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Investigation of the capacity of the mononuclear phagocyte system to remove immune complexes from the circulation was performed by the administration of 125I-labelled aggregates of human immunoglobulin G (AIgG) to patients with seropositive rheumatoid arthritis and healthy volunteers. It was found that the rate at which AIgG disappeared from the circulation was significantly prolonged in patients with RA, t1/2 61 +/- 49 min, versus 26 +/- 8 min in healthy volunteers (p less than 0.01). We were not able to establish a correlation between the t1/2 of AIgG and immune complex levels in the circulation, or between t1/2 and articular disease activity (Ritchie index). The sites of removal of AIgG from the circulation were analysed by determining radioactivity levels detectable over liver, spleen and heart. No correlation was found between t1/2 and liver/spleen uptake ratios. We have demonstrated that the removal of AIgG from the circulation of patients with RA is abnormal, though the biological significance of this finding remains to be determined.

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In the present study, we tested mononuclear phagocyte system function in nine healthy controls and 15 SLE patients with complement activating 123I-labelled aggregates of human IgG (AIgG). Clearance half-time of AIgG was 26 +/- 8 min in controls, compared to 58 +/- 27 min in patients (P less than 0.005). Binding of AIgG to erythrocytes was significantly lower in patients, 9.3 +/- 8.1 vs 24 +/- 20% (P less than 0.05). The increase of C3a-levels in plasma was significantly lower in patients than in controls (P less than 0.05 at 3 and 8 min), suggesting less complement activation. Liver and spleen uptake of 123I-AIgG was measured with a gamma camera and expressed as liver/spleen uptake ratios. In patients, the liver/spleen uptake ratios were significantly higher than in controls at 15 min, 3.8 +/- 2.0 vs 2.31 +/- 0.7 (P less than 0.05), due to less splenic uptake of AIgG. Correlations between clearance half-time or liver/spleen uptake ratios and immune complex levels or disease activity were not found. This study indicates that clearance of soluble AIgG is abnormal in patients with SLE, due to decreased splenic uptake of AIgG.

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Using aggregates of IgG (AIgG) obtained by heat aggregation of a 16 g% human IgG solution, we sought a method for measuring the function of the mononuclear phagocyte system with a probe that bears more resemblance to soluble immune complexes than erythrocytes coated with anti-rhesus IgG (EIgG). It was found that intravenous administration of 10 micrograms AIgG/kg body weight did not cause any detectable side effects in chimpanzees. In nine healthy volunteers, a dose of 10 micrograms AIgG/kg body weight was used without any adverse reactions. AIgG is cleared from the human circulation with a t1/2 of 26 +/- 8 min (mean +/- SD). The site of clearance is predominantly the liver, as shown by an average liver spleen uptake ratio of 230:100. In whole blood obtained from the volunteers, it was found that erythrocytes bound significant amounts of AIgG, suggesting that CR1 on erythrocytes is involved in the clearance of complement activating immune complexes in humans. In five of the volunteers, clearance studies with EIgG had been done in a previous study. EIgG was cleared from the circulation with a t1/2 of 30 +/- 6.2 min (mean +/- SD). The predominant site of clearance of EIgG was the spleen. These data indicate that sensitized red blood cells are cleared from the circulation differently from soluble IgG aggregates.

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Previously, we have shown that enucleated human neutrophils (PMN cytoplasts), when activated by particulate or fluid stimuli, generate superoxide and hydrogen peroxide at rates comparable (per unit area of plasma membrane) to those observed with intact neutrophils. Moreover, PMN cytoplasts also ingest and, to a certain extent, kill bacteria. We now report that PMN cytoplasts can be cryopreserved with maintenance of their functional activity. The PMN cytoplasts were frozen in a medium with 10% (v/v) fetal calf serum and 10% (v/v) dimethyl sulfoxide, and stored at -70 degrees C. After thawing and washing, the recovery was 75%. The content of alkaline phosphatase and lactate dehydrogenase, the consumption of oxygen and generation of hydrogen peroxide, and the rate of phagocytosis of Staphylococcus aureus bacteria was the same for fresh and cryopreserved PMN cytoplasts. Identical values were obtained after preservation in liquid nitrogen. These results open possibilities to store neutrophil material, allowing longitudinal follow-up of patients, comparative studies between different patients, exchange of material between laboratories, and storage of reference material for experiments in series.

