by hypertonicity is a time-dependent, reversible phenomenon and is independent of calcium, 2) the increase in intracellular potassium concentration associated with hypertonic exposure is an important factor contributing to this response, and 3) interactions between calcium and components of the sodium-potassium transport system may account for the enhanced uptake of calcium produced by hypertonicity.     

Opsonic activity of human immune serum on in vitro phagocytosis of Plasmodium falciparum infected red blood cells by monocytes.

In vitro human monocytes from normal blood donors ingest red blood cells infected with Plasmodium falciparum more efficiently than normal red blood cells (NRBC). The phagocytic activity of human monocytes for infected red blood cells (IRBC) is greatly enhanced by the addition of immune sera obtained from individuals living in areas with endemic malaria. In contrast, the addition of sera obtained from individuals recovering from a first infection, or pooled normal sera, does not result in increased phagocytosis of IRBC. The phagocytosis enhancing activity of immune sera is associated with the IgG fraction and IgG depleted sera do not stimulate phagocytosis. Enhanced immune serum mediated phagocytosis occurs as a result of opsonization of IRBC. This was demonstrated by experiments in which monocytes or IRBC were preincubated with immune serum prior to the phagocytic assay. The opsonic activity could be absorbed by IRBC but not by NRBC. The opsonization of IRBC and subsequent phagocytosis were also dependent on the stage of development of the intracellular parasite. IRBC containing schizonts and trophozoites were preferentially phagocytosed as compared with ring forms. The role of malaria induced surface alterations and/or malaria surface antigens in the opsonization of IRBC by immune sera is discussed. These experiments suggest that phagocytosis of P. falciparum IRBC by monocytes may play a role in the immune elimination of malaria infection in humans.