Aurin Tricarboxylic Acid Protects against Red Blood Cell Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinemia

Objectives

Paroxysmal nocturnal hemoglobinemia (PNH) is a rare but serious condition characterized by complement-mediated red blood cell (RBC) hemolysis and episodic thrombotic attack. It results from decay accelerating factor (CD55), and protectin (CD59), becoming attached to RBC and other cell surfaces. Absence of these protective proteins leaves such cells vulnerable to self attack at the C3 convertase and membrane attack complex (MAC) stages of complement activation. We have previously reported that aurin tricarboxylic acid (ATA) is an orally effective agent that selectively blocks complement activation at the C3 convertase stage as well as MAC formation at the C9 insertion stage.

Design and Methods

We used a CH50 assay method and western blot analysis to investigate the vulnerability to complement attack of PNH RBCs compared with normal RBCs. Zymosan was used as the activator of normal serum and PNH serum. ATA was added to the sera to determine the concentration necessary to protect the RBCs from lysis by the zymosan-activated sera.

Results

We found that erythrocytes from PNH patients on long term treatment with eculizumab were twice as vulnerable as normal erythrocytes to lysis induced by complement activated serum. Western blot data showed the presence of both C3 and C5 convertases on the PNH patient erythrocyte membranes. These data indicate persistent vulnerability of PNH erythrocytes to complement attack due to deficiencies in CD55 and CD59. ATA, when added to serum in vitro, protected PNH erythrocytes from complement attack, restoring their resistance to that of normal erythrocytes.

Conclusions

We conclude that ATA, by protecting PNH erythrocytes from their decay accelerating factor (CD55) and protectin (CD59) deficiencies, may be an effective oral treatment in this disorder.     

Analysis of the effects of activation of the alternative pathway of complement on erythrocytes with an isolated deficiency of decay accelerating factor.

E from individuals with the Inab blood group phenotype have an isolated deficiency of decay accelerating factor (DAF, CD55). DAF is a glycosyl phosphatidylinositol anchored membrane protein that inhibits activation of both the classical and alternative pathways of complement. Deficiency of DAF from the E of paroxysmal nocturnal hemoglobinuria (PNH) is thought to contribute to their greater sensitivity to complement-mediated lysis. Unlike PNH E, however, Inab cells are not susceptible to acidified serum lysis, a process that is mediated through activation of the alternative pathway. This observation led us to hypothesize that membrane constituents other than DAF control susceptibility to acidified serum lysis. To investigate this hypothesis, Inab E were incubated in acidified serum, and hemolysis and C3 deposition (as a measure of alternative pathway activation) were quantitated. C3 deposition of Inab cells was approximately 20 times greater than normal, however, hemolysis was not observed. Inab E expressed a normal amount of membrane inhibitor of reactive lysis (MIRL, CD59), a glycosyl phosphatidylinositol anchored protein that is also deficient in PNH. When MIRL function was blocked with antibody, C3 deposition markedly increased, and 100% of the Inab cells hemolyzed in acidified serum. These studies demonstrate that susceptibility to acidified serum lysis is controlled primarily by MIRL, and that in addition to its regulatory affect on the membrane attack complex, MIRL also modulates the activity of the C3 convertase of the alternative pathway by a mechanism that remains to be determined.     

H19, a surface membrane molecule involved in T-cell activation, inhibits channel formation by human complement.

Here we compare the properties of leukocyte antigens H19 and CD59 with those of the PI-linked 18,000-20,000 Mr molecules which inhibit lysis of human cells by the autologous terminal complement components C5b-9. H19, a 19,000 Mr protein found on human erythrocytes, monocytes, neutrophils, T-lymphocytes and other cells, is one of the ligands involved in the spontaneous rosette formation between human T-lymphocytes and erythrocytes. Recent evidence indicates that H19 also participates in T-cell activation. CD59 is a widely distributed 18,000-25,000 Mr protein anchored to the cell membrane by phosphatidylinositol (PI). The function of CD59 is unknown. Affinity-purified H19 incorporates into cell membranes and inhibits channel formation by human C5b-9 on guinea pig erythrocytes. Significant inhibition is achieved with picogram quantities of H19, corresponding to approximately 600 molecules per erythrocyte. H19 is most effective when C9 is limiting but quite active when C5b-7 or C8 are limiting, indicating that it may interact with several of the structurally related terminal complement components. The inhibitory activity is blocked by mAbs to either CD59 or to H19. H19 is PI-anchored: it is released from the cell membrane by treatment with PI-specific phospholipase C, and it is absent from cells from a patient with paroxysmal nocturnal hemoglobinuria (PNH). Analysis of PNH erythrocytes after treatment with terminal complement proteins shows that the H19-negative erythrocytes are more susceptible to C5b-9-mediated lysis. Treatment of normal human erythrocytes with either anti-H19 or anti-CD59 renders them more susceptible to lysis by human C5b-9. We conclude that H19 and CD59 are probably the same molecule and are identical or closely related to the recently described inhibitors of C5b-9 channel formation.     

