Human lymphocyte complement receptors. Quantitative requirements for C3 of normal and chronic lymphocyte leukemia lymphocytes.

Erythrocytes coated with varying amounts of human complement were used to detect lymphocytes with complement receptors from normal subjects and patients with chronic lymphocytic leukemia. The relationship between the percentage of lymphocytes rosetting and the quantity of C3 present on complement-coated erythrocytes were studied. Small quantities of C3 (less than 5 fg/erythrocyte) caused maximal rosetting of normal lymphocytes. Maximal rosetting with chronic lymphocytic leukemia lymphocytes was not reached until much greater amounts of C3 were used to coat the erythrocytes. This difference in sensitivity to erythrocyte-bound complement was not due to an increased fraction of complement receptor-bearing cells in the leukemic patients. This loss of sensitivity of the chronic lymphocytic leukemia lymphocyte for complement may play a role in the immune deficiency present in this disease.

Mechanisms of immune lysis of red blood cells in vitro. I. Paroxysmal nocturnal hemoglobinuria cells.

The effect of five different reactions which activate complement (antibody activation, reduction in ionic strength, acidification, cobra venom factor (CoF) activation, and inulin activation) upon normal and PNH cells was investigated, using normal serum and serum devoid of the fourth component of complement (C4) activity from patients with hereditary angioneurotic edema (HANE) as a source of complement. Both normal and HANE serum lysed paroxysmal nocturnal hemoglobinuria (PNH) cells when complement was activated by acidification, CoF and inulin, indicating that the terminal steps of complement were activated in the absence of C4, presumably by the alternate or properdin pathway. Normal but not HANE serum lysed cells coated with anti-I, indicating that complement was activated by the C1-dependent classic pathway. HANE serum partially supported lysis by serum at reduced ionic strength, indicating that the activation of terminal complement components had occurred through both of the pathways of activation. The amount of the third component of human complement (C3) which was bound to the membrane of lysed and unlysed cells by these procedures was determined by anti-C3 absorption and was found to differ for each method of complement activation. In general, more C3 was bound to lysed cells than to unlysed cells. For given conditions, more was bound to PNH cells than to normal cells. However, very much less bound C3 was required for lysis of the PNH cells than for lysis of normal cells. These two phenomena, especially the latter, account for the marked lysis of PNH cells when complement is activated. Normal cells treated with AET (aminoethylisothiouronium bromide) did not bind more C3 than untreated cells and the lysed cells had less bound C3 than lysed PNH cells.

Measurement of the third component of complement bound to red blood cells in patients with the cold agglutinin syndrome.

The amount of the third component of complement (C3) bound to red cells of patients with the cold agglutinin syndrome was determined by a quantitative assay, measuring the fixation of the first component of complement by anti-C3. Abrupt reduction in the serum concentration of cold agglutinin by plasmapheresis markedly decreased the hemolytic rate, but the amount of C3 bound to circulating cells did not change appreciably. When this patient was transfused with normal cells. C3 accumulated on the transfused cells within 48 h to the level present on his own cells, but selective destruction of the transfused cells did not occur. When patients were subjected to acute cold stress, cell-bound C3 rose abruptly and intravascular hemolysis occurred. These studies suggest that most of the C3 detected on the circulating red cells of cold agglutinin patients is in an inactive form, and that the rate of attachment of C3 to the membrane is important in determining hemolysis.

Quantitative influence of antibody and complement coating of red cells on monocyte-mediated cell lysis.

Monocyte-mediated lysis in vitro of human red cells coated with measured amounts of immunoglobulin G (IgG) or complement were studied. 1,000-1,500 molecules of IgG anti-D are necessary to effect measurable lysis, and lysis increases linearly with increasing levels of antibody sensitization. 100 microgram/ml of IgG1 abolished lysis even at maximal levels of anti-D sensitization (15,000 molecules/cell). Two isoimmune IgG anti-A or anti-B antisera were 5 to 10-fold less efficient in promoting phagocytosis or lysis per molecule of IgG bound; however, because of the greater antigen density of A or B, more than 100,000 molecules IgG/cell could be bound, producing equivalent lysis to anti-D-coated cells. Although inhibition by IgG1 was similar at equivalent levels of sensitization with anti-A, anti-B, or anti-D at high levels of coating with anti-A or anti-B (not attainable with anti-D), lysis was not inhibited by IgG1. Cells coated with human complement components alone were not lysed by monocytes; however, complement coating augmented IgG-mediated lysis and reduced the quantity of anti-D necessary to produce lysis to less than 1,000 molecules/cell. After thorough degradation of C3b by serum to C3d, complement augmentation persisted.

