Characterization of alternative pathway inhibition by a serum derived, low molecular weight complement inhibitor.

Normal human serum contains an inhibitor of complement which is distinguished by its small size of 500 daltons, the low molecular weight inhibitor (LMWI). When LMWI was present during incubation of zymosan or cobra venom factor with serum, formation of complement reactive complexes was blocked as measured by failure of these mixtures to lyse susceptible erythrocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH). Addition of LMWI to pre-formed complexes had no effect on their subsequent haemolytic activity. When dialysis was used to remove LMWI from reaction mixtures, it was shown that LMWI had not irreversibly altered any of the complement components. Purified components were used to demonstrate that LMWI prevented factor D activation of cobra venom factor-factor B complexes. LMWI also strongly inhibited binding of 125I-factor B to human erythrocytes bearing C3b and had little or nor effect on binding of 125I-factor H to the C3b bearing cells. Factor B binding to C3b was equally inhibited on normal and PNH erythrocytes. Thus, a dialysable fraction from normal human serum prevents activation of human complement by blocking formation, but not the activity of the C3/C5 convertase. These low molecular weight inhibitors result in inhibition of factor B binding to C3b and inhibition of factor D activation of C3bB complexes.

Interactions of human, cultured kidney cells with the complement system.

When heat-killed, cultured human kidney cells were incubated with normal human serum, complement (C) activation occurred with moderate consumption of C4, C2, C3, and C5 hemolytic activity. No loss of C1 activity and no, or only slight, reduction in C6 activity was detectable until high cell concentrations were reached. C4 and C2 consumption could not be prevented by blocking the primary C pathway through prior EGTA chelation of the serum. Both living and heat-killed kidney cells were incubated with normal serum and examined for surface-bound C components using immunofluorescent techniques. Heat-killed kidney cells were strongly positive for C3, which was distributed in a diffuse, speckled pattern over the entire cell surface. These cells were also weakly positive for IgG and Clq immunofluorescence, but were negative for surface albumin, C5, and beta 1H. In contrast, living cell suspensions showed only occasional cells positive for C3, IgG, or Clq and all cells were negative for albumin, C5, and beta 1H. Viability staining revealed that the few C3 positive cells in living cell suspensions belonged to a small, nonviable subpopulation. These data indicate that dead cells can initiate limited C activation, which can result in binding of C3 to the cell surface.

Isolation and characterization of a low molecular weight complement inhibitor present in normal human serum.

Normal human serum and urine were found to contain a low molecular weight complement inhibitor (LMWI). LMWI was separated from serum by dialysis in membrane tubing or through an Amicon PM10, and then concentrated on an Amicon UM05 membrane. On Bio-Gel P-2 filtration, LMWI was eluted just after the column calibration marker, stachyose hydrate (6666 . 6 daltons), and was estimated to be 500 daltons. Both pathways of complement activation were susceptible to modulation by LMWI. Addition of LMWI reduced the haemolysis of sheep erythrocytes sensitized with antibody, rabbit erythrocytes and guinea-pig erythrocytes bearing human C3 and C4. Formation of EAC142 from EAC14 and guinea-pig C2 was blocked, indicating a failure to generate the classical pathway C3 convertase: however, the lysis of preformed EAC142 was not suppressed. Conversion of factor B and C3 did not occur when LMWI was present during zymosan activation of serum. This indicates that the inhibitor either prevented, or acted at a step prior to, the cleavage of factor B by factor D. LMWI did not prevent formation of erythrocyte C567 intermediates nor their subsequent lysis by C8 and C9. Thus, serum contains a 500-dalton inhibitor which modulates the activities of both complement pathways at an early step in each of the activation sequences. LMWI may serve as a regulator of the inflammatory process by suppressing C3 convertase formation and generation of complement-derived, biologically reactive molecules.

Rhabdomyolysis in a patient with nonoliguric renal failure: similarities to the toxic-shock syndrome.

We have described a 13-year-old girl with idiopathic rhabdomyolysis, myoglobinuria, and nonoliguric renal failure. The biochemical abnormalities and enzyme and isoenzyme values and their interrelationship to the hosptial course are stressed. We believe our patient's condition had marked similarities to the toxic-shock syndrome. The case illustrates the importance of the rapid recognition of myoglobinuria so that its potentially fatal biochemical abnormalities may be expeditiously identified and treated.

Nephritis in children and young adults with systemic lupus erythematosus and normal urinary sediment.

