Ann E. Stitzel

Activity of the alternative pathway of complementin the newborn infant

Levels of C3, properdin, factor B, and C3 to C9 activity were markedly reduced in cord sera taken from 94 normal newborn infants. Nevertheless, cord serum supported complete activation of its own alternative pathway by zymosan or CoF. Lysis of a target cell, however, was defective; nearly 75% of cord sera had reduced rabbit erythrocyte CH50 titers. These were partially increased by the addition of factor B and properdin, and totally restored by adding factor B, properdin, and C3 to C9. Therefore, although the alternative pathway of the neonate is intact, it appears to be limited in its ability to generate an adequate number of stable and active enzymatic sites on a target cell membrane.

Evidence that production of autoantibody to the alternative pathway C3 convertase is a normal physiologic event

The origin of autoantibody production was studied with the use of antibody to the alternative pathway C3 convertase (C3 nephritic factor (C3NeF), as a model. Pokeweed mitogen stimulation of peripheral mononuclear cells from newborn infants, normal adults, and patients with membranoproliferative glomerulonephritis indicated that the ability to make C3NeF is apparently present in everyone from the time of birth. In addition, C3NeF appeared to express a single or very limited idiotope (21/21 isolates). The data also suggest that the elaboration of C3NeF may approximate an antibody response after immunization. Thus the C3NeF fraction of the total IgG or IgM produced in culture by pokeweed mitogen-stimulated mononuclear cells from normal neonates and adults, as well as from patients, was in the range of the production of specific antibody. Further, both IgG and IgM C3NeF produced by cells from these normal individuals, including newborn infants, had an affinity for antigen (10(8) to 10(9) L/mol) that was also in the range of specific antibody. Most of the autoantibody molecules (5/7) from serum were IgG3; two B cell clones producing C3NeF were CD5-negative. These experiments indicate that unmutated germline genes are used in the production of C3NeF and that a limited spectrum of antiidiotypic antibodies regulate its production.

Production of IgG and IgM autoantibody to the alternative pathway C3 convertase in normal individuals and patients with membranoproliferative glomerulonephritis

To understand the origin of autoantibody production, we studied the ontogeny of antibody to the alternative pathway C3 convertase (C3 nephritic factor or C3NeF). Peripheral blood mononuclear cells from newborns, normal adults, and patients with membranoproliferative glomerulonephritis produced IgM and IgG C3NeF after culture for 14 days with pokeweed mitogen. Both IgM and IgG moieties appear to have the same paratope and are able to inhibit each others binding and function. The affinity constant for each of the C3NeF molecules was moderately high (10(8) liters/mol) and there appeared to be little difference between the Ka values for the IgG and the IgM autoantibodies or between Ka values for autoantibodies isolated from newborns, adults, and patients. These data, then, indicate that the ability to produce C3NeF autoantibody is present from the time of birth in normal individuals. The high affinity of these autoantibodies under normal conditions suggests that C3NeF may play a more important physiological role than previously anticipated.

On the origin of C3 nephritic factor (antibody to the alternative pathway C3 convertase): Evidence for the Adam and Eve concept of autoantibody production

The antibody to the alternative pathway C3 convertase, designated C3 nephritic factor or C3NeF, is an autoantibody that is produced in everyone from the time of birth. The elaboration of C3NeF utilizes germline V-region genes which undergo antigen-driven affinity maturation, resulting in an autoantibody that is produced in large amounts with high affinity and narrow specificity. Our data also suggest that under normal conditions, the idiotypic network may play an important part in the control of this autoantibody. Further, a defect in the network with loss of control or inappropriate stimulation may be an underlying mechanism in the unrestricted production of C3NeF in patients with membranoproliferative glomerulonephritis.

Alteration of the complement system in children with Henoch Schönlein Purpura

Abstract The complement system was studied in detail in 24 patients with Henoch Schonlein Purpura. Serum levels of C3 and C3–C9 hemolytic titers were normal or elevated. Classical pathway activity was determined by quantitation of serum C1, C4, and C2 titers as well as by the ability of serum to support C3–C9 consumption with an immune precipitate and lysis of sensitized erythrocytes (CH 50 ). All of these studies were normal. Alternative pathway activity was determined by measurement of serum levels of properdin, properdin convertase, and factor B as well as the ability of serum to support C3–C9 activation with zymosan or cobra venom factor and the lysis of unsensitized rabbit erythrocytes. Only the serum levels of properdin and properdin convertase were abnormal. In 11 of 12 patients studied within the first week of the disease, levels of serum properdin (P) were reduced more than 3 SD from the normal mean; similarly, 5 of 12 patients had reduced serum levels of properdin convertase (PC). In addition, of five patients whose initial blood was obtained between the first and second week of the disease, four had low levels of either P or PC. Finally, seven samples were obtained 14 days or more after onset; four had low serum levels of P or PC and three of these were patients assayed from 1–5 years after onset. The length of time required for the P or PC to return to normal was extremely variable.

