Blood group A immunodeterminants on human red cells differ in biologic activity and sensitivity to alpha-N-acetylgalactosaminidase.

BACKGROUND: Epitopes of blood group A antigen can be enzymatically cleaved from red cells (RBCs), but the extent of cleavage required for normal survival in allogeneic blood transfusion recipients is unknown. Therefore, the cleavage rates were studied for A antigen epitope binding of 1) complement-activating anti-A, 2) Dolichos biflorus anti-A, lectin, and 3) hemagglutinating anti-A during incubation with a purified alpha-N-acetylgalactosaminidase, E.C. 3.2.1.49 (alpha-GalNAc'ase). STUDY DESIGN AND METHODS: Suspensions of group A RBCs were incubated with alpha-GalNAc'ase. Cells were removed at intervals, washed, and tested for loss of binding by monoclonal, polyclonal, and complement-activating anti-A, D. biflorus anti-A1 lectin, and Ulex europaeus anti-H lectin. RESULTS: A epitopes binding D. biflorus lectin were highly susceptible to alpha-GalNAc'ase; simultaneously with their loss, binding with U. europaeus lectin emerged. Loss of complement-mediated hemolysis was slower. A epitopes binding hemagglutinating anti-A were most resistant. Cleavage of A epitopes from membrane glycosphingolipids with short oligosaccharide chains was similarly resistant. Rates of cleavage from A1 and A2 RBCs were similar. CONCLUSION: RBC epitopes of blood group A differ in susceptibility to cleavage and biologic reactivity, which suggests that subsets mediating important biologic functions exist on functionally and topographically distinct membrane glycoconjugates.

Unique C1 inhibitor dysfunction in a kindred without angioedema. I. A mutant C1 INH that inhibits C1-s but not C1-r.

We have described hereditary incomplete deficiency of the fourth component of complement (C4) in 10 members of a large kindred. C4 deficiency in this kindred is not linked to C4 loci in the HLA region. C4 synthesis is decreased, and C4 catabolism is normal in kindred members with low serum C4 levels. We have discovered a uniquely dysfunctional C1 inhibitor in all C4-deficient members of this kindred. C1 inhibitor dysfunction is revealed by incubating sera of affected members with EDTA, which destroys all C4 activity in these sera, but not in normal sera or sera from individuals with partial C4 deficiencies. The M(r) of C1 inhibitor purified from affected members is normal, but approximately 50% of this C1 inhibitor resists cleavage by trypsin (0.14 microM) at arg444, suggesting a substitution at this position. Moderate increases in trypsin, however, result in cleavage of the resistant molecules, which would not be expected if arg444 were the site of the mutation. All molecules in C1 inhibitor purified from affected members' plasma bind to activated C1s (C1-s), but approximately 50% of molecules in these preparations do not bind to activated C1r (C1r). These findings show that affected kindred members have a unique mutation in C1 inhibitor. The mutant C1 inhibitor does not prevent the activation of C1s by C1-r when serum Ca2+ is chelated by EDTA, but its inhibition of C1-s is normal in vivo, as shown by normal C2 levels, normal C4 catabolism, and absence of angioedema in C4-deficient members. The nature of the mutation, its selective failure to inhibit C1-r, and its relationship to decreased C4 synthesis remain to be defined.

Serum IgG antibodies to C1q in hypocomplementemic urticarial vasculitis syndrome.

Urticaria, angioedema, and arthritis are cardinal features of hypocomplementemic urticarial vasculitis syndrome (HUVS). Considered to be an immune complex-mediated disorder, HUVS has been differentiated from systemic lupus erythematosus (SLE), based on its clinical manifestations and the C1q precipitin (C1q-p) reaction, which is manifested as gel precipitation of C1q by a small percentage of HUVS IgG molecules. This phenomenon has been attributed to an Fc region abnormality, and the responsible IgG molecules are said to possess C1q-p activity. We purified IgG from 4 HUVS patients and confirmed that HUVS IgG contains C1q binding activity. F(ab')2 fragments from these patients also bound to C1q, as measured by 2 different C1q binding methods at physiologic ionic strength; HUVS IgG Fc fragments did not bind to C1q. Preincubation of HUVS F(ab')2 fragments with antibody to human F(ab')2 prevented subsequent binding to C1q. We conclude that IgG antibodies to C1q are present in HUVS serum, and it is likely that these antibodies are C1q-p. Because the clinical manifestations of HUVS and the presence of anti-C1q antibodies have been described in patients with SLE, our findings support the concept that HUVS is an autoimmune syndrome related to SLE.