One male and seven female patients (aged 6 to 26 years) with systemic lupus erythematosus (SLE), normal urinalyses, and normal biochemical tests of renal function, had renal biopsies to determine if significant nephropathy existed. Several had active SLE in other body systems at the time, either clinically or as evidenced by low serum complement and high native DNA antibody levels. The renal biopsy specimens were studied by light, fluorescent antibody, and electron microscopy. Three patients had a generalized segmental, two had a focal segmental, and one had a generalized diffuse proliferative glomerulonephritis. In addition, one patient had minimal glomerular findings with interstitial inflammation. All eight patients were found to have moderate immune complex deposition by immunofluorescence and/or electron microscopy studies. The absence of clinical renal involvement in patients with SLE does not preclude ongoing active and "silent" glomerular damage with moderately severe proliferative changes.

Inherited deficiency of the seventh component of complement associated with nephritis. Propensity to formation of C56 and related C7-consuming activity.

A 46-yr-old female with chronic pyelonephritis was found to lack complement (C) activity by the use of hemolytic screen assays in agarose gels. These assays also revealed a propensity of patient serum to form an activated complex of the fifth and sixth components of C, C56. Each of the C component hemolytic activities was present in normal or elevated amounts with the exception of C7, which was undetectable; addition of purified C7 led to the restoration of hemolytic activity. C-dependent phagocytosis, immune adherence, and neutrophil chemotaxis were normal. Family studies demonstrated that the defect was transmitted as an autosomal codominant apparently not linked with alleles at the HLA-A or HLA-B loci. Persisting C56 was readily formed in this as compared to normal serum upon incubation with multiple C activators including zymosan, inulin, immune complexes, heat-aggregated human gamma globulin, endotoxin, and agarose. A heat-stable (56 degrees C, 30 min) activity which consumed C7 with time-and temperature-dependent kinetics was detected in plasma and serum, and seemed to be similar to a "C7 inactivator" previously described in another C7-deficient individual. However, this activity was found to have properties identical to those of C56 during low ionic strength precipitation and chromatography on Sephadex G-200, to be specifically removed upon passage through an anti-C5 immunoadsorbent column, and to be associated with a small amount of C56, suggesting that it represents an expression of small amounts of C56 rather than a new C-inhibitory activity. Thus, an individual with chronic nephritis lacking C7 is reported; the utility of a hemolytic screen assay in agarose plates for the detection of such patients is emphasized; persisting C56 is shown readily to be formed in this serum; and the presence of C7-consuming activity which is associated with and in all likelihood attributable to C56 is shown.

Hereditary deficiency of the third component of complement in a child with fever, skin rash, and arthralgias: response to transfusion of whole blood.

A previously well 34-month-old male presenting with fever, skin rash, and arthralgias was found to lack C3 by immunochemical (undetectable) and hemolytic (1% normal) assays. No infectious agent could be demonstrated. Protein levels of Clq. C4, C5, properdin, and C3b-INA and hemolytic activities of complement components C1 to C9 except C3 were normal or elevated; total hemolytic complement activity was 13% of normal and was reconstituted by purified C3. Properdin factor B was 702 (normal 175 to 275) mug/ml, and was not cleaver upon addition of zymosan or cobra venom factor. The serum had normal immune adherence activity, but was deficient in ability to opsonize Candida albicans for uptake and Escherichia coli for killing by neurophils, generate neutrophil chemotactic factors and inhibit the growth of E. coli; these activities were restored by purified C3. A transfusion of 320 ml 1-hour-old normal whole blood on the fifty-second day resulted in transitory elevation of the C3 level to 25 mg/dl with a fall-off (approximately 2 1/2% per hour) to undetectable levels by 69 hours; it was followed by disappearance of the skin rash and arthralgias and return to normal of the previously elevated temperature and CRP levels. C3 levels in family members (seven of 24 half-normal), lack of anti-C3 activity, normal C3b-INA levels and a normal rate of catabolism of transfused C3 indicated that the deficiency was inherited with autosomal codominance and involved decreased synthesis of C3. Thus, this child is a unique individual with inherited C3 deficiency presenting with absence of repeated infections, whose symptoms of fever, skin rash, and arthralgia were abated by whole blood transfusion.

Cyclophosphamide treatment in steroid-sensitive nephrotic syndrome of childhood.

An 8-week course of 3 mg. per kg. body-weight per day of cyclophosphamide was administered to eighty-two children with steroid-senstitive nephrotic syndrome. One died, and of the remainder, 69% were in remission after 1 year and 44% after 4-years. Older children and those in whom cyclophosphamide was given during a steroid-maintained remission fared better.