Joan R Urmson

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.

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.

743 UTILIZATION OF PROPERDIN IN CHILDREN WITH VASCULITIS AND MILK ALLERGY

Patients with various types of vasculitis have serologic findings which indicate that properdin is consumed during disease activity. In all of 22 patients with documented Henoch Schönlein Purpura, the level of serum properdin was reduced more than 3 standard deviations below the mean in the first week of the disease. Patients with predominant signs of abdominal pain and glomerulonephritis had more pronounced reductions in serum properdin than did those patients with arthritis as a presenting symptom. Serum levels of properdin convertase were also reduced in 12 of the 22 patients. As the disease subsides, levels of properdin and properdin convertase return to normal. All other serum complement components and alternative pathway proteins were normal. Similarly, six patients with nonspecific vasculitis had isolated reductions in serum properdin which correlated well with disease activity. Of great interest is the fact that 3 patients with milk allergy also had isolated low levels of serum properdin. The level of properdin returned to normal on a milk-free diet and fell on challenge with milk. In addition, these latter patients demonstrate a functional inadequacy of the alternative pathway as judged by incomplete C3-C9 consumption on the addition of zymosan to their serum. These findings suggest that properdin and properdin convertase may be involved in those disorders effecting vascular inflammation.

1105 EVIDENCE THAT IMMUNE COMPLEX GLOMERULONEPHRITIS ASSOCIATED WITH INDOLENT BACTERIAL SEPSIS MAY LEAD TO PROGRESSIVE RENAL DISEASE

Glomerulonephritis (GN) associated with subacute bacterial endocarditis (SBE) or an infected ventricular jugular shunt (IVJS) is due to the development of immune complexes and complement fixation via the classical pathway. This process is thought to be self limiting once the infection is eradicated. Two children, one with SBE and one with IVJS, developed membranoproliferative glomerulonephritis (determined by biopsy) accompanied by azotemia, hematuria, proteinuria and hypocomplementemia (C1, C4, C2, C3). Serum levels of properdin, but not factor B, were also markedly depressed. After elimination of infection by antibiotics (and removal of the V-J shunt in the patient with hydrocephalus), C3 levels rapidly returned to normal. Serum levels of C4 and P, however, remained low for 6 months suggesting continued complement involvement. Hematuria, proteinuria, reduced GFR and hypertension also persisted. Both patients were normocomplementemic by 9 months but even after 2 years had residual proteinuria and hypertension. These data suggest that complement fixation may occur for long periods in the absence of new immune complex formation. During that time, glomerular inflammation apparently continues and may eventually result in irreparable renal damage. (Supported in part by USPHS grant #AI 12721 and New York State Health Research Council grant #352.)

742 IMPLICATIONS CONCERNING THE COMPLEMENT SYSTEM IN A PATIENT WITH GLOMERULONEPHRITIS AND IMMUNOCHEMICAL FINDINGS OF HEREDITARY ANGIONEUROTIC EDEMA

An 11-year-old white female with focal glomerulonephritis was found to have an absence of functional Cl esterase inhibitor, 5% of normal levels of serum C2 and C4, as well as evidence of circulating, active C1 esterase. C1 esterase inhibitor measured by immunochemical means, however, was only reduced by 25%. Several family members had similar findings. Neither the patient nor her family had clinical signs of hereditary angioneurotic edema (HANE) despite continued and persistent complement consumption for at least 18 months. These data suggest that mechanisms controlled by C1 esterase inhibitor, other than those related to complement, might be responsible for the clinical features of HANE. In addition, evidence from immunofluorescent studies of the deposition of IgG and C3 on the kidney of this patient suggests the participation of an immune complex and terminal complement activity in the genesis of the glomerulonephritis. The prolonged and marked depression of C2 and C4, however, precludes any utilization of the classical pathway in that process. Further, it would appear that the hypocomplementemia antedated the onset of the nephritis and, therefore, may be a predisposing factor in the persistence of the immune complex and its presumed activity via the alternative pathway.