Complement-mediated hemodynamic depression in the early postburn period.

This study examines the influence of complement on systemic hemodynamics following severe thermal injury in rats. Animals were injected intraperitoneally at t = -36 and t = -24 hours with either cobra venom factor (20 units/kg/dose; n = 56) to delete circulating complement or with saline alone (n = 64). Rats within each subset were then subjected to either a 50% TBSA full-thickness scald burn or sham burn. Cardiac output (CO), mean arterial pressure (MAP), heart rate, systemic vascular resistance (SVR), stroke volume, and cardiac power as well as hematocrit and the change in per cent complement activity were determined at various time periods between 15 minutes and 25 hours after the burn. In normocomplementemic animals the burn produced a marked early (t = 3-6 hours) depression in CO and MAP with a rise in SVR. Over time the hemodynamics returned to normal (t = 12 hours) and eventually approached a hyperdynamic response (t = 24 hours). Serum hemolytic complement activity fell immediately and progressively after the burn, indicating significant complement activation. Complement depletion attenuated the early decline in CO and sharply lowered the rise in SVR in the early postburn period. In addition, complement depletion improved heart rate and stroke volume and appeared to preserve/enhance late (t = 24 hours) cardiac function. This study suggests that complement activation contributes to the depression in cardiac output in the early postburn period.

Visceral perfusion abnormalities following complement activation. Clues to the mediators of organ ischemia in trauma and sepsis. First place winner: Conrad Jobst Award.

Complement, activated during infection and injury, has been implicated as a mediator of microvascular injury and obstruction. This study examines how two potent activators of complement, zymosan, and cobra venom factor (CVF), affect systemic and visceral perfusion. Rats were injected with either saline (1 ml/kg), zymosan (5 mg/kg) or CVF (5 units/kg) at t = 0 and 30 minutes. Thermodilution cardiac output, mean arterial pressure, heart rate, systemic vascular resistance, and hematocrit were determined at t = 2 hours. Effective hepatic and renal blood flows, by clearance of galactose and p-aminohippurate respectively, were determined over the next hour. The per cent change in total hemolytic complement from t = 0 to t = 3 hours was determined by immune hemolysis of sheep erythrocytes. There was no difference in systemic hemodynamic parameters between the three groups. Hepatic blood flow was depressed in both the zymosan (3.83 +/- 0.23 ml/min/100 g) and CVF (3.72 +/- 0.20 ml/min/100 g) groups compared with controls (4.62 +/- 0.19 ml/min/100 g, P less than 0.05). Renal blood flow in the zymosan-treated group (6.40 +/- 0.24 ml/min/100 g) increased over control (4.80 +/- 0.40 ml/min/100 g, P less than 0.05) but was unchanged in the CVF group (5.06 +/- 0.23 ml/min/100 g). The amount of complement activated correlated with the change in hepatic (r = -0.419, P less than 0.05) but not renal (r = -0.008, P = 0.917) flow. Complement activation may occupy a proximal position in the pathogenesis of hepatic ischemia associated with trauma and sepsis.

Contribution of toxic oxygen intermediates to complement-induced reductions in effective hepatic blood flow.

This study examines the effects of complement activation and of complement-induced oxygen radical production on the principal determinant of hepatic function, i.e., effective hepatic blood flow (EHBF). Female Sprague-Dawley rats received cobra venom factor, 40 units/kg, in two divided doses at 30-minute intervals. At t = 2 hours, thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, and EHBF by galactose clearance were determined. Complement activation produced a significant depression in EHBF independent of changes in systemic perfusion. To determine whether oxygen radicals participated in the insult, additional animals were pretreated with superoxide dismutase, 6 mg/kg, plus catalase, 15 mg/kg, immediately before complement activation. Concomitant treatment with the oxygen radical scavengers attenuated the degree of complement-induced hepatic ischemia, again independent of effects on systemic perfusion. This study suggests that the reduction in hepatic blood flow that accompanies animal models of trauma and sepsis may result, in part, from the sequelae of complement activation with oxygen radicals as secondary mediators.

Allopurinol and lodoxamide in complement-induced hepatic ischemia.