Decreased opsonic and chemotactic activities in sera of postburn patients and partial opsonic restoration with properdin and properdin convertase

Abstract Defective phagocytic functions may play a prominent role in the susceptibility of burn patients to bacterial infections. Serially collected sera from 12 patients in this study, with 1° to 3° burns of between 20 and 85% body surface, showed significantly depressed opsonic and chemotactic activities for prolonged periods. Sera from 3 of the 12 patients were assayed for C 3 , C 4 , C 3 -C 9 hemolytic activity, factor B, and properdin and properdin convertase. All factors were decreased initially, but quickly returned to normal, except properdin and properdin convertase which remained at levels of less than 50% of normal for prolonged periods. Significant restoration of yeast opsonic activity by the addition of either purified properdin or properdin convertase to opsonically deficient sera suggests that a defect of the alternate pathway of complement activation is present in patients with burns.

Interaction of properdin convertase and properdin in the alternative pathway of complement activation

Abstract When activated properdin convertase ( PC ) is added to normal human serum, properdin is altered as manifested by a shift in electrophoretic mobility and a decrease in the amount of protein measurable by radial immunodiffusion. The degree of alteration is dependent on the concentration of PC and the time of incubation. Thus, PC at a concentration of 5 × 10 −2 μg/ml will interact with serum properdin in 5–10 min at 37°C. Subsequent to this interaction, 95 per cent of serum C3–C9 hemolytic activity is consumed. This consumption appears to be mediated by the generation of two distinct enzymatic specificaties, one for C3 and one for C5. The consumption of C7 requires the prior activation of C5 either directly or via C3. Both the C3 and C5 reactivity are independent of C142 or factor B activation. Nevertheless, factor B can be activated subsequent to these fluid phase interactions, presumably those involving C3 alteration. Activation of factor B in this fashion requires factor D but is relatively inefficient and may reflect a change in C3 other than cleavage and formation of C3 b . In the presence of zymosan, properdin convertase mediated C3–C9 consumption is blocked. The extent of this inhibition depends on the relative amounts of PC and zymosan; the site of inhibition appears to involve the interaction of PC altered properdin and C3.

Hereditary angioneurotic edema: immunochemical 'activity' without clinical expression.

An 11-year-old white female with focal glomerulonephritis was found to have an absence of functional C1 esterase inhibitor. C1 esterase inhibitor measured by immunochemical means, however, was only slightly reduced. After an initial time period marked by variable hypocomplementemia, presumably due to immune complex formation associated with the nephritis, immunochemical signs of active and severe hereditary angioneurotic edema (HANE) developed. These have been unremitting for 3 1/2 years. Clinical signs of HANE, however, have never developed. Thus, during this time, there have been no clinical abnormalities despite the fact that free C1 esterase activity has been persistently present in this patients serum and serum levels of functional C1 esterase inhibitor, C2 and C4 have been continuously less than 2% of normal. It appears, therefore, that this patient has an unusual form of HANE mainifested solely by the complement alterations seen during symptomatic attacks but without clinical expression of that serologic activity.

Inhibition of the alternative pathway of complement activation by a serum factor generated during transplant rejection.

Abstract During rejection of an allograft, a T-cell dependent factor is generated which appears in the serum and is deposited on the graft. This factor is capable of inhibiting the alternative pathway of complement activation; thus, in the presence of this material, the rate and extent of C3–C9 hemolytic consumption is markedly diminished when zymosan, C3NeF, C3b or CoF are added to normal human serum. The inhibitor appears to function by blocking the activation of C3 thereby preventing the conversion of factor B by factor D as well as the more terminal C3 utilization by activated factor B. With zymosan, both sites are involved; with CoF, factor B activation is unimpaired, but its effect on C3 is blocked.

Components of the Alternative Pathway of Complement in Otitis Media with Effusion

An evaluation of the alternative pathway of complement was undertaken in patients with otitis media with effusion (OME). Middle ear fluid (MEF) and serum specimens were obtained from 34 patients at the time of elective myringotomy for OME. Bacterial, viral, and mycoplasma cultures were made for all specimens of the fluids. Immunochemical determinations by radial immunodiffusion were performed for C3, C5, factor B, properdin, factor H, factor I, and albumin. Each patients recent clinical course and past history were reviewed. The results of all viral and mycoplasma cultures were negative. Three of 55 bacterial cultures were positive for type B Haemophilus Influenzae. All components of the alternative pathway measured were found to be present in varying amounts in MEF. When the levels of the complement components were compared to the clinical factors studied, there were no observable differences. These data suggest that components of the alternative pathway Of complement are present in OME and are not useful in predicting the clinical course or outcome of this disorder.