Activation of human complement by immunoglobulin G antigranulocyte antibody.

The ability of antigranulocyte antibody to fix the third component of complement (C3) to the granulocyte surface was investigated by an assay that quantitates the binding of monoclonal anti-C3 antibody to paraformaldehyde-fixed cells preincubated with Felty's syndrome serum in the presence of human complement. The sera from 7 of 13 patients with Felty's syndrome bound two to three times as much C3 to granulocytes as sera from patients with uncomplicated rheumatoid arthritis. The complement-activating ability of Felty's syndrome serum seemed to reside in the monomeric IgG-containing serum fraction. For those sera capable of activating complement, the amount of C3 fixed to granulocytes was proportional to the amount of granulocyte-binding IgG present in the serum. Thus, complement fixation appeared to be a consequence of the binding of antigranulocyte antibody to the cell surface. These studies suggest a role for complement-mediated injury in the pathophysiology of immune granulocytopenia, as has been demonstrated for immune hemolytic anemia and immune thrombocytopenia.

Mechanisms of immune lysis of the red cells in hereditary erythroblastic multinuclearity with a positive acidified serum test and paroxysmal nocturnal hemoglobinuria.

The red cells of patients with hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS), a form of congenital dyserythropoietic anemia, and the cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are lysed more readily than normal cells by certain antibodies, notably cold agglutinins (anti-I) and complement. With some but not other examples of anti-I, HEMPAS and PNH cells adsorbed more antibody than normal cells. Equal quantities of adsorbed antibody bound equal quantities of the first component of complement (C1) to normal, PNH, and HEMPAS cells. However, for a given quantity of bound antibody and C1, much more of the fourth component of complement (C4) was bound to HEMPAS cells than to normal cells. This resulted in the binding of proportionately larger quantities of the third component of complement (C3) to these cells. The same amount of bound C3 was found on the membranes of normal and HEMPAS cells for a given degree of lysis. Hence, the marked increase in lysis of HEMPAS cells is due to the increased adsorption of antibody and/or increased binding of C4.PNH cells bound the same amount of C4 per bound C1 as normal cells but bound more C3 than normal cells. However, the mean concentration of C3 on the membrane of PNH cells was one-third to one-fifth that on normal cells for a given degree of lysis. Hence, the increased lysis of PNH cells is due to the increased binding of C3 and increased hemolytic effectiveness of the bound C3.

Recurrent thrombocytopenia following idiopathic thrombocytopenic purpura. The importance of platelet-bound IgG in establishing cause.

A 20-year-old man experienced two separate thrombocytopenic illnesses. The first episode represented classic idiopathic thrombocytopenic purpura (ITP) and was associated with elevated platelet-bound IgG values. Adequate control of thrombocytopenia could not be obtained with prednisone therapy, and splenectomy produced a clinical remission. Four weeks after splenectomy, an acute febrile illness typical of cytomegalovirus (CMV) infection developed, and CMV grew grom a sample of the patient's blood. Thrombocytopenia recurred during the CMV infection but was not associated with elevated platelet-bound IgG levels. Since the second episode of thrombocytopenia was associated with normal amounts of platelet-bound IgG, it was not ascribed to relapse of the ITP, and the thrombocytopenia resolved rapidly, without specific therapy. There are various therapeutic implications of an accurate causative diagnosis of thrombocytopenia.

Flecanide-induced immune neutropenia. Documentation of a hapten-mediated mechanism of cell destruction.