Intravascular complement activation with either zymosan or cobra venom factor (CVF) impairs hepatic blood flow. Oxygen radical scavengers given at the time of complement activation attenuate the resulting hepatic ischemia. It is not clear whether complement-stimulated phagocytes or transiently ischemic then reperfused endothelial and parenchymal cells generated the toxic oxygen radicals. In this study, a group of rats were given allopurinol (50 mg/kg/day postoperatively X 3 days plus 100 mg/kg iv at t = 0), a specific inhibitor of xanthine oxidase, prior to complement activation with CVF (20 units/kg iv at t = 30 and 60 min) to determine whether xanthine oxidase-derived oxygen radicals contributed significantly to the hepatic perfusion abnormalities. Additional rats received lodoxamide tromethamine (10 mg/kg iv bolus at t = 0 followed by 20 mg/kg/hr iv infusion), a novel and potent inhibitor of mast cell release and inhibitor of xanthine oxidase, prior to the same CVF challenge to determine whether mast cell mediators were involved in the flow disturbance. Thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, and effective hepatic blood flow (EHBF) by galactose clearance were determined at t = 2 hr. The percentage change in total hemolytic complement activity (% delta CH50) was determined between serum obtained prior to sacrifice and at t = 0. Systemic hemodynamics and HCT were for the most part unaffected regardless of pretreatment group or challenge with CVF or saline. CVF challenge produced a 25% reduction (P less than 0.05) in EHBF in vehicle-pretreated rats compared to saline challenge. Neither allopurinol nor lodoxamide tromethamine significantly improved EHBF when given prior to CVF challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic complement activation produces hemodynamic changes characteristic of sepsis.

Zymosan was administered intravenously in graded doses to Sprague-Dawley rats to investigate the hemodynamic effects of systemic complement activation. At two hours, thermodilution cardiac output, mean arterial pressure, heart rate, systemic vascular resistance, hematocrit, effective hepatic and renal blood flows, and percent change in total hemolytic complement activity were measured on all animals. Progressively increasing doses of zymosan produced characteristic hemodynamic changes of progressively deteriorating stages of hyperdynamic sepsis. In addition, complement activation resulted in a redistribution of systemic blood flow with hepatic hypoperfusion similar to that seen in sepsis. Renal blood flow was unaffected early after complement activation. Additional rats were studied from the control and a representative zymosan-treated group at 24 and 48 hours to determine if the hemodynamic changes observed at two hours persisted or resolved. All systemic hemodynamic measures returned to normal at both 24 and 48 hours. Liver blood flow, however, remained depressed and actually deteriorated over time. Renal perfusion, which was stable at both two and 24 hours, fell below control values in the zymosan-treated group at 48 hours. We conclude that complement may be a mediator of both systemic and visceral flow abnormalities that precede, and perhaps precipitate, organ failure in trauma and sepsis.

Complement activation in peritonitis. Association with hepatic and renal perfusion abnormalities. First place winner: Conrad Jobst award.

The authors have shown that systemic activation of the complement system with either zymosan or cobra venom factor produces some of the hemodynamic changes characteristic of sepsis, specifically, a reduction in hepatic perfusion despite a normal or hyperdynamic systemic circulation. This study was undertaken to determine whether complement activation accompanied reductions in effective hepatic and renal blood flow (EHBF and ERBF, respectively) in a septic murine model previously demonstrated to be associated with flow redistribution. Rats underwent either cecal ligation and puncture (CLP) or sham laparotomy after a baseline blood sample was collected for complement assay. Eighteen hours later, thermodilution cardiac output, mean arterial pressure, heart rate, hematocrit, EHBF by galactose clearance, and ERBF by p-aminohippurate (PAH) clearance were determined. A second blood sample was collected for measurement of total hemolytic complement (CH50) by immune hemolysis of sheep erythrocytes and was compared to the t = 0 sample for calculation of per cent change in CH50. The cardiac output and hematocrit were normal in the CLP group relative to sham. The septic animals were tachycardic and slightly hypotensive, suggesting a diminished systemic vascular resistance. EHBF and ERBF fell dramatically in the septic group despite the normal cardiac output. Residual hemolytic complement activity was reduced to less than 40% of preseptic levels in the CLP group while sham values were no different than baseline, indicating massive complement activation in the septic animals. This study demonstrates an association between complement activation and hepatic and renal perfusion abnormalities in murine peritonitis. Work is underway to establish the temporal relationship between complement activation and visceral flow changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolism of C4 and linkage analysis in a kindred with hereditary incomplete C4 deficiency.

We studied a kindred in which C4 deficiency had been discovered. Unlike families with total absence of C4, in this kindred C4 deficiency was found to be incomplete, autosomal dominant, not caused by null alleles, and not associated with a high incidence of systemic lupus erythematosus. The deficient state was caused by hyposynthesis of C4, not by hypercatabolism. The locus for incomplete C4 deficiency was not closely linked to the major histocompatibility complex. The abnormal autosomal dominant allele is, apparently, rare, and how it causes decreased synthesis of C4 is unknown.

