Protectin Conjugates in Tissue Regeneration 1 Inhibits Macrophage Pyroptosis by Restricting NLRP3 Inflammasome Assembly to Mitigate Sepsis via the cAMP-PKA Pathway.

Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel anti-inflammatory and proresolving lipid mediator biosynthesized from docosahexaenoic acid. Excessive activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and consequent pyroptosis are involved in diverse inflammatory diseases. However, how PCTR1 affects NLRP3 inflammasome activation and pyroptosis are still unclear. Here, we demonstrated that PCTR1 inhibited NLRP3 inflammasome activation and pyroptosis. These results show that PCTR1 dose-dependently inhibited gasdermin D cleavage in lipopolysaccharide (LPS)-primed murine primary macrophages upon nigericin stimulation. Additionally, PCTR1 treatment after LPS priming inhibited caspase-1 activation and subsequent mature interleukin-1ß release independent of the nuclear factor-kappa B pathway. PCTR1 exerted its inhibitory effects by blocking NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC) interaction and ASC oligomerization, thereby restricting NLRP3 inflammasome assembly. However, the inhibitory effect of PCTR1 could be reversed by KH7 and H89, which are the inhibitors of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Moreover, PCTR1 treatment alleviated lung tissue damage and improved mouse survival in LPS-induced sepsis. Our study unveils the molecular mechanism of negative regulation of NLRP3 inflammasome activation and pyroptosis by a novel lipid mediator and suggests that PCTR1 may serve as a potential treatment option for NLRP3-inflammasome driven diseases.

PCTR1 improves pulmonary edema fluid clearance through activating the sodium channel and lymphatic drainage in lipopolysaccharide-induced ARDS.

Acute respiratory distress syndrome (ARDS) is a lethal clinical syndrome characterized by damage of the epithelial barriers and accumulation of pulmonary edema fluid. Protectin conjugates in tissue regeneration 1 (PCTR1), an endogenously produced lipid mediator, are believed to exert anti-inflammatory and pro-resolution effects. PCTR1 (1 µg/kg) was injected at 8 hr after lipopolysaccharide (LPS; 14 mg/kg) administration, and the rate of pulmonary fluid clearance was measured in live rats at 1 hr after PCTR1 treatment. The primary type II alveolar epithelial cells were cultured with PCTR1 (10 nmol/ml) and LPS (1 µg/ml) for 8 hr. PCTR1 effectively improved pulmonary fluid clearance and ameliorated morphological damage and reduced inflammation of lung tissue, as well as improved the survival rate in the LPS-induced acute lung injury (ALI) model. Moreover, PCTR1 markedly increased sodium channel expression as well as Na, K-ATPase expression and activity in vivo and in vitro. In addition, PCTR1i also upregulated the expression of LYVE-1 in vivo. Besides that, BOC-2, HK7, and LY294002 blocked the promoted effect of PCTR1 on pulmonary fluid clearance. Taken together, PCTR1 upregulates sodium channels' expression via activating the ALX/cAMP/P-Akt/Nedd4-2 pathway and increases Na, K-ATPase expression and activity to promote alveolar fluid clearance. Moreover, PCTR1 also promotes the expression of LYVE-1 to recover the lymphatic drainage resulting in the increase of lung interstitial fluid clearance. In summary, these results highlight a novel systematic mechanism for PCTR1 in pulmonary edema fluid clearance after ALI/ARDS, suggesting its potential role in a therapeutic approach for ALI/ARDS.

A mathematical model of cellular swelling in Neuromyelitis optica.