Immune-mediated granulocytopenia due to cardiac antiarrhythmic medications is a rare, but potentially dangerous, event. This article characterizes the first case, to our knowledge, of severe granulocytopenia associated with the administration of flecanide acetate, a new class I antiarrhythmic drug. Immunologic studies determined that flecanide was capable of binding to the surface of normal neutrophils. The patient's serum contained an IgG antibody that could specifically bind to the haptenized neutrophils, presumably mediating enhanced destruction of mature granulocytes both in the serum and within the bone marrow. Cessation of flecanide therapy resulted in resolution of the granulocytopenia. The titer of antineutrophil antibody in the patient's serum decreased to background levels within the next five months. Similar antibodies were not found in serum from nonsensitized individuals. The capacity of flecanide to bind to normal neutrophils may prove to be a significant risk factor for the subsequent development of antineutrophil antibodies and agranulocytosis in patients receiving this drug. Careful hematologic monitoring of all patients who are receiving this medication is, therefore, strongly urged.

Autoimmune neutropenia in Hodgkin's disease.

A patient was profoundly neutropenic at the time of diagnosis of stage IIIB Hodgkin's disease. The neutropenia was not due to infection or bone marrow involvement by tumor. It did not respond to discontinuation of medication or to splenectomy, done for pathologic staging of Hodgkin's disease. The patient's serum contained abnormally increased granulocyte-binding antibody, which reacted with his own cells. The neutropenia resolved with high-dose prednisone therapy, and has not recurred after chemotherapy. Thus, immune neutropenia--as well as autoimmune hemolytic anemia and immune thrombocytopenic purpura--can be associated with Hodgkin's disease. Recognition and treatment of such immune processes assume major importance in planning cytotoxic therapy for the underlying malignancy.

Felty's syndrome without splenomegaly.

A woman with chronic rheumatoid arthritis and severe granulocytopenia but without splenic enlargement by physical examination or radionuclide scanning was studied for granulocyte-bound immunoglobulin G (IgG) and serum antigranulocyte antibodies. Prior to splenectomy 73 to 110 X 10(-14) g/cell of IgG were detected on the patient's granulocytes, a value in the range (20 to 220 X 10(-14) g) found in 16 patients with classic Felty's syndrome. Granulocyte-bound IgG in 21 patients with rheumatoid arthritis without Felty's syndrome was less than 20 X 10(-14) g. Following splenectomy, the patient had a partial correction of her peripheral granulocyte count, and granulocyte bound IgG was repeated less than 20 X 10(-14) g/cell. When paraformaldehyde-fixed granulocytes, obtained either from normal donors or from the patient after splenectomy, were incubated in the patient's serum obtained before splenectomy, more IgG was bound than with control serums from patients with rheumatoid arthritis. Similar results were obtained when serums from patients with classic Felty's syndrome were incubated with paraformaldehyde-fixed granulocytes. Thus, patients with rheumatoid arthritis without overt splenic enlargement may have pathophysiologic Felty's syndrome, and in vitro studies such as these may be used to define this process.

Complement-activating antineutrophil antibody in systemic lupus erythematosus.

Serum samples from 18 patients with systemic lupus erythematosus (SLE) were tested for neutrophil C3-fixing ability and neutrophil-binding lgG by the binding of radioiodinated monoclonal anti-C3 antibody and staphylococcal protein A to paraformaldehyde-fixed allogeneic neutrophils sensitized with serum. Serum from patients with SLE resulted in the binding of significantly greater amounts of lgG to neutrophils than normal serum, but this lgG binding did not correlate with the degree of neutropenia. In contrast, serum samples from 10 neutropenic patients with SLE resulted in the binding of significantly greater amounts of C3 to neutrophils when compared with serum samples from eight non-neutropenic patients with SLE. Fixation of C3 to neutrophils by serum from patients with SLE appeared to be due to the binding of complement-activating monomeric antineutrophil lgG autoantibody. A significant negative correlation (r = -0.78) between the neutrophil count and the C3-fixing ability of serum from patients with SLE suggested that antineutrophil antibody-mediated activation of complement may be important in the pathophysiology of neutropenia in SLE.

Idiopathic neutropenia: antineutrophil antibodies and clinical correlations.