Biochemical background of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by paroxysms of intravascular hemolysis. A considerable part of erythrocytes in patient blood is susceptible to autologous complement activation because of the deficiency of CD59, which is a glycosylphosphatidylinositol (GPI)-anchored protein and inhibits the formation of the membrane attack complex (MAC) of complement. The deficiency of CD59 is derived from the inability of GPI-anchor synthesis. Although more than 10 proteins are involved in the GPI-anchor synthesis, the mutation of only one protein, PIG-A, causes the defect in about 200 patients with PNH who have been analyzed. The reason why only PIG-A causes the deficiency of GPI anchor is due to the location of its gene on X chromosome. The clonal stem cell mutated with PIG-A gene in the bone marrow loses the capability of the synthesis of GPI-anchor. The mutation of PIG-A gene alone, however, seems to be insufficient to account for the survival of the PIG-A-deficient cells in the bone marrow. Thus, a fraction of the mutant stem cells probably gain a survival advantage by some additional changes, either additional mutations or changes in immunological circumstances. The release of the surviving cells into blood stream results in a clinical syndrome with PNH.     

Flow cytometric analysis of CD55 and CD59 expression on blood cells in paroxysmal nocturnal haemoglobinuria

PNH is a rare clonal disorder of hematopoietic stem cells, therefore all blood cells lineages are involved. The main feature is an increased sensitivity of erythrocytes to complement-mediated cell lysis due to deficiency of membrane-bound GPI (glycosylphosphatidylinositol)-anchored proteins which normally function as inhibitors of reactive hemolysis. In the present study, we performed flow cytometric analysis using monoclonal antibodies against CD55 and CD59 for the detection of PNH-type clone in the blood of 50 patients (28 females and 22 males, age range 7-67 yrs). In one patient only we found a large population (95%) of granulocytes with decreased expression of both CD55 and CD59 molecules (type I PNH) and in two others with partial loss of CD55 expression (type II PNH). The expression was determined chiefly on granulocytes which in the control group showed reliable and high expression of CD55 and CD59.     

Use of some snake venoms in the diagnostics of paroxysmal nocturnal hemoglobinuria (PNH)

The crude toxin of Agkistrodon piscivorus was found to produce hemolysis of PNH erythrocytes by way of complement activation, however the degree of PNH blood cell lysis was lower than in other techniques used. The crude toxins of Naja naja and Naja oxiana cause much higher hemolysis though lower hemolysis takes place in normal blood cells. In some normal persons and different blood diseases it is responsible for high hemolysis even when the complement is absent. From Naja naja toxin a fraction can be isolated which has a "specific" complement effect only on PNH blood cells. This fraction can thus be utilized in a specific test for this disease.     

Mesenchymal stromal cells engage complement and complement receptor bearing innate effector cells to modulate immune responses

Infusion of human third-party mesenchymal stromal cells (MSCs) appears to be a promising therapy for acute graft-versus-host disease (aGvHD). To date, little is known about how MSCs interact with the body's innate immune system after clinical infusion. This study shows, that exposure of MSCs to blood type ABO-matched human blood activates the complement system, which triggers complement-mediated lymphoid and myeloid effector cell activation in blood. We found deposition of complement component C3-derived fragments iC3b and C3dg on MSCs and fluid-phase generation of the chemotactic anaphylatoxins C3a and C5a. MSCs bound low amounts of immunoglobulins and lacked expression of complement regulatory proteins MCP (CD46) and DAF (CD55), but were protected from complement lysis via expression of protectin (CD59). Cell-surface-opsonization and anaphylatoxin-formation triggered complement receptor 3 (CD11b/CD18)-mediated effector cell activation in blood. The complement-activating properties of individual MSCs were furthermore correlated with their potency to inhibit PBMC-proliferation in vitro, and both effector cell activation and the immunosuppressive effect could be blocked either by using complement inhibitor Compstatin or by depletion of CD14/CD11b-high myeloid effector cells from mixed lymphocyte reactions. Our study demonstrates for the first time a major role of the complement system in governing the immunomodulatory activity of MSCs and elucidates how complement activation mediates the interaction with other immune cells.     