Neuromyelitis Optica (NMO) is a severe neuro-inflammatory disease of the central nervous system characterized by predominant damage to the optic nerve and of the spinal cord. The pathogenic antibody found in the majority of patients targets the AQP4 channels on astrocytic endfeet and causes the cells to swell. Although, the pathophysiology of the disease is broadly known, there are no specific targeted treatments for this process clinically available nor accurate prognostic markers both during attacks and for predicting long term neuronal damage. This lack is, in part, due to the rarity of the disease and its relatively recent pathogenic clarity. Hence, the ability to mathematically model the progress of the condition to test prospective therapies in silico would be a step forward. This paper combines state of the art models of cellular metabolism and cytotoxic oedema in neurons and astrocytes and augments it with a detailed characterization of water transport across the cellular membrane. In particular, we capture the process of perforation of the cell through the human complement cascade and resulting water and ionic fluxes. Simulating NMO by injecting its antibody and human complement into the extracellular space showed a 25% increase of the astrocytic volume after 12 h from onset. Most of the volume change occurred during the first 30 min of simulation with a peak volume change of 38%. The model was further adapted to simulate the therapeutic potential of CD59. It was found that there is a threshold of CD59 concentration that can prevent the swelling of astrocytes. Since the astrocyte volume changes mostly during the first hour, further experimental work should focus on this time scale to provide data for further model refinement and validation.

An anticomplement agent that homes to the damaged brain and promotes recovery after traumatic brain injury in mice.

Activation of complement is a key determinant of neuropathology and disability after traumatic brain injury (TBI), and inhibition is neuroprotective. However, systemic complement is essential to fight infections, a critical complication of TBI. We describe a targeted complement inhibitor, comprising complement receptor of the Ig superfamily (CRIg) fused with complement regulator CD59a, designed to inhibit membrane attack complex (MAC) assembly at sites of C3b/iC3b deposition. CRIg and CD59a were linked via the IgG2a hinge, yielding CD59-2a-CRIg dimer with increased iC3b/C3b binding avidity and MAC inhibitory activity. CD59-2a-CRIg inhibited MAC formation and prevented complement-mediated lysis in vitro. CD59-2a-CRIg dimer bound C3b-coated surfaces with submicromolar affinity (KD). In experimental TBI, CD59-2a-CRIg administered posttrauma homed to sites of injury and significantly reduced MAC deposition, microglial accumulation, mitochondrial stress, and axonal damage and enhanced neurologic recovery compared with placebo controls. CD59-2a-CRIg inhibited MAC-induced inflammasome activation and IL-1ß production in microglia. Given the important anti-infection roles of complement opsonization, site-targeted inhibition of MAC should be considered to promote recovery postneurotrauma.

Effect of polyunsaturated fatty acids and their metabolites on bleomycin-induced cytotoxic action on human neuroblastoma cells in vitro.

In the present study, we noted that bleomycin induced growth inhibitory action was augmented by all the polyunsaturated fatty acids (PUFAs) tested on human neuroblastoma IMR-32 (0.5 × 10(4) cells/100 µl of IMR) cells (EPA > DHA > ALA = GLA = AA > DGLA = LA: ∼ 60, 40, 30, 10-20% respectively) at the maximum doses used. Of all the prostaglandins (PGE1, PGE2, PGF2α, and PGI2) and leukotrienes (LTD4 and LTE4) tested; PGE1, PGE2 and LTD4 inhibited the growth of IMR-32 cells to a significant degree at the highest doses used. Lipoxin A4 (LXA4), 19,20-dihydroxydocosapentaenoate (19, 20 DiHDPA) and 10(S),17(S)-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid (protectin: 10(S),17(S)DiHDoHE), metabolites of DHA, significantly inhibited the growth of IMR-32 cells. Pre-treatment with AA, GLA, DGLA and EPA and simultaneous treatment with all PUFAs used in the study augmented growth inhibitory action of bleomycin. Surprisingly, both indomethacin and nordihydroguaiaretic acid (NDGA) at 60 and 20 µg/ml respectively enhanced the growth of IMR-32 cells even in the presence of bleomycin. AA enhanced oxidant stress in IMR-32 cells as evidenced by an increase in lipid peroxides, superoxide dismutase levels and glutathione peroxidase activity. These results suggest that PUFAs suppress growth of human neuroblastoma cells, augment growth inhibitory action of bleomycin by enhancing formation of lipid peroxides and altering the status of anti-oxidants and, in all probability, increase the formation of lipoxins, resolvins and protectins from their respective precursors that possess growth inhibitory actions.

