Charles J. Parker

Isolation and characterization of a membrane protein from normal human erythrocytes that inhibits reactive lysis of the erythrocytes of paroxysmal nocturnal hemoglobinuria.

The observation that type III erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are susceptible to hemolysis initiated by activated cobra venom factor complexes (CoFBb), whereas normal erythrocytes are resistant, implies that the PNH III cells are deficient in a membrane constituent that regulates this process. To isolate the inhibitory factor from normal erythrocytes, membrane proteins were first extracted with butanol and then subjected to sequential anion exchange, hydroxylapatite, and hydrophobic chromatography. Analysis by SDS-PAGE and silver stain of the inhibitory fractions showed a single band corresponding to a protein with an apparent Mr of 18 kD. PNH erythrocytes were incubated with incremental concentrations of the radiolabeled protein and then washed. In a dose-dependent fashion, the protein incorporated into the cell membrane and inhibited CoFBb-initiated lysis. This protein inhibitor functioned by restricting the assembly of the membrane attack complex at the level of C7 and C8 incorporation. By using a monospecific antibody to block the function of the inhibitor, it was shown that normal erythrocytes are rendered susceptible to CoFBb-initiated hemolysis. Analysis by Western blot of membrane proteins revealed that PNH III erythrocytes are deficient in the 18-kD protein. By virtue of its molecular weight and inhibitory activity, the 18-kD protein appears to be discrete from other previously described erythrocyte membrane proteins that regulate complement. These studies also indicate that the susceptibility of PNH III erythrocytes to reactive lysis is causally related to a deficiency of the 18-kD membrane inhibitor.

Human immunodeficiency virus type 1 incorporates both glycosyl phosphatidylinositol-anchored CD55 and CD59 and integral membrane CD46 at levels that protect from complement-mediated destruction.

Human immunodeficiency virus type 1 (HIV-1) can be either resistant or sensitive to complement-mediated destruction depending on the host cells. Incorporation of different levels of host cell CD46, CD55 and CD59 may account for this differential sensitivity to complement. However, it has not been determined whether CD46, CD55 and CD59 can all be incorporated at levels which protect virions. To determine whether each of these proteins can protect HIV-1, virions were derived from CHO cells expressing either human CD46, CD55 or CD59. Virions were shown to incorporate both glycosyl phosphatidylinositol (GPI)-anchored CD55 and CD59 as well as transmembrane CD46. Importantly, all three virus preparations were significantly more resistant to complement lysis than control virus. This study demonstrates that HIV-1 incorporates both transmembrane and GPI-anchored complement control proteins from host cells and that both types of protein increase complement resistance of virus.

Relationship between the membrane inhibitor of reactive lysis and the erythrocyte phenotypes of paroxysmal nocturnal hemoglobinuria.

Susceptibility to hemolysis initiated by activated cobra venom factor (CoF) complexes is a characteristic that distinguishes the most complement-sensitive type III erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) from the intermediately sensitive type II and the normally sensitive type I cells. Recently we isolated a membrane constituent from normal erythrocytes that inhibits CoFBb-initiated hemolysis, and this protein was designated membrane inhibitor of reactive lysis (MIRL). To investigate the molecular basis of the variability in complement sensitivity among PNH erythrocytes, the surface expression of MIRL and decay accelerating factor (DAF) on the three phenotypes of PNH was quantified immunochemically. Both complement regulatory proteins were markedly deficient on the erythrocytes from a patient with predominately type III cells. The erythrocytes from patients with a majority of either type II or I cells were also significantly deficient in both MIRL and DAF. While cytofluorometric analysis confirmed the quantitative deficiencies, segregation of erythrocytes into discrete subpopulations that expressed either no MIRL or normal amounts of MIRL was not observed. The results of immunoprecipitation studies were consistent with quantitative, but not qualitative abnormalities of MIRL and DAF. Selective removal of the sensitive erythrocytes indicated that approximately 20% of the normal amount of MIRL is sufficient to protect cells from CoF-initiated lysis. These studies suggest that relatively subtle quantitative differences in membrane complement regulatory proteins underlie the variability in complement sensitivity of PNH erythrocytes.

Eculizumab for paroxysmal nocturnal haemoglobinuria.

