E J Brown

Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

We used monoclonal antibodies and flow cytometry to study the expression of the receptors for the complement fragments C3bi (CR3) and C3b (CR1) on human polymorphonuclear neutrophil leukocytes (PMN). Expression of both receptors was minimal on cells stained in anticoagulated whole blood incubated at 0 degree or 37 degrees C. PMN isolated with Percoll density gradients and held at 0 degree C also had only minimal expression of both receptors. With the isolated cells, however, a spontaneous increase in expression of both receptors occurred upon warming to 37 degrees C. This did not represent complete expression of either receptor since additional increments in surface expression could be induced upon stimulation with N-formyl-methionyl-leucyl-phenylalanine or Raji cell supernatant. The increases in complement receptor (CR) expression appeared to be specific since there were no changes in expression of the Fc gamma receptor or beta-2-microglobulin under any of these conditions. The increased CR expression seems to involve translocation from an intracellular pool since it is complete within minutes and is not blocked by puromycin or cycloheximide. These results demonstrate that both CR3 and CR1 expression increase rapidly upon activation of PMN and that isolated cells can be used to study this phenomenon, which may be a critical part of neutrophil function in vivo.

Opsonic Requirements for Intravascular Clearance after Splenectomy

We investigated the opsonic requirements for intravascular clearance of pneumococci in guinea pigs and of sensitized erythrocytes in human beings after splenectomy. The impaired clearance of injected pneumococci in splenectomized guinea pigs was corrected by immunization. This improvement in clearance was due to increased hepatic sequestration of organisms. There was a significant delay in antibody-mediated clearance of autologous erythrocytes sensitized with IgG (P < 0.001), although the rate of complement-mediated clearance in splenectomized patients was normal. A fourfold increase in sensitizing antibody resulted in a significant improvement in clearance that was due to increased hepatic sequestration (P < 0.005). One patient who had an intact spleen and who had previously received Thorotrast (thorium oxide) had impaired antibody-mediated clearance despite increased sensitization. These observations suggest that, after splenectomy the remaining macrophages of the reticuloendothelial system require increased amounts of antibody to mediate efficient intravascular clearance of opsonized particles.

Profile of endothelial and leukocyte activation in Fabry patients.

Fabry disease is an X‐linked recessive disorder resulting in the deposition of globotriaosylceramide in numerous cell types including vascular endothelial cells. Because this disease is associated with vascular injury and a high recurrence rate of thrombotic events, measurements of factors regulating endothelium and leukocyte interaction may provide insight into the mechanisms leading to a prothrombotic state. Twenty‐five patients with Fabry disease and 25 control subjects participated in the study. Plasma from all 25 Fabry patients and 15 of the 25 controls were studied for multiple endothelial factors. Leukocyte integrins were measured by flow cytometry in 21 Fabry patients and 10 controls. The concentrations of soluble intercellular adhesion molecule‐1, vascular cell adhesion molecule‐1, P‐selectin, and plasminogen activator inhibitor were significantly higher and thrombomodulin was significantly lower in Fabry patients. Expression of the integrin CD11b on monocytes was also significantly higher in the Fabry patients. This study reveals measurable evidence for endothelium and leukocyte activation that is consistent with a prothrombotic state in Fabry patients compared with controls. Further investigations of these findings may help to understand the mechanism of stroke in Fabry disease and provide indicators (or markers) of efficacy of future therapeutic intervention. Ann Neurol 2000;47:229–233

Immunoglobulin G Fc Receptor-Mediated Clearance in Autoimmune Diseases

The reticuloendothelial system is thought to play an important role in removing immune complexes and other immunologically active substances from the circulation via interaction with specific cell-surface receptors. The function of the reticuloendothelial system in humans with autoimmune diseases was studied in vivo by measuring the rate of removal of IgG-coated, radio-labeled autologous erythrocytes. Such cells are removed by phagocytic cells of the spleen, and the process depends on the presence of an intact IgG Fc fragment. Studies in patients with active systemic lupus erythematosus show a profound defect in Fc-receptor-specific clearance that correlates with disease activity. Patients with other autoimmune diseases have defects in Fc receptor functional activity when their illness is characterized by tissue deposition of immune complexes. Normal patients with HLA-B8/DRw3, an HLA type associated with an increased incidence of autoimmune disease, also have an increased incidence of defective Fc receptor-specific functional activity, suggesting that this defect may predispose patients with this haplotype to develop manifestations of immune complex-mediated disease.