Cellular resolution of inflammation--catabasis.

Controlled inflammation has become a central focus in chronic disease therapeutics. Exaggerated inflammation is a common factor that contributes to matrix destruction, cellular senescence, and nonhealing in a variety of disease processes. Efforts at controlling inflammation have traditionally concentrated on systemic antagonists to inflammation such as nonsteroidal anti-inflammatory agents. More recently, following increased appreciation of local biological cellular and molecular events occurring at the wound interface, efforts are being focused on local targeted approaches to catabasis, the resolution of inflammation. These efforts relate to the isolation and understanding of the mechanisms of actions of various "stop signal mediators." These lipoxins, resolvins, and protectins are produced and stimulated by cellular interactions in the blood stream, extracellular matrix, and in cells themselves. Transmission of these signals between cells and the extracellular matrix and between cells themselves occurs via a variety of mechanisms including through intracellular gap junctions, connexins, and cadherins. The existence of these mediators, signals, and channels of communication all provide new therapeutic options for achieving catabasis in a more defined and targeted fashion.

[Resolution of inflammation--pharmacological aspects]. / Rezolutia inflamatiei--implicatii farmacologice.

Inflammation is a fundamental biologic process evolutionally preserved by a germ line code. The interplay of the epigenetic with the environment directs the code to temporally distinct inflammatory responses, which can be acute or chronic. The aim of this study is to present new aspects regarding the resolution of inflammation. Acute inflammation normally resolves by mechanisms still somewhat elusive. Current evidence suggests that an active coordinated program initiated the first few hours after the inflammatory response begins and its failure lead to chronic inflammation. This process is essential for appropriate host responses, tissue protection and the return to homeostasis. Prostaglandins and leukotrienes are lipid mediators that play important roles in host defense and acute inflammation. Granulocytes promote the switch of arachidonic acid-derived prostaglandins and leukotrienes to lipoxins, active antiinflammatory and pro-resolution mediators. The apoptosis of the neutrophils coincides with the biosynthesis of resolvins and protectins from omega-3 polyunsaturated fatty acids and releases anti-inflammatory and reparative cytokines. This information could lead to new treatments for inflammatory diseases.

Complement component c1q mediates mitochondria-driven oxidative stress in neonatal hypoxic-ischemic brain injury.

Hypoxic-ischemic (HI) brain injury in infants is a leading cause of lifelong disability. We report a novel pathway mediating oxidative brain injury after hypoxia-ischemia in which C1q plays a central role. Neonatal mice incapable of classical or terminal complement activation because of C1q or C6 deficiency or pharmacologically inhibited assembly of membrane attack complex were subjected to hypoxia-ischemia. Only C1q(-/-) mice exhibited neuroprotection coupled with attenuated oxidative brain injury. This was associated with reduced production of reactive oxygen species (ROS) in C1q(-/-) brain mitochondria and preserved activity of the respiratory chain. Compared with C1q(+/+) neurons, cortical C1q(-/-) neurons exhibited resistance to oxygen-glucose deprivation. However, postischemic exposure to exogenous C1q increased both mitochondrial ROS production and mortality of C1q(-/-) neurons. This C1q toxicity was abolished by coexposure to antioxidant Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thus, the C1q component of complement, accelerating mitochondrial ROS emission, exacerbates oxidative injury in the developing HI brain. The terminal complement complex is activated in the HI neonatal brain but appeared to be nonpathogenic. These findings have important implications for design of the proper therapeutic interventions against HI neonatal brain injury by highlighting a pathogenic priority of C1q-mediated mitochondrial oxidative stress over the C1q deposition-triggered terminal complement activation.

[Effect of peptide seals specific to CD59 on the expression of apoptosis-related genes in HeLa cells].