The complement system plays a central part in both innate and acquired immunity, but the contribution of complement activation to pathobiology is largely ancillary. An exception to the non-dominant role of complement in disease is the haemolytic anaemia of paroxysmal nocturnal haemoglobinuria (PNH). The intravascular haemolysis that is the clinical hallmark of PNH is a consequence of deficiency of the complement inhibitory proteins decay accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL, CD59). Eculizumab is a humanised monoclonal antibody that binds and prevents activation of complement C5 and the subsequent formation of the cytolytic membrane attack complex of complement. Eculizumab inhibits the intravascular haemolysis of PNH, reduces transfusion requirements, stabilises haemoglobin concentration, and improves quality of life. Although chronic treatment with eculizumab increases the risk of infections with Neisseria meningitides, the drug is generally safe and well tolerated. But as is the case with other drugs developed for treatment of ultra-orphan diseases, eculizumab is expensive, and treatment must continue indefinitely because C5 inhibition does not affect the process (ie, clonal proliferation of haemopoietic stem cells with a mutant phosphatidylinositol glycan complementation class A [PIGA] gene) that underlies PNH. Moreover, due to the heterogeneous nature of the disease, treatment with eculizumab is not appropriate for all patients with PNH.

Vitronectin is a substrate for transglutaminases

Vitronectin (VN) was found to be a substrate for both plasma transglutaminase (Factor XIIIa) and guinea pig liver transglutaminase (TG). Incorporation of [3H]-putrescine indicated the presence of reactive glutaminyl residues in VN. When VN was incubated with TG or Factor XIIIa, in the absence of putrescine, multimeric covalent complexes were identified, indicating that VN can also contribute lysyl residues to the bond catalyzed by transglutaminases. Cross-linking of VN by TG and Factor XIIIa may modulate the effects of VN on the complement and coagulation systems in hemostatic plugs and extracellular matrix.

Biomedical polymers differ in their capacity to activate complement.

Conventionally, complement activation by biomedical polymers has been evaluated by determining the C3a concentration in the fluid phase only. According to this criterion, biomaterials such as hemodialysis membranes made from cellulosic or various synthetic polymers were classified as activators or nonactivators of complement. Since certain membranes bind large quantities of C3a from the fluid phase, classification based on fluid-phase C3a concentration has in some instances been inaccurate. As follows from the comparison of complement activation by cuprophane and polyacrylonitrile membranes, the capacity of a biomedical polymer to activate complement is not determined by the number of potential covalent binding sites on its surface. Biomaterial itself may lack hydroxyl and/or amino groups, and yet it may activate C3 in human serum very efficiently. Some of the biomaterials may also bind unactivated/unfragmented C3 whether in the absence or presence of other serum proteins. In addition, binding of factor B (a promotor of C3 activation) and binding of factor H (an inhibitor of C3 activation) to certain biomaterials have been found to be independent of complement activation and unaffected by the presence or absence of C3. Thus, it is becoming apparent that the requirements for the formation and stability of the C3 convertase on artificial surfaces differ from those on biological membranes, and that the relative magnitude of binding of factor B and factor H to the surface per se cannot be used as a reliable indicator of the capacity of the biomaterial to activate complement. Further studies are necessary to elucidate the molecular mechanisms of C3 and C5 activation on the surfaces of biomedical polymers.(ABSTRACT TRUNCATED AT 250 WORDS)

The erythrocytes in paroxysmal nocturnal haemoglobinuria of intermediate sensitivity to complement lysis

The sensitivity to lysis by complement of the erythrocytes of 56 patients with paroxysmal nocturnal haemoglobinuria (PNH) was compared to the membrane expression of decay accelerating factor (DAF, CD55), membrane inhibitor of reactive lysis (MIRL, CD59) and acetylcholinesterase (AChE). Most patients (36/50 72% in whom the analysis could be made) appeared to have erythrocytes of intermediate sensitivity to complement in the blood. These cells appeared as a discrete population of cells (PNH II cells), as a ‘tail’ of cells slightly less sensitive than the predominant PNH III cells (previously called PNH IIIb cells), or as a continuous spectrum of cells sensitive to complement. The PNH III cells totally lacked all three proteins (DAF, MIRL, AChE) by flow cytometric analysis whereas PNH I cells appeared to have normal or nearly normal amounts of each. The cells of intermediate sensitivity (PNH II) had coordinately decreased expression of all three proteins; the level of expression of DAF and MIRL paralleled the sensitivity of the cells to the haemolytic action of complement.