Long-Term Therapy of Hereditary Angioedema with Danazol

We treated 69 patients who had hereditary angioedema with danazol to alleviate attacks of mucocutaneous angioedema involving the skin, oropharynx, and gastrointestinal tract, and we documented the continued efficacy of danazol for long-term treatment (1 to 6 years) of hereditary angioedema. Significant dose-related, adverse reactions occurred, including weight gain, myalgias, headaches, microscopic hematuria, abnormal liver function tests, anxiety, altered libido, alopecia, dizziness, and nausea. Alterations in menstrual function were consistently observed. About 10% of patients noted masculinizing side effects, such as acne, hirsutism, and voice deepening. We recommend downward titration of danazol dosage to achieve control of attacks and minimize adverse reactions. Periodic monitoring of patients on long-term danazol therapy is essential to avoid undesirable toxicity.

A quantitative analysis of the interactions of antipneumococcal antibody and complement in experimental pneumococcal bacteremia.

The mechanism of protection of type-specific antipneumococcal antibody and complement in bacteremia was investigated with purified rabbit antibody and a guinea pig model of pneumococcal bacteremia. IgG and IgM were isolated from the sera of rabbits immunized with type 7 pneumococci (Pn), and their binding to Pn was quantitated. The number of antibody-binding sites on the pnuemococcal capsule was also determined. Pn were incubated with various amounts of the immunoglobulin preparations before intravenous injection into nonimmune guinea pigs. Whereas 120 molecules of IgM per Pn were sufficient to enhance bloodstream clearance of Pn, 1,400 molecules of IgG per bacterium were required to produce this effect. As the amount of either IgG or IgM added to the Pn was increased, the rate of bloodstream clearance accelerated. In striking contrast, greater than 1,000 molecules of IgM had no effect on the rate of clearance in C4-deficient guinea pigs, which cannot activate complement via the classic pathway. Similarly, 5,000 molecules of IgG had only minimal effect in C4-deficient guinea pigs, and 24,000 molecules of IgG had no effect in guinea pigs depleted of complement by cobra venom factor. Thus, the in vivo opsonic effects of both IgG and IgM anticapsular antibody are mediated via their ability to activate complement. IgG anti-pneumococcal cell wall antibody, raised by intravenous injection of rabbits with unencapsulated Pn, had no effect on the rate of bloodstream clearance of Pn or on the polymorphonuclear leukocyte killing of type 7 Pn in an in vitro bacterial assay. Because the opsonic effects of anticapsular antibody required complement activation, the ability of anticell wall IgG to activate complement was compared with the two classes of anticapsular antibody. As judged by depletion of C3 and C4 from guinea pig serum, as well as by the fixation of radiolabeled C3 to Pn, IgM anticapsular antibody was the best complement activator. However, anticell wall IgG was somewhat more active than anticapsular IgG in each of these tests of complement activation and fixation. When equivalent amounts of C3 were fixed to Pn by each of the three antibodies, Pn sensitized with IgG and IgM anticapsular antibodies caused immune adherence, whereas Pn sensitized with anticell wall IgG did not. This may explain the failure of anticell wall antibody of mediate complement-dependent phagocytosis of Pn in vivo or in vitro. Although anticell wall IgG is capable of activating complement and fixing C3 to Pn, it is not opsonic; the most likely reason is that the nonopsonic antibody mediates C3 deposition in sites on the Pn that cannot interact efficiently with phagocytic cell C3 receptors.

The role of the spleen in experimental pneumococcal bacteremia.

The importance of the spleen in host defense against pneumococcal bacteremia has been suggested by a number of experimental models as well as the occurrence of the syndrome of overwhelming pneumococcal sepsis in asplenic individuals. We studied the mechanism of splenic protection against pneumococcal bacteremia using a guinea pig model. Rates of removal of pneumococci from the blood stream in normal and splenectomized guinea pigs were compared with the extent of hepatic and splenic sequestration of radiolabeled organisms for three different types of pneumococci. A relationship was found between the virulence of a pneumococcus for normal guinea pigs, the extent to which it is cleared by the spleen, and the magnitude of the defect in blood stream sterilization induced by splenectomy. The spleen plays an increasingly important role in the clearance of progressively more virulent organisms, for which hepatic clearance cannot compensate. Thus, the division between hepatic and splenic clearance of bacteremia is a key determinant of the outcome of experimental pneumococcal infection.