AIM: To study the effect of peptide seals specific to CD59 on the expression of apoptosis-related survivin, caspase-3 and bax in HeLa cells and investigate the mechanism of peptide seals specific to CD59 in inducing apoptosis of HeLa cells. METHODS: The peptide seals specific to CD59 were put into HeLa cells and HeLa cells with CD59-transfected gene, respectively. After 24 hours of interaction, the inhibitory ratio of cell proliferation was measured by MTT and the expression of survivin, caspase-3 and bax was detected by immunohistochemistry. RESULTS: The inhibitory ratio of HeLa cells with CD59-transfected gene + peptide seal group was higher than that of HeLa cells + peptide seal group. There was a significant difference in depressing the proliferation between two groups (P < 0.01). Compared with HeLa cells + peptide seal group, the survivin expression of HeLa cells with CD59-transfected gene + peptide seal group was decreased (P < 0.05) while caspase-3 expression was increased (P < 0.05). There was no significant difference in the expression of bax in four groups. CONCLUSION: The peptide seal specific to CD59 can down-regulate the expression of survivin, activate caspase-3 and enhance the apoptosis of HeLa cells.

Novel chemical mediators in the resolution of inflammation: resolvins and protectins.

Resolvins and protectins are new families consisting of distinct chemical series of lipid-derived mediators, each with unique structures and apparent complementary anti-inflammatory actions. Both families of compounds, Rv and protectins, are also generated when aspirin is given in mammalian systems in their respective epimeric forms. The resolvins and protectins each dampen inflammation and PMN-mediated injury from within, which is a key culprit in many common human diseases. The results of these initial studies underscore the roles of resolvins and protectins in inflammation resolution as well as catabasis and spotlight the therapeutic potential for this new arena of immunomodulation and host protection. It is likely that the resolvins, protectins, and their AT-related forms may play roles in other tissues and organs. Moreover, it is noteworthy that fish (eg, trout) generate lipoxygenase products such as LXAs from endogenous EPA and also biosynthesize RvDs and protectins from endogenous DHA. Taken together, these findings suggest that these novel lipid mediators (eg, resolvins and protectins) are conserved in evolution as self-protective and host-protective chemical mediators. In view of the essential roles of DHA and EPA in human biology and medicine uncovered to date, the physiologic relevance of the resolvins and protectins is likely to extend beyond our current appreciation.

Protection of erythrocytes from human complement-mediated lysis by membrane-targeted recombinant soluble CD59: a new approach to PNH therapy.

Paroxysmal nocturnal hemoglobinuria (PNH) results from the expansion of a hematopoietic clone that is deficient in glycosylphosphatidylinositol-anchored molecules. PNH is characterized by chronic hemolysis with acute exacerbations due to the uncontrolled activity of complement on PNH cells, which lack the inhibitor of homologous complement, CD59. Symptoms include severe fatigue, hemoglobinuria, esophageal spasm, erectile dysfunction, and thrombosis. We report the use of a novel synthetically modified recombinant human CD59, rhCD59-P, a soluble protein that attaches to cell membranes. In vitro treatment of PNH erythrocytes with rhCD59-P resulted in levels of CD59 equivalent to normal erythrocytes and effectively protected erythrocytes from complement-mediated hemolysis. The administration of rhCD59-P to CD1 mice resulted in levels of CD59 on erythrocytes, which protected them from complement-mediated lysis. Thus, rhCD59-P corrects the CD59 deficiency in vitro and can bind to erythrocytes in an in vivo murine model, protecting the cells from the activity of human complement, and represents a potential therapeutic strategy in PNH.

Effects of CD59 on antitumoral activities of phycocyanin from Spirulina platensis.