686 COMPLEMENT-MEDIATED DESTRUCTION OF LYMPHOCYTES AND LYMPHOBLASTS: INHIBITION IN PATIENTS WITH ACUTE LYMPHOBLASTIC LEUKEMIA (ALL)

Previous data have shown that sera from patients with ALL contain a material which will block the activation of C3 by both the classical and alternative pathways. An in vitro assay was established to test whether or not this “inhibitor” will prevent complement-mediated killing of normal peripheral lymphocytes (L) or bone marrow-derived malignant lymphoblasts (LB). This assay utilizes L or LB as a target cell, antilymphocyte globulin, and serum; cell death is measured by the uptake of 5% eosin. With normal human serum (NHS), the extent of killing of L or LB depends upon the concentrations of target cells, antibody, and serum as well as the time of incubation at 37° C. Using optimal conditions with ALL sera or with NHS to which purified “inhibitor” has been added, the average killing was decreased by 64%. Increasing Ab concentration two fold, serum concentration five fold or the time of incubation three fold did not completely correct the defect with ALL sera. This “inhibitor” is present in NHS and appears to function by modulating the biologic activity of C3. At diagnosis or relapse, patients with ALL may be deregulated in the elaboration of this material. The resultant excess of the “inhibitor” apparently leads to blockade of the effects of antibody and complement in eliminating tumor cells.

676 ACTIVITY OF THE ALTERNATIVE PATHWAY OF COMPLEMENT (AP) IN THE NEWBORN

Cord sera (CS) from 94 normal newborns were studied. Concentrations of properdin convertase, never before measured in the neonate, were at adult levels. In accord with other studies, serum levels of C3, properdin, factor B and C3-C9 activity were markedly depressed. Despite these low levels, however, CS was able to support complete activation of its own AP when incubated with zymosan or cobra venom factor. Thus, formation and stabilization of sufficient factor B-containing enzyme can occur to mediate complete consumption of that amount of C3-C9 contained in CS. This latter process, however, does not result in effective lysis of a target cell. Nearly 75% of CS had rabbit erythrocyte CH50 titers more than 2 S.D. below the mean for adult sera. This deficiency is only partially corrected by the addition of excess purified C3-C9 to CS. Addition of factor B and P is more effective but reconstitution with all of the above is necessary to achieve maximal lysis. These data suggest that although the AP of the neonate is completely intact, its activity is suboptimal. Further, although the entire pathway is limited, the major deficit appears to be in the ability to generate an adequate number of stable and active enzymatic sites on a target cell membrane.

336 UTILIZATION OF THE ALTERNATIVE PATHWAY OF COMPLEMENT IN PATIENTS WITH THYROID DISEASE: PRESUMPTIVE EVIDENCE AGAINST A ROLE FOR ANTIBODY

Complement studies were performed on 21 patients, 2 months to 16 years of age, with various types of thyroid disease. Circulating antithyroglobulin or antimicrosomal antibodies were present in 12/21 (titers > 1:40). All sera were quantitated for C4 (classical pathway); factor B, properdin and properdin convertase (alternative pathway); C3 and C3-C9 activity (both path-ways). In addition, functional assays were done by determining the extent of complement activation by an immune complex (classical pathway) or by zymosan, cobra venom factor, or rabbit erythrocytes (alternative pathway). Abnormalities were found in 16 patients, 11/12 with antithyroid antibodies and 5/9 without detectible antibodies. All but one of the abnormalities occurred in the alternative pathway. These consisted primarily of low serum levels (> 3 SD below the mean for age) of factor B and properdin or ineffective function with CoF. Such abnormalities suggest in vivo utilization of this pathway. No association, however, exists between the titers of antibody and any abnormality. Since C3 is deposited on the thyroid gland in many of these disorders, it would appear that it is the alternative pathway which is responsible for that deposition. Further, since antibody is not usually associated with alternative pathway activity, its role in that process is open to question.