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Enucleated human polymorphonuclear leukocytes (PMN) were prepared by centrifuging isolated, intact PMN over a discontinuous Ficoll gradient that contained 20 microM cytochalasin B. The enucleated cells (PMN cytoplasts) contained about one-third of the plasma membrane and about one-half of the cytoplasm present in intact PMN. The PMN cytoplasts contained no nucleus and hardly any granules. The volume of the PMN cytoplasts was about one-fourth of that of the original PMN. Greater than 90% of the PMN cytoplasts had an "outside-out" topography of the plasma membrane. Cytoplasts prepared from resting PMN did not generate superoxide radicals (O2-) or hydrogen peroxide. PMN cytoplasts incubated with opsonized zymosan particles or phorbol-myristate acetate induced a respiratory burst that was qualitatively (O2 consumption, O2- and H2O2 generation) and quantitatively (per unit area of plasma membrane) comparable with that of intact, stimulated PMN. Moreover, at low ratios of bacteria/cells, PMN cytoplasts ingested opsonized Staphylococcus aureus bacteria as well as did intact PMN. At higher ratios, the cytoplasts phagocytosed less well. The killing of these bacteria by PMN cytoplasts was slower than by intact cells. The chemotactic activity of PMN cytoplasts was very low. These results indicate that the PMN apparatus for phagocytosis, generation of bactericidal oxygen compounds, and killing of bacteria, as well as the mechanism for recognizing opsonins and activating PMN functions, are present in the plasma membrane and cytosol of these cells.

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During phagocytosis neutrophils from 8 patients with chronic granulomatous disease released 2-3 times more activity of lysozyme and beta-glucuronidase than did normal neutrophils. This difference was caused by the partial inactivation of these enzymes by normal neutrophils. The inactivation of granule enzymes depends on oxidative products and takes place mainly in the phagolysosomes. Myeloperoxidase is involved in this phenomenon.

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A diminished chemotactic response was observed with the neutrophils of a patient with the Chediak-Higashi syndrome, who was not in the accelerated phase of the disease. An abnormally low release of myeloperoxidase from these cells during phagocytosis was also noted; this resulted in a decreased iodination capacity and probably also caused the defect in the intracellular killing of bacteria by the neutrophils. The level of cyclic AMP in these cells was elevated, but decreased after treatment with ascorbate either in vitro or in vivo. During ascorbate therapy, the bactericidal activity of the neutrophils normalized, whereas the chemotactic response remained low. Nevertheless, the patient had significantly less infections during ascorbate therapy. The bleeding tendency, due to a storage-pool disorder of the Chediak-Higashi platelets, was unaffected by treatment with ascorbate. The patient's lymphocytes did not display any activity in antibody-dependent lymphocytotoxicity. This defect was not affected by treatment with ascorbate either.

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During phagocytosis, neutrophils generate reactive oxygen metabolites and release lysosomal enzymes into the extracellular medium. We have investigated the possibility that these enzyme are inactivated by the oxygen compounds. Phagocytosing neutrophils from 12 patients with chronic granulomatous disease, which do not generate these oxygen metabolites, released two to three times more activity of lysozyme and beta-glucuronidase than did normal neutrophils. This difference proved to be due to a decrease of approximately 20% of the total activity of these enzymes in normal neutrophils, but not in neutrophils of patients with chronic granulomatous disease. This inactivation of enzymes took place during phagocytosis of opsonized zymosan particles as well as during stimulation of normal cells with phorbol myristate acetate. The inactivation was not due to formation of inhibitors. The lysosomal enzymes were not activated when the neutrophils were stimulated under anaerobic conditions. Addition of catalase, superoxide dismutase, or albumin gave no protection against the oxidative damage; reduced glutathione gave partial protection. The oxidative inactivation was more pronounced in the presence of azide. Measurement of the activity and the amount of protein of acid alpha-glucosidase in the cells showed that the specific activity of this enzyme decreased by approximately 50% during 30 min of phagocytosis. This indicates that the inactivation of the lysosomal enzymes takes place in the phagolysosomes, before the enzymes have leaked into the extracellular medium.

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This report reviews some of our work on the relative importance of the glutathione redox system and catalase in protecting human neutrophils in vitro against hydrogen peroxide, generated either by these cells during phagocytosis or artificially in the medium by an enzyme system. Neutrophils deficient in glutathione reductase were rapidly inactivated during phagocytosis, unless protected by scavengers of oxidative products in the medium. In contrast, normal neutrophils remained functionally active. Thus, despite the presence of a normal catalase activity, a defect in the glutathione system totally impairs the protection of neutrophils against their own metabolic products. In catalase-inhibited or catalase-deficient neutrophils, no damage was observed during phagocytosis. We conclude that the glutathione redox system is the most important protection system against damage by oxidative products of neutrophils. During incubation of neutrophils with glucose + glucose oxidase, an extracellular system that generates hydrogen peroxide, we found that both catalase and the glutathione redox system were needed for adequate protection against oxidative injury. Apparently, this extracellular stress cannot be efficiently dealt with by the glutathione system alone: co-operation with catalase is needed in this situation. Under certain conditions, oxidative damage was observed even when the level of reduced glutathione was still relatively high, indicating that perhaps catalase and glutathione each protect different cell structures, and that both systems are needed together for the preservation of the total cell function.

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