The regulatory effect of phycocyanin (PC) from Spirulina platensis on cluster of differentiation 59 (CD59) gene expression of Hela cells and antitumoral mechanism of PC was investigated in this study. PC was purified by hydroxylapatite (HA) and sephacrylHR-200 gel-filtration columns chromatography. The molecular weight of PC was determined by SDS-PAGE electrophoresis. The CD59 cDNA was inserted into the eukaryotic expression plasmid pALTER-MAX, and the recombinant vector pALTER-MAX-CD59 was successfully constructed. By using cationic liposome (Lipfectamine-2000)-mediated transfection method, the recombinant plasmid pALTER-MAX-CD59 and the selective marker PcDNA were cotransfected into Hela cells and normal Chinese hamster ovary (CHO) cells. Stable positive cell clones were sorted out and disposed with different concentrates of PC. The expression of CD59 protein was determined by in situ hybridization, immunofluorescence and enzyme linked immunosorbent assay (ELISA). In addition, the effect of PC on the proliferation of Hela cells was determined by MTT method and the expression of Fas protein was by immunohistochemistry. Results showed that PC can promote the expression of CD59 protein in Hela cells, hold back it is reproductions of Hela cells, and moreover, a dosage effect was found between them. Namely, with the ascendance of PC concentration, the expression quantities of CD59 protein and apoptosis-inducing Fas protein increased and the multiplication activity of Hela cells declined, whereas PC was of no use to CD59 and Fas protein expression, and reproduction of normal CHO cells as well. Besides an imaginable antitumoral molecular immune mechanism of PC was brought forward and discussed.

Deletion of the gene encoding CD59a in mice increases disease severity in a murine model of rheumatoid arthritis.

OBJECTIVE: To investigate the roles of CD59a in the protection of joint tissue in the context of murine antigen-induced arthritis (AIA). METHODS: AIA was triggered in CD59a-deficient (CD59a(-/-)) mice and in CD59a-sufficient (CD59a(+/+)) controls; the course and severity of disease were compared between groups. The effects on arthritis of restoring CD59 to the joint in CD59a(-/-) mice by use of a membrane-targeted recombinant CD59 were also explored. RESULTS: Disease, as assessed clinically by measurement of joint swelling on day 1 (P < 0.0001), day 2 (P < 0.01), and day 7 (P < 0.02) and histologically from indicators of joint damage on day 21 (P < 0.02), was significantly enhanced in CD59a(-/-) mice compared with CD59a(+/+) wild-type controls. Membrane attack complex (MAC) deposition in the arthritic joints of CD59a(-/-) mice was also increased compared with that in the joints of CD59a(+/+) controls. Restitution of CD59 activity in joints of CD59a(-/-) mice was attempted with soluble recombinant rat CD59 (sCD59) or with a novel membrane-targeted rat CD59 derivative (sCD59-APT542). Strong immunohistochemical staining of the synovial membrane and subsynovial tissue was apparent in sCD59-APT542-injected joints, but not in joints injected with untargeted sCD59. Intraarticular administration of sCD59-APT542 markedly ameliorated disease severity in CD59a(-/-) mice, knee swelling was significantly reduced over the time course of the disease, and joint damage, assessed histologically, was significantly milder on day 21 (P < 0.05). CONCLUSION: These data firmly implicate the MAC of complement as a major effector of joint damage in the murine AIA model of rheumatoid arthritis (RA), and they provide a rationale for the inhibition of MAC assembly as a therapeutic strategy for RA.

Adenovirus-mediated expression of human CD55 or CD59 protects adult porcine islets from complement-mediated cell lysis by human serum.

BACKGROUND: Protection against complement activation may reduce acute islet damage in pig-to-human islet xenotransplantation. Expression of the human complement regulatory proteins decay-accelerating factor (DAF, CD55) or CD59 was induced on intact adult porcine islets (APIs) by adenoviral transduction. The functional capacity of the transgenes was examined in vitro after exposure to fresh human serum. METHODS: Intact APIs were transduced with adenoviral vectors Ad.hDAF or Ad.hCD59 or a control vector. After 3 days, the islets were trypsin dissociated to a single-cell suspension. A cytotoxicity assay was performed in which the islet cells were incubated with human complement active AB serum. Flow cytometry and immunohistochemistry were used to evaluate transgene expression. RESULTS: APIs could be transduced to express hDAF or hCD59. Flow cytometry analysis of islet single cells revealed that only a fraction of the cells expressed the transgene; immunohistochemical staining of transduced islets demonstrated that mainly cells located in the periphery of the islets were expressing the protein. Cells from nontransduced islets or islets expressing the control protein were sensitive to lysis in human sera (66+/-4.0% and 73+/-3.7% cytotoxicity, respectively). Single cells from islets transduced with hDAF and hCD59 were partially protected from lysis. Islet cells expressing hCD59 were slightly less sensitive to lysis (33+/-3.3%) than cells expressing hDAF (45+/-3.5%). CONCLUSIONS: These data show that intact pig islets can be transduced to express human regulators of complement activation on the surface and that pig islet cells expressing hDAF or hCD59 are less sensitive to complement-mediated lysis.