PURPOSE: The present study was done to evaluate the clinical characteristics of a large series of adult patients with chronic idiopathic neutropenia, and correlate the presence of antineutrophil antibodies, their class (IgG or IgM), and their ability to fix complement with clinical parameters, including other hemocytopenias, splenomegaly, and infections. PATIENTS AND METHODS: One hundred twenty-one adult patients with chronic idiopathic neutropenia were studied. Serum neutrophil-binding antibodies were measured using paraformaldehyde-fixed granulocytes (PFGs) from normal volunteers as target cells. 125I-labeled staphylococcal protein A was used to detect IgG antibodies while IgM antibodies were detected by using 125I-labeled mouse monoclonal anti-IgM antibody. Sera containing antineutrophil antibodies were tested for their ability to fix complement on donor PFGs by using 125I-labeled monoclonal antibody to the third component of complement. RESULTS: Of the 121 patients with chronic idiopathic neutropenia, 71 patients had isolated neutropenia, while 50 had neutropenia combined with either anemia and/or thrombocytopenia. Among the 71 patients with isolated neutropenia, there were 51 females (72%), compared with 28 females (56%) among the 50 patients with combined hemocytopenias (p = 0.083). Patients with multiple hemocytopenias were significantly older (p less than 0.01), were more likely to demonstrate splenomegaly (p = 0.001), and may have had more infectious complications. From all the patients, 36% of sera were shown to have antineutrophil antibodies, with a non-significant trend for these to be found more frequently in patients with multiple hemocytopenias. Sera with mixed IgG-IgM antineutrophil antibodies were significantly more likely to fix complement than those with isolated IgG or IgM antibodies, and among the patients with antineutrophil antibodies, complement-fixing antibodies were significantly associated with multiple hemocytopenias. Splenomegaly was significantly associated both with antineutrophil antibodies (p = 0.008) and with infections (p = 0.007). Antineutrophil antibodies were not associated with infections. CONCLUSIONS: Approximately one third of adult patients with idiopathic neutropenia have IgG and/or IgM antineutrophil antibodies demonstrable in their serum. There is a subset of patients with idiopathic neutropenia with multiple hemocytopenias who tend to be older, less likely to show female predominance, more likely to have splenomegaly and infections, and more likely to have antineutrophil antibodies, especially mixed IgG-IgM and complement-fixing antibodies.

Complement activation by heparin-protamine complexes during cardiopulmonary bypass: effect of C4A null allele.

OBJECTIVES: The first objective was to determine the effect of inherited differences in the classic pathway complement protein C4 on complement activation by heparin-protamine complexes in cardiac surgery. Specifically, we hypothesized that patients with heterozygous C4A null phenotype (A0BB), who have decreased amounts of C4A, may have increased complement activation because of reduced clearance of heparin-protamine complexes. The second objective was to determine whether heparin-protamine-induced complement activation correlated with postoperative pulmonary shunt fractions. METHODS: C4 typing was performed by agarose gel immunofixation and crossed immunoelectrophoresis. Complement activation was measured by radioimmunoassay of C3a and C4a before cardiopulmonary bypass, after bypass, and after protamine infusion. Shunt fractions were calculated from blood gases. RESULTS: Of the 79 patients, 18 expressed heterozygous C4A null allele (A0BB), 16 had heterozygous C4B null allele (AAB0), three had homozygous C4B null alleles (AA00), and the rest expressed both C4A and C4B alleles (AABB). Patients with heterozygous C4A null allele had significantly increased plasma levels of C4a after protamine neutralization of heparin (C4a of 2862 +/- 375 ng/ml; mean +/- standard error of the mean) when compared with patients with normal expression of C4 alleles (AABB) (C4a of 1580 +/- 141 ng/ml) or heterozygous C4B null allele (C4a 1526 +/- 208 ng/ml). Pulmonary shunt fractions obtained after the operation correlated with the classic pathway complement activation by heparin-protamine complexes, but not with alternative pathway complement activation during cardiopulmonary bypass. CONCLUSIONS: Patients with heterozygous C4A null phenotype have increased complement activation by heparin-protamine complexes during cardiac operations, possibly because of their defective clearance. The classic pathway complement activation by heparin-protamine interaction correlates with postoperative pulmonary shunt fractions.

Acquired factor VIII inhibitor with squamous cell cancer of the epiglottis.

A 73-year-old woman developed an acquired factor VIII inhibitor in association with squamous cell carcinoma of the epiglottis. The inhibitor was an IgG antibody that reacted with factor VIII in vitro and in vivo. Intravenous gamma-globulin therapy was successful in reducing the inhibitor so that curative surgery could be undertaken. With surgical resection of the tumor the inhibitor did not recur. The relevance of this type of coagulation disorder to the surgical management of the patient's head and neck cancer is discussed.

