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.

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.

A comparative study of digital images versus 35-millimeter images.

Digital imaging is an alternative to 35-mm Kodachromes and may be a useful tool to the physician. The purpose of this study was to compare the quality of digital images to 35-mm images and then compare the two methods based on the ability to make the correct diagnosis. Dermatology patients were photographed with both a digital camera and a 35-mm camera. Images from 22 patients were compared and graded on four criteria (sharpness of image, contrast, depth of field, and trueness of color). Diagnoses were then made from 30 pairs of images of different patients. In all graded areas the 35-mm images scored significantly higher. The correct diagnosis was made in 69.6% of the digital images compared to 77.3% of the 35-mm images, a difference that was not statistically significant. One may conclude that digital images hold promise for present and future use in dermatology and other areas of medicine.

Analysis of the binding of human C3b to glycoproteins on rabbit and sheep erythrocytes.

To investigate the possibility that activation of the human alternative pathway of complement is influenced by cellular constituents that interact with C3b, the membrane proteins on rabbit and sheep erythrocytes that are associated with cell-bound human C3b have been analyzed. For both types of erythrocytes, activated C3b bound in a diffuse pattern via hydroxylamine-sensitive ester bonds. By using a homobifunctional crosslinker, a membrane component that has an apparent Mr of 35 kDa was shown to be noncovalently associated with C3b on sheep erythrocytes, but rabbit erythrocytes lacked a predominant C3b-associated protein. These studies suggest that regulation of human alternative pathway activity may be influenced by membrane glycoproteins that interact with cell-bound C3b.

Induction of the paroxysmal nocturnal hemoglobinuria phenotype in normal human erythrocytes: effects of 2-aminoethylisothiouronium bromide on membrane proteins that regulate complement.

To investigate the mechanism by which treatment of normal human erythrocytes with the sulfhydryl reagent 2-aminoethylisothiouronium bromide (AET) induces susceptibility to complement mediated lysis, the effects of AET on the structural and functional integrity of decay accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and complement receptor type 1 (CR1) were examined. Following treatment with AET, erythrocyte MIRL and CR1 were no longer recognized in situ by antibodies, and antibody binding to DAF was diminished by approximately 50%. These studies indicated that the structural integrity of the three complement regulatory proteins was either partially (DAF) or completely (MIRL and CR1) disrupted by AET. Subsequent experiments showed that functional inactivation paralleled the structural disruption. Treatment of normal erythrocytes with AET induced susceptibility to cobra venom factor-initiated hemolysis, indicating that the functional activity of MIRL had been destroyed. The capacity of erythrocyte CR1 to serve as a cofactor for factor I-mediated cleavage of iC3b to C3c and C3dg was lost following treatment with AET. C3 convertase activity increase markedly following treatment of erythrocytes with AET, but convertase activity on AET cells was approximately 50% less than that observed when DAF function on normal cells was completely inhibited by antibody. Susceptibility of AET cells to acidified serum lysis was shown to be due primarily to inactivation of MIRL. Unexpectedly, in acidified serum the activity of the amplification C3 convertase of the APC was found to be controlled by MIRL as well as by DAF. These studies show that AET induces susceptibility to complement-mediated lysis by disrupting the structural and functional integrity of membrane constituents that regulate the activity of both the C3 convertases and the membrane attack complex of complement.

Activation of complement by hemodialysis membranes: polyacrylonitrile binds more C3a than cuprophan.

Conventionally, complement activation by hemodialysis membranes has been determined by measuring fluid phase C3a. Based on such measurements, polyacrylonitrile (PAN) membranes have been classified as weak activators compared to cuprophan. Previous studies have demonstrated, however, that PAN adsorb fluid phase C3a. Based on that observation, we hypothesized that complement activation by PAN might be artifactually underestimated if relatively large amounts of C3a remained membrane bound. In the present study, a method that allows the simultaneous quantification of both fluid phase and membrane bound C3a was used to assess complement activation by PAN and cuprophan. Pieces of membrane were incubated with C3-depleted serum that had been repleted with radiolabeled C3. Subsequently, the supernates and membranes were subjected to SDS-PAGE, and complement activation was quantified by determining the radioactivity of the C3a bands in the gel. The results showed that while the serum exposed to cuprophan membranes contained almost five times more C3a than that exposed to PAN, approximately 80 times more C3a was bound to the PAN membranes. Consequently, the total amount of C3a generated in the presence of PAN was higher than that generated in the presence of cuprophan. We conclude that assessment of complement activation by hemodialysis membranes using fluid phase C3a measurements alone may be misleading.

Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum.

When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay-accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane-associated activation of the APC.

Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell-associated protein.

The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol-specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.

CD11b/CD18 integrin and a beta-glucan receptor act in concert to induce the synthesis of platelet-activating factor by monocytes.

We determined the mechanism by which opsonized zymosan particles, which are derived from yeast and composed of carbohydrate polymers, stimulate platelet-activating factor (PAF) synthesis by monocytes. A role for CD11b/CD18 was demonstrated because antibodies to this integrin decreased PAF synthesis, zymosan bearing only a ligand for CD11b/CD18 (iC3b) induced the synthesis of PAF, and monocytes that did not express CD11b/CD18 produced much less PAF than control monocytes. Ligation of CD11b/CD18 was not sufficient for PAF synthesis suggesting that an additional receptor was involved. Monocytes are known to bind beta-glucan which is a major component of zymosan. Opsonized beta-glucan particles stimulated the synthesis of PAF, and a soluble form of beta-glucan partially inhibited PAF synthesis in response to opsonized zymosan. Two lines of evidence suggested that the beta-glucan receptor mediating this response was distinct from CD11b/CD18. First, CD11b/CD18-deficient monocytes produced PAF when stimulated by zymosan opsonized with isolated C3b, a molecule that binds to complement receptor type 1 (CD35). Second, inducing contact of monocytes with zymosan by centrifugation resulted in PAF synthesis that was not inhibited by antibodies to CD11b/CD18. The combination of soluble beta-glucan and antibodies to CD11b/CD18 completely blocked PAF synthesis in response to opsonized zymosan. Together, these results demonstrate that induction of maximal PAF synthesis by serum-opsonized zymosan requires the concerted interactions of monocyte receptors for iC3b and beta-glucan. Additionally, they suggest that CD11b/CD18 facilitates binding of the particle and that a beta-glucan receptor transduces the activation signal.