Immune complexes and complement abnormalities in patients with cystic fibrosis. Increased mortality associated with circulating immune complexes and decreased function of the alternative complement pathway.

Serum samples from 139 patients with cystic fibrosis (CF) were tested for complement abnormalities and circulating immune complexes (CIC). We found no consistent changes in whole complement activity. However, we found CIC in 29% of these patients and decreased activity of the alternative complement pathway (ACP) in 36%. During 5 yr of observation, mortality was much higher in patients whose sera contained CIC (p less than 0.001) or decreased ACP activity (p less than 0.01). Of patients with both abnormalities, 31% died; however, no deaths occurred in patients with normal ACP activity and negative tests for CIC (p less than 0.001). During a subsequent 2.5-yr period, 55% of patients greater than or equal to 21 yr old with both findings died. In contrast, no deaths occurred in older patients lacking this combination (p = 0.0062). Circulating immune complexes but not decreased ACP activity were an independent risk factor for death. Our findings support the hypothesis that humoral immune mechanisms may contribute to morbidity and mortality in CF.

Terminal complement component deficiencies and rheumatic disease: development of a rheumatic syndrome and anticomplementary activity in a patient with complete C6 deficiency.

Hereditary deficiencies of early complement components have usually been associated with the development of rheumatic diseases like systemic lupus erythematosus (SLE), while terminal component deficiency is well known to predispose to recurrent neisserial infection. In contrast, only recently have patients been reported with rheumatic disease and hereditary deficiency of a terminal component. The clinical syndrome in these patients has been characterised as 'SLE-like'. We describe here a third patient with complete C6 deficiency and a systemic rheumatic illness characterised by fever, anaemia, lymphadenopathy, hepatosplenomegaly, episcleritis, and asymmetric arthritis. After blood transfusion her serum became anticomplementary; IgG antibody to human C6 was found to be the cause of anticomplement activity. Persistent absence of C6 in this patient and production of anti-C6 antibody after antigenic challenge indicate hereditary C6 deficiency. This case supports an association between hereditary deficiency of a terminal complement protein and the development of systemic rheumatic disease.

Inhibition of zymosan-induced alternative complement pathway activation by concanavalin A.

Zymosan, a polysaccharide composed primarily of glucan and mannan residues, activates the complement system through the alternative complement pathway. We showed that zymosan-induced complement activation is inhibited by zymosan-bound lectins with carbohydrate specificities for mannosyl and glycosyl residues. Lectins unable to bind mannosyl or glucosyl residues did not inhibit zymosan-induced complement activation.

The rabbit properdin system: I. Identification of a new factor and purification of rabbit properdin.

Pure preparations of rabbit properdin were obtained from rabbit serum by ion-exchange chromatography. These preparations functioned as properdin when they were measured with the zymosan assay or with a serum reagent selectively depleted of properdin by a specific immunoabsorbent. Properdin in these preparations was in its activated state. A new serum factor was required to measure properdin activity when purified preparations of rabbit properdin were tested with the zymosan assay. This factor was designated as ZBP, or zymosan-binding protein. ZBP appeared to be distinct from known components of the alternative complement pathway and the classical complement system, and it did not appear to be an immunoglobulin.

Activation of protein mediators of inflammation and evidence for endotoxemia in Borrelia recurrentis infection.

Fifteen patients with Borrelia recurrentis infection were studied to evaluate the role of certain plasma proteins and endotoxin in the pathophysiology of both the acute illness and the Jarisch-Herxheimer-like reaction. The causative spirochetes disappeared from the blood during the Jarisch-Herxheimer-like reaction, which occurred about 2 hours after antibiotic therapy. The mean titers of Hageman factor, plasma prekallikrein and serum hemolytic complement activity were decreased at the time of admission and 2 hours after treatment, and rose to normal values during convalescence. Serum properdin titers were decreased in 14 patients at the time of admission, in 12 patients 2 hours after treatment, and in none during convalescence. The frequency of elevated levels of fibrinogen-related antigens increased from three patients at the time of admission to 12 patients 2 hours after treatment. Results of plasma limulus tests for endotoxin-like material were positive in 11 patients at the time of admission and in 13 patients 2 hours after treatment. These findings demonstrated that Hageman factor, prekallikrein and proteins of the complement system are activated in B. recurrentis infection and that endotoxin may play a role in both the acute illness and in the development of the Jarisch-Herxheimer-like reaction after treatment.