In vitro activation of human complement by polyacrilonitrile dialyzer membrane assayed by complement binding to bystander red cells.

Quantitation of complement activation by polyacrilonitrile (PAN) dialyzer membrane is complicated by the high adsorptive capacity of the membrane for fluid phase anaphylotoxins. Assays for these anaphylotoxins, therefore, underestimate the degree of complement activation produced by this membrane. Alternative methods of measuring in vitro complement activation by the PAN and Cuprophan membranes were explored by incubating normal human erythrocytes with the membranes in the presence of serum. This led to deposition of C3d on these "innocent bystander" red cells, and provided an independent parameter for measuring complement activation. The PAN membrane caused significantly more C3d deposition on red cells, and thus more complement activation than Cuprophan. The possible significance of complement activation by PAN membrane, in consideration of its property of binding the resultant anaphylotoxins, is discussed.

Neutrophil lactoferrin release during open heart surgery is unrelated to complement activation.

The relationship between neutrophil activation and complement activation during open heart surgery was evaluated by measuring plasma lactoferrin and C3a des Arg (C3a) in 30 adult patients undergoing coronary artery surgery. Measurements were made before induction of anesthesia, before cardiopulmonary bypass, at the end of bypass, and 10 min after protamine infusion. Changes in the measured parameters thus reflected activation during the three distinct phases of cardiac surgery: pre cardiopulmonary bypass, the bypass phase, and the heparin neutralization phase. A major rise in lactoferrin occurred in the pre bypass period, from 99 +/- 13 (mean +/- SEM) to 647 +/- 48 ng/ml (p < 0.001). During this time, C3a levels did not rise significantly (384 +/- 22 to 439 +/- 36 ng/ml). The bypass procedure resulted in a rise of both lactoferrin and C3a levels, with lactoferrin reaching 1,092 +/- 69 ng/ml and C3a increasing to 1,884 +/- 179 ng/ml after bypass (p < 0.001 for both). In individual patients, however, the changes in lactoferrin during bypass did not correlate with changes in C3a levels (r = -0.06). After protamine infusion, C3a levels reached 3,301 +/- 324 ng/ml, while the plasma lactoferrin levels declined to 522 +/- 57 ng/ml. Thus, during open heart surgery, neutrophil activation, as measured by plasma lactoferrin concentration, does not correlate with complement activation resulting from the bypass procedure or the protamine neutralization of heparin. The potential clinical relevance of the neutrophil granular release of lactoferrin is discussed.

Diving decompression fails to activate complement.

The present study evaluated complement activation during decompression after air dives in a hyperbaric chamber. Intravascular bubbles were quantified by Doppler ultrasound scoring. Eighteen subjects completed 92 dives, of which 74 produced bubbles. Complement activation was assessed by plasma C3a des Arg and red-cell-bound C3d before and after each dive. These parameters of in vivo complement activation failed to show significant activation. In vitro complement activation susceptibility tests on pre-dive sera were performed to explore their association with in vivo complement activation and intravascular bubbles. Such tests failed to identify a distinct complement-sensitive group and did not correlate with in vivo complement activation during the dives and/or intravascular bubble appearance. Two subjects developed decompression sickness but were not different from the rest of the group regarding in vitro complement sensitivity or complement activation during dives.

Effect of heparin on complement activation and lysis of paroxysmal nocturnal hemoglobinuria (PNH) red cells.

The effect of heparin upon the binding of the third component of complement (C3) to PNH red cells in vitro and their subsequent hemolysis is described. Heparin, in increasing concentrations, progressively inhibits membrane C3 fixation and hemolysis when the classic complement pathway is activated by anti-red cell antibodies. Heparin has a biphasic effect upon membrane C3 fixation and hemolysis when complement is activated in serum at decreased ionic strength (sucrose lysis) or in serum at decreased pH (Ham test). Heparin in concentrations above 2 U/ml inhibits C3 binding and hemolysis while lower concentrations of heparin enhance the consequences of complement activation by these two procedures. This enhanced complement activation may explain the increased hemolysis sometimes reported in PNH patients treated with heparin, and suggests that heparin may aggravate the consequences of pathologic alternative pathway complement activation in other diseases.