Human NK cell-mediated cytotoxicity triggered by CD86 and Gal alpha 1,3-Gal is inhibited in genetically modified porcine cells.

Delayed xenograft rejection is a major hurdle that needs to be addressed to prolong graft survival in pig-to-primate xenotransplantation. NK cell activation has been implicated in delayed xenograft rejection. Both Ab-dependent and independent mechanisms are responsible for the high susceptibility of porcine cells to human NK cell-mediated cytotoxicity. Previous reports demonstrated a role of Galalpha1,3-Gal Ag in triggering the Ab-independent responses. We hypothesize that expression of CD80 and/or CD86 on porcine cells may also play a role in NK cell activation as human NK cells express a variant of CD28. Our initial analysis showed that porcine endothelial cells and fibroblasts express CD86, but not CD80. Genetic engineering of these cells to express hCD152-hCD59, a chimeric molecule designed to block CD86 in cis, was accompanied by a reduction in susceptibility to human NK cell-mediated cytotoxicity. The use of a specific anti-porcine CD86-blocking Ab and the NK92 and YTS cell lines further confirmed the involvement of CD86 in triggering NK cell-mediated lysis of porcine cells. Maximal protection was achieved when hCD152-hCD59 was expressed in H transferase-transgenic cells, which show reduced Galalpha1,3-Gal expression. In this work, we describe two mechanisms of human NK cell-mediated rejection of porcine cells and demonstrate that genetically modified cells resist Ab-independent NK cell-mediated cytotoxicity.

CD59 prevents human complement-mediated injuries in isolated guinea pig hearts.

OBJECTIVE: To assess complement-mediated myocardial injury on isolated guinea pig working hearts and cardioprotective effects of CD59. METHODS: Using a modified Langendorff apparatus, isolated guinea-pig working hearts were perfused with a modified Krebs Henseleit buffer containing 3% heat-inactivated human plasma and zymosan (IPZ) (control) (n = 10), 3% normal human plasma and zymosan (NPZ) (n = 10), or 3% normal human plasma and zymosan and 1.5 microg/ml CD59 (NPZC) (n = 10), respectively. Epicardial electrocardiogram (ECG), cardiac output (CO), coronary arterial flow (CF), maximum left ventricular developed pressure (LVP(max)), maximum left ventricular developed pressure increase rate (+ dp/dt(max)), maximum left ventricular developed pressure decrease rate (- dp/dt(max)) and heart rate (HR) were recorded at 0, 15, 30, 45 and 60 min of treatment. After the experiment, immunohistochemical examination was performed to detect the presence of C3a or C5b-9 in the myocardium of the isolated hearts. RESULTS: Compared the IPZ group, hearts treated with NPZ showed a slight depression on ST segments of epicardial ECG at 15 min, a significant elevation between 30 min to 60 min, a decrease in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max), and an increase in HR at 15 min. The observed alterations in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max) remained decreased, while the HR remained increased until the end of the protocol. The all above parameters of hearts treated with NPZC were similar to the control group (IPZ) at any given time. Immunohistochemical examination showed positive signals of C3a and C5b-9 in the myocardium of hearts treated with NPZ. C3a was positive in NPZC, and C3a and C5b-9 were negative in IPZ. CONCLUSIONS: Activated human complements directly damage isolated guinea pig working hearts, and CD59 offers a significant protection against the injuries.