Analysis of PI (phosphatidylinositol)-anchoring antigens in a patient of paroxysmal nocturnal hemoglobinuria (PNH) reveals deficiency of 1F5 antigen (CD59), a new complement-regulatory factor.

FACS analysis together with PIPLC treatment was applied to PI-anchoring antigens such as DAF (decay-accerelating factor, CD55), 1F5 antigen (CD59), CD14 and CD16 on the cell surfaces of blood cells from a normal adult and a male patient with paroxysmal nocturnal hemoglubinuria (PNH). Through the extensive analysis, this patient proved to be completely defective in 1F5 antigen, a newly found complement-regulatory protein, on all the blood cells tested. In normal blood cells such as lymphocytes, monocytes and granulocytes, 1F5 antigen was expressed as one of PI-anchoring proteins. In contrast to most of PNH patients, this patient reserved DAF, CD14 and CD16 at normal levels in his erythrocytes, monocytes and granulocytes. Also, there were no significant differences between the normal adult and the patient in the activities of erythrocyte acetylcholinesterase and granulocyte alkaline phosphatase which were also known to be PI-anchoring enzymes. Thus, deficiency of 1F5 antigen must be deeply related to the clinical symptoms of PNH in this patient.     

Soluble CD59 expressed from an adenovirus in vivo is a potent inhibitor of complement deposition on murine liver vascular endothelium

Inappropriate activation of complement on the vascular endothelium of specific organs, or systemically, underlies the etiology of a number of diseases. These disorders include atypical hemolytic uremic syndrome, membranoproliferative glomerulonephritis, atherosclerosis, age-related macular degeneration, diabetic retinopathy, and transplant rejection. Inhibition of the terminal step of complement activation, i.e. formation of the membrane attack complex, using CD59 has the advantage of retaining the upstream processes of the complement cascade necessary for fighting pathogens and retaining complement's crucial role in tissue homeostasis. Previous studies have shown the necessity of membrane targeting of soluble CD59 in order for it to prove an effective inhibitor of complement deposition both in vitro and in vivo. In this study we have generated an in vivo model of human complement activation on murine liver vascular endothelium. This model should prove useful for the development of anti-complement therapies for complement-induced pathologies of vascular endothelium. Using this model, we have demonstrated the viability of a non membrane-targeted soluble CD59 to significantly inhibit complement deposition on the endothelium of murine liver vasculature when expressed in vivo from an adenovirus. This result, unanticipated based on prior studies, suggests that the use of non membrane-targeted sCD59 as an anti-complement therapy be re-visited.     

Flow cytometric analysis of glycosylphosphatidyl-inositol-anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by total or partial deficiency of membrane proteins anchored to the cell surface through a glycosylphosphatidyl-inositol (GPI) moiety. The relationship between the size of the PNH clone, determined by the expression of GPI-anchored proteins (AP; CD14, CD48, CD55, CD59, and CD66b) on erythrocytes, lymphocytes, monocytes, and granulocytes using forward and side scatter analysis, and severity of the disease was evaluated in 19 PNH patients. CD55 antigen expression did not delineate abnormal erythrocytes as well as did anti-CD59.The proportion of monocytes deficient in CD55, CD59, CD48, and CD14 (48-97%) and of granulocytes deficient in CD55, CD59, and CD66b (60-99%) was greater than the proportion of erythrocytes deficient in CD59 (24-95%) and the proportion of lymphocytes deficient in CD55 and CD59 (30-98%). There were no significant correlations among reticulocyte, leukocyte, and platelet counts and GPI-AP-deficient immunophenotypes in red and white blood cells. However, high coefficients of determination were seen between hemoglobin levels and granulocytes deficient in CD59 (r(2) = 0.76), CD55 (r(2) = 0.74), and CD66b (r(2) = 0.74) antigens and between hemoglobin and monocytes deficient in CD55 (r(2) = 0.73), CD59 (r(2) = 0.80), and CD14 (r(2) = 0.75) antigens. These results are interpreted as indicating that the size of PNH clone is better assessed by immunophenotypic analysis of monocytes and granulocytes rather than of lymphocytes and erythrocytes.     