A quantitative fluorescent method for measurement of bacterial adherence and phagocytosis.

We have developed a new two-color fluorescent method for the quantitative measurement of adherence and ingestion of Streptococcus pneumoniae by human monocytes. The method employs a fluorescent naphthalimide, Lucifer Yellow VS, that has been covalently linked to the bacterial cell wall. Bacteria were opsonized and allowed to adhere to monocytes. Lucifer Yellow did not alter the bacterial interaction with complement in serum or with the phagocytic cell. The ability of monocytes to ingest the adherent bacteria was tested under a variety of conditions. Rabbit antibody to Lucifer Yellow derivatized with Texas Red was used to detect monocyte-bound, but uningested bacteria. Dual laser flow cytometry simultaneously quantitated the total number of monocyte-associated S. pneumoniae and the number that remained surface adherent. This method allows separate analysis of the opsonins and receptors involved in bacterial adherence to phagocytes and in the ingestion process.

The Role of Complement in Host Resistance to Bacteria

Some of the earliest observations that led to our current understanding of the function of the complement system were made just before the turn of the century when it was noted that fresh blood had the power to kill certain types of bacteria [3, 17]. Analysis of the bactericidal activity of blood led to the recognition of factors called into play by the presence of specific antibodies to the bacterial surface that were responsible for the lytic of bactericidal event. Because early investigators thought that this activity occurred only after the binding of antibody, it was called complement. More detailed analysis of these factors elucidated the protein components active in the “classic” complement pathway, or cascade, and later that in the “alternative” pathway. As our understanding of this system developed, it became clear that complement plays a major role in protecting the individual from overwhelming bacterial infection and participates in both “specific” and “nonspecific” immunity. Complement mediates its effects through at least two important mechanisms. The first to be recognized was the bactericidal reaction, that is, the direct complement killing of microorganisms; later, it became clear that complement proteine act as opsonins, coating bacteria with protein fragments which are recognized with great specificity by phagocytic cells and which facilitate the phagocytic process.

Interaction of desialated guinea pig erythrocytes with the classical and alternative pathways of guinea pig complement in vivo and in vitro.

We examined the fate of desialated autologous erythrocytes injected intravenously into guinea pigs (GP). Desialated GP erythrocytes (E) were lysed directly or cleared by the reticuloendothelial system in normal GP (NIH-GP) and cleared by the reticuloendothelial system in GP genetically deficient in the classical complement pathway component C4 (C4D-GP), which activate complement only via the alternative pathway. Desialated E were also cleared in cobra venom factor-treated GP (CVF-GP), which had less than 1% of normal C3 levels, but were not cleared at all in C4D-CVF-GP. Preinjection of asialoorosomucoid (ASOR) and ovalbumin (OVA) had no effect on the rate of E clearance. These in vivo studies indicated that complement activation is essential for clearance of desialated E and that clearance is unaffected by blockade of galactose or mannose receptors. Inhibition of complement-mediated clearance required blockade of both classical and alternative complement pathways. In vitro studies showed that lysis of desialated E could occur in NIH-GP serum (GPS) but not in C4D-GPS. Surprisingly, CVF-GPS also caused lysis of desialated E. Lysis was dependent on both natural antibody to desialated E and classical pathway activation; natural antibody was of both the IgG and IgM classes. C3 uptake studies demonstrated that almost 10 times as many C3 molecules/E were deposited by NIH-GPS as by C4D-GPS or CVF-GPS onto desialated E. Approximately equal numbers of C3 molecules were deposited by CVF-GPS, which did lyse desialated E, and by C4D-GPS, which did not. We suggest that the molecular mechanism of in vivo clearance and in vitro lysis of desialated E by CVF-GP is via classical pathway deposition of C3b into sites on the erythrocyte surface protected from inactivation by H (beta 1H) and I (C4b/3b inactivator). Deposition of C3b into these sites by alternative pathway activation is sufficient to cause clearance but not lysis of desialated E. CVF-GPS may not represent an adequate reagent for testing the complement dependence of various biologic phenomena, particularly if the question involves surfaces that can provide protected sites for C3b molecules.