A novel approach to preventing the hemolysis of paroxysmal nocturnal hemoglobinuria: both complement-mediated cytolysis and C3 deposition are blocked by a monoclonal antibody specific for the alternative pathway of complement

The clinical hallmark of paroxysmal nocturnal hemoglobinuria (PNH) is chronic intravascular hemolysis that is a consequence of unregulated activation of the alternative pathway of complement (APC). Intravascular hemolysis can be inhibited in patients by treatment with eculizumab, a monoclonal antibody that binds complement C5 thereby preventing formation of the cytolytic membrane attack complex of complement. However, in essentially all patients treated with eculizumab, persistent anemia, reticulocytosis, and biochemical evidence of hemolysis are observed; and in a significant proportion, their PNH erythrocytes become opsonized with complement C3. These observations suggest that PNH patients treated with eculizumab are left with clinically significant immune-mediated hemolytic anemia because the antibody does not block APC activation. With a goal of improving PNH therapy, we characterized the activity of anti-C3b/iC3b monoclonal antibody 3E7 in an in vitro model of APC-mediated hemolysis. We show that 3E7 and its chimeric-deimmunized derivative H17 block both hemolysis and C3 deposition on PNH erythrocytes. The antibody is specific for the APC C3/C5 convertase because classical pathway-mediated hemolysis is unaffected by 3E7/H17. These findings suggest an approach to PNH treatment in which both intravascular and extravascular hemolysis can be inhibited while preserving important immune functions of the classical pathway of complement.

Paroxysmal nocturnal hemoglobinuria.

Purpose of reviewThe aim is to report on recent observations related to the natural history of paroxysmal nocturnal hemoglobinuria (PNH) and to review new therapeutic strategies for controlling the hemolysis of PNH. Recent findingsThis review focuses on studies designed to characterize the long-term outcome of patients with PNH treated with eculizumab and to define the relationship between PNH and bone marrow failure syndromes. New therapeutic strategies aimed at controlling extravascular as well as intravascular hemolysis are also examined. SummaryLong-term safety and efficacy of eculizumab was observed in a large group of patients. Survival for the group was not different from that of a sex-matched and age-matched control group from the general population. Thrombotic complications were rare and deaths due to PNH or complications of therapy were not observed. These studies suggest that patients with clinical PNH who are treated with eculizumab have a benign clinical course. Patients with bone marrow failure who have PNH cells detected by high-sensitivity flow cytometry have aplastic anemia or low-risk myelodysplastic syndrome. For patients with a percentage of PNH cells that is below the threshold for producing laboratory evidence of hemolysis (subclinical PNH), expansion of the clone to a size sufficient to produce clinical PNH is not observed. Approximately 50% of patients with bone marrow failure who have clinical evidence of PNH at presentation will require PNH-specific therapy. Novel reagents that target the alternative pathway of complement C3 convertase are being developed with a goal of inhibiting both the extravascular and the intravascular hemolysis of PNH.

A Role for C5 and C5a-ase in the Acute Neutrophil Response to Group B Streptococcal Infections

Congenic C5-deficient and C5-sufficient mice were infected with group B streptococci (GBS) to determine if the polymorphonuclear leukocyte (PMNL) chemoattractant C5a contributes to PMNL recruitment in GBS infection and if GBS C5a-ase reduces C5a-induced PMNL recruitment in vivo. PMNL accumulation was greater in the peritoneum and air spaces of C5-sufficient mice than in C5-deficient mice. Administration of human C5 to C5-deficient mice caused a significant increase in PMNL recruitment following infection with C5a-ase-negative GBS. GBS C5a-ase did not reduce PMNL accumulation in C5-sufficient mice but reduced PMNL recruitment in C5-deficient mice reconstituted with human C5. These data indicate that C5a is important for rapid PMNL recruitment to sites of GBS infection and that GBS C5a-ase inactivates human, but not murine, C5a in vivo. Reduction of the acute inflammatory response by C5a-ase likely contributes to GBS virulence in human neonates.