CD59 blocks not only the insertion of C9 into MAC but inhibits ion channel formation by homologous C5b-8 as well as C5b-9.

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b-9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b-9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59(-) human B-cells, which formed non-selective ion channels with a total conductance of 360 +/- 24 pS as measured at the beginning of the steady-state phase of the inward currents. C5b-8 and small-size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59(+) human B-cells in spite of small-size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59(-) cells inhibited the serum-evoked inward current. The ion channels formed by the small-size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b-8 as well as that of small-size C5b-9. These results offer an explanation as to why CD59-expressing cells are not leaky in spite of a buildup of homologous C5b-8 and small-size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.

Complement-mediated cell death induced by rituximab in B-cell lymphoproliferative disorders is mediated in vitro by a caspase-independent mechanism involving the generation of reactive oxygen species.

Mechanisms involving the in vitro effect of rituximab in cells from 55 patients with B-cell lymphoproliferative disorders were investigated. No cytotoxic effect was observed when cells were incubated with rituximab alone, but in the presence of human AB serum rituximab induced complement-dependent cell death (R-CDC). A cytotoxic effect was observed in cells from 9 of 33 patients with B-cell chronic lymphocytic leukemia, 16 of 16 patients with mantle-cell lymphoma, 4 of 4 patients with follicular lymphoma, and 2 of 2 patients with hairy-cell leukemia. R-CDC was observed in cells from patients expressing more than 50 x 10(3) CD20 molecules per cell, and directly correlated with the number of CD20 molecules per cell. Preincubation with anti-CD59 increased the cytotoxic effect of rituximab and sensitized cells from nonsensitive cases. Neither cleavage of poly-ADP ribose polymerase (PARP) nor activation of caspase-3 was observed in R-CDC. In addition, no cells with a hypodiploid DNA content were detected and R-CDC was not prevented by a broad-spectrum caspase inhibitor, suggesting a caspase-independent mechanism. Incubation with rituximab in the presence of AB serum induced a rapid and intense production of reactive oxygen species (ROS). R-CDC was blocked by the incubation of cells with N-acetyl-L-cysteine (NAC) or Tiron, 2 ROS scavengers, indicating that the cytotoxic effect was due to the generation of superoxide (O) radicals. In conclusion, the results of the present study suggest that CD20, CD59, and complement have a role in the in vitro cytotoxic effect of rituximab, which is mediated by a caspase-independent process that involves ROS generation.

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement.

BACKGROUND: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS: Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS: Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS: When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.

CD2-mediated CD59 stimulation in keratinocytes results in secretion of IL-1alpha, IL-6, and GM-CSF: implications for the interaction of keratinocytes with intraepidermal T lymphocytes.

Normal epidermal keratinocytes are here shown to express membrane-associated complement inhibitory protein CD59 in vitro that protects keratinocytes from damage by complement because preincubation with blocking antibodies to CD59 renders the cells susceptible to complement mediated lysis. CD59 expression in keratinocytes is constitutive and not modulated by inflammatory cytokines, phorbol myristate acetate (PMA), and a number of other agents tested. Antibody mediated cross-linking of CD59, however, revealed an additional function of CD59: keratinocytes in vitro are activated to secrete the cytokines IL-1alpha, IL-6, and GM-CSF. CD59 mediated induction of these cytokines is regulated at the transcriptional level. Binding of keratinocytes to HL60 cells that express CD59 ligand CD2 induced the same pattern of secreted cytokines whereas binding to CD2-negative HL60 cells did not. Induction of cytokine secretion was completely blocked by preincubation of keratinocytes with both anti-CD58 and anti-CD59 antibodies together. The results demonstrate that CD2-mediated CD59 stimulation in human keratinocytes leads to synthesis of a particular set of cytokines implying a potential activation pathway in the interaction of keratinocytes with intraepithelial CD2+ T cells.