Down-regulation of human complement factor H sensitizes non-small cell lung cancer cells to complement attack and reduces in vivo tumor growth

Malignant cells are often resistant to complement activation through the enhanced expression of complement inhibitors. In this work, we examined the protective role of factor H, CD46, CD55, and CD59 in two non-small cell lung cancer cell lines, H1264 and A549, upon activation of the classical pathway of complement. Complement was activated with polyclonal Abs raised against each cell line. After blocking factor H activity with a neutralizing Ab, C3 deposition and C5a release were more efficient. Besides, a combined inhibition of factor H and CD59 significantly increased complement-mediated lysis. CD46 and CD55 did not show any effect in the control of complement activation. Factor H expression was knockdown on A549 cells using small interfering RNA. In vivo growth of factor H-deficient cells in athymic mice was significantly reduced. C3 immunocytochemistry on explanted xenografts showed an enhanced activation of complement in these cells. Besides, when mice were depleted of complement with cobra venom factor, growth was recovered, providing further evidence that complement was important in the reduction of in vivo growth. In conclusion, we show that expression of the complement inhibitor factor H by lung cancer cells can prevent complement activation and improve tumor development in vivo. This may have important consequences in the efficiency of complement-mediated immunotherapies.     

Further evidence that paroxysmal nocturnal haemoglobinuria is a disorder of defective cell membrane lipid rafts

The glycolipid glycosylphosphatidylinositol anchor (GPI-A) plays an important role in lipid raft formation, which is required for proper expression on the cell surface of two inhibitors of the complement cascade, CD55 and CD59. The absence of these markers from the surface of blood cells, including erythrocytes, makes the cells susceptible to complement lysis, as seen in patients suffering from paroxysmal nocturnal haemoglobinuria (PNH). However, the explanation for why PNH-affected hematopoietic stem/progenitor cells (HSPCs) expand over time in BM is still unclear. Here, we propose an explanation for this phenomenon and provide evidence that a defect in lipid raft formation in HSPCs leads to defective CXCR4- and VLA-4-mediated retention of these cells in BM. In support of this possibility, BM-isolated CD34⁺ cells from PNH patients show a defect in the incorporation of CXCR4 and VLA-4 into membrane lipid rafts, respond weakly to SDF-1 stimulation, and show defective adhesion to fibronectin. Similar data were obtained with the GPI-A⁻ Jurkat cell line. Moreover, we also report that chimeric mice transplanted with CD55^−/− CD59^−/− BM cells but with proper GPI-A expression do not expand over time in transplanted hosts. On the basis of these findings, we propose that a defect in lipid raft formation in PNH-mutated HSPCs makes these cells more mobile, so that they expand and out-compete normal HSPCs from their BM niches over time.     

Complement sensitivity of erythroid and myeloid precursors in paroxysmal in nocturnal hemoglobinuria

To test the hypothesis that paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder, the growth of BFU-e and CFU-gm and the complement sensitivity of cultured cells from BFU-e and CFU-gm colonies, as well as of unipotential progenitor cells (CFU-gm and BFU-e), were examined in five PNH patients. BFU-e growth was reduced in the three patients examined, and poor CFU-gm growth was noted in three of the five patients. Compared to normals, BFU-e and CFU-gm colonies in all patients demonstrated an increased susceptibility to the lytic action of complement when the release of 59Fe and myeloperoxidase was measured as specific markers for monitoring membrane damage. Compared to the growth of normal bone marrow cells, CFU-gm growth was significantly inhibited by pretreatment of bone marrow mononuclear cells with monoclonal OKIal antibody and complement. These findings support the proposition that a membrane defect predisposing blood cells to complement-mediated lysis may occur at the level of unipotential progenitor cells.     

Lysis of red blood cells in some haemolytic anaemias--the lytic effect of agkistrodon piscivorus venom in vitro.

The lysis of red blood cells induced by the Agkistrodon piscivorus venom (APV) in vitro in the presence and/or in the absence of plasma was examined in autoimmune haemolytic anaemia (AIHA), hereditary spherocytosis (HS) and paroxysmal nocturnal haemoglobinuria (PNH). Virtually haemolysis did not differ from that of normals in AIHA, it was slightly increased in HS and significantly higher in PNH, however, only in the presence of autologous or normal homologous plasma. The mechanism of PNH blood cells lysis which is probably related to the activation of the third complement component is discussed.     

Targeting of functional antibody-decay-accelerating factor fusion proteins to a cell surface

Recombinant soluble complement inhibitors hold promise for the treatment of inflammatory disease and disease states associated with transplantation. Targeting complement inhibitors to the site of complement activation and disease may enhance their efficacy and safety. Data presented show that targeting of decay-accelerating factor (DAF, an inhibitor of complement activation) to a cell surface by means of antibody fragments is feasible and that cell-targeted DAF provides significantly enhanced protection from complement deposition and lysis compared with soluble untargeted DAF. An extracellular region of DAF was joined to an antibody combining site with specificity for the hapten dansyl, at the end of either C(H)1 or C(H)3 Ig regions. The recombinant IgG-DAF chimeric proteins retained antigen specificity and bound to dansylated Chinese hamster ovary cells. Both soluble C(H)1-DAF and C(H)3-DAF were effective at inhibiting complement-mediated lysis of untargeted Chinese hamster ovary cells at molar concentrations within the range reported by others for soluble DAF. However, when targeted to a dansyl-labeled cell membrane, C(H)1-DAF was significantly more potent at inhibiting complement deposition and complement-mediated lysis. Cell-bound C(H)1-DAF also provided cells with protection from complement lysis after removal of unbound C(H)1-DAF. Of further importance, the insertion of a nonfunctional protein domain of DAF (the N-terminal short consensus repeat) between C(H)1 and the functional DAF domain increased activity of the fusion protein. In contrast to C(H)1-DAF, C(H)3-DAF was not significantly better at protecting targeted versus untargeted cells from complement, indicating that a small targeting vehicle is preferable to a large one. We have previously shown that for effective functioning of soluble complement inhibitor CD59, binding of CD59 to the cell surface close to the site of complement activation is required. Significantly, such a constraint did not apply for effective DAF function.     

Expression of CD55 and CD59 on peripheral blood cells from systemic lupus erythematosus (SLE) patients

CD55 and CD59 are glycosylphosphatidylinositol-anchored proteins with complement inhibitory properties. CD55 inhibits the formation of C3 convertases, and CD59 prevents the terminal polymerisation of the membrane attack complex. It has been reported that SLE patients seems to have an acquired deficiency of these proteins associated with secondary autoimmune haemolytic anaemia and lymphopenia. The aim of this study was to evaluate the presence of altered CD55 and CD59 expression on peripheral blood cells from SLE patients. Flow cytometric analyses were performed on red and white blood cells from 23 SLE patients and 23 healthy controls. We observed more CD55- and CD59-lymphocytes (p = 0.005 and p = 0.019, respectively), and CD59-granulocytes (p = 0.045) in SLE patients than in controls. These results suggest there is an altered pattern of CD55 and CD59 expression on the peripheral blood cells of SLE patients, and it may play a role in the cytopenias in these patients.     

Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria

Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells. Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells. To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum. We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level. However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells. It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells. As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI. Thus, we constructed several mutants. Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin. These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death. Nevertheless, these mutants almost completely failed to kill host cells. It was revealed that the substitutions affect the events that follow oligomerization. The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells. Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.     

Expression of complement regulatory proteins [membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59)] in endometrial stressed cells

In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.     

Expression of the membrane complement regulatory protein CD59 (protectin) is associated with reduced survival in colorectal cancer patients

It has been known for some time that the immune system can recognise growing tumours, and that tumours may respond by modulation of molecules, which make them resistant to further attack. Expression, over-expression, or loss of these molecules may function as markers of tumour progression and prognosis. Among such molecules are the membrane-bound complement regulatory proteins (mCRP), which protect cells from bystander attack by autologous complement. These include CD59 (protectin), which prevents formation of the MAC complex in the terminal stages of complement activation. In the present study, we evaluated immunohistochemical expression of CD59 in a series of over 460 well-characterised colorectal cancers using tissue microarrays (TMA), and related this information to known tumour and patient variables and to survival. The CD59 expression was observed in 69 (15%) of cases overall, and was significantly associated with tumour grade. In contrast, no associations were noted with tumour site, stage or histological type. On survival analysis, a further correlation was observed between expression of CD59 by the colorectal tumours and a reduction in disease-specific patient survival. This observation was strongest for patients with early stage disease. However, a negative impact on survival was also seen in those patients with late stage disease. These results indicate that TMA linked to good clinicopathological databases with good long term follow up are useful tools for determining new prognostic indicators that can be used in future patient management. Immune surveillance may result in immune–editing that induces variable expression of a range of target antigens, and these may be useful prognostic markers. This study has identified CD59 expression as a marker of poor prognosis in colorectal cancer patients.This article is a symposium paper from the "Robert Baldwin Symposium: 50 years of Cancer Immunotherapy", held in Nottingham, Great Britain, on 30th June 2005.