Functional neurokinin 1 receptors for substance P are expressed by human vascular endothelium.

Substance P (SP), a neurotachykinin, is important in a number of inflammatory processes in which the endothelial cell also plays a critical role. SP receptors have previously been identified only on arterial endothelium, and the scant in vitro evidence for direct effects of SP on human endothelium is based on studies using nonarterial cells. To better understand SP's role in inflammation, we sought to identify functional SP receptors on human endothelium in situ and in culture. Autoradiographic ligand binding to human umbilical cord sections demonstrates the presence of SP binding sites with characteristics of the neurokinin 1 (NK-1) receptor (displacement by GTP analogues and the NK-1 specific antagonist CP-96,345) on human umbilical arterial, but not venous, endothelium. In culture, human umbilical venous endothelial cells (HUVECs) and human aortic endothelial cells express low levels of available SP binding sites. However, HUVECs, which are serum starved and refed, undergo a dramatic increase in SP binding. SP binding to starved/refed HUVECs induces a transient increase in intracellular calcium. This calcium flux is dose dependent over appropriate SP concentrations and can be blocked by NK-1 specific antagonists. The proinflammatory effects of SP may be mediated in part through the NK-1 receptor on endothelium.

Tumor necrosis factor alpha/cachectin stimulates eosinophil oxidant production and toxicity towards human endothelium.

Eosinophils (EOs) participate in a variety of inflammatory states characterized by endothelial cell damage, such as vasculitis, pneumonitis, and endocarditis. We find that 100 U/ml TNF-alpha/cachectin (TNF), a concentration attainable in the blood of humans with parasitic infestations, stimulates highly purified populations of EOs to damage human umbilical vein endothelial cells (HUVEC), a model of human endothelium. This TNF-dependent EO cytotoxicity is strongly inhibited by heparin and methyprednisolone but unaffected by the platelet-activating factor antagonist BN52012 or scavengers of superoxide anion and H2O2, superoxide dismutase and catalase. However, addition of a physiologically relevant concentration of Br- (100 microM) enhances EO/TNF damage to HUVEC, implicating the possible participation of EO peroxidase (EPO) in the killing mechanism. EOs adherent to FCS-coated plastic wells more than double their production of superoxide anion and the cytotoxic EPO-derived oxidant HOBr when exposed to TNF, showing that TNF activates the respiratory burst of EOs attached to a "physiologic" surface. Unlike PMNs, EOs were not irreversibly activated to kill unopsonized endothelium by previous exposure to TNF, and did not degranulate or upregulate CR3 expression as detected by Mo1 in the presence of 100 U/ml TNF. HUVEC exposed 18 h to TNF were considerably more susceptible to lysis by PMA-activated EOs and reagent H2O2, demonstrating a direct effect of TNF upon endothelium, perhaps through inhibition of antioxidant defenses. These findings suggest that abnormally elevated serum levels of TNF may provoke EOs to damage endothelial cells and thereby play a role in the pathogenesis of tissue damage in hypereosinophilic states.

Release of heparan sulfate from endothelial cells. Implications for pathogenesis of hyperacute rejection.

Heparan sulfate proteoglycan associated with endothelial cells in normal blood vessels inhibits intravascular coagulation and egress of blood cells and plasma proteins, key features of hyperacute rejection. It was shown herein that exposure of cultured porcine endothelium to human serum as a source of natural antibodies and complement caused cleavage and release of 5% of endothelial cell proteoglycans within 4 min and greater than 50% within 1 h. Proteoglycan release depended on activation of the classical complement pathway and preceded irreversible cell injury. These findings suggest that loss of endothelial cell proteoglycan may be a critical step in the pathogenesis of hyperacute rejection and in diseases involving humoral injury to endothelial cells.

Protection against lethal hyperoxia by tracheal insufflation of erythrocytes: role of red cell glutathione.

Intact erythrocytes placed into the tracheobronchial tree of hyperoxic rats dramatically improved their chances for survival. Over 70 percent of the animals so treated survived more than 12 days during continuous exposure to 95 percent oxygen, whereas all of the control animals died within 96 hours. Lungs from erythrocyte-protected rats showed almost none of the morphologic damage suffered by untreated animals. Erythrocytes containing cyanomethemoglobin were as beneficial as normal erythrocytes, but cells in which glutathione was partially blocked were significantly less protective. Analogous results were obtained in vitro: 51Cr-labeled target cells released 70 to 90 percent of their label when exposed briefly to hydrogen peroxide or to toxic oxygen species generated by phorbol ester-stimulated neutrophils. Addition of intact erythrocytes decreased release by approximately 75 percent, but significantly less than this if red blood cell glutathione was partially blocked. These results suggest that insufflated erythrocytes, through their recyclable glutathione, protect rats from toxic oxygen species engendered by hyperoxia.

Donor and recipient plasma follistatin levels are associated with acute GvHD in Blood and Marrow Transplant Clinical Trials Network 0402.

Follistatin is an angiogenic factor elevated in the circulation after allogeneic hematopoietic cell transplantation (HCT). Elevations in follistatin plasma concentrations are associated with the onset of and poor survival after acute GvHD (aGvHD). Using data from the Blood and Marrow Transplant Clinical Trials Network 0402 study (n=247), we sought to further quantify the longitudinal associations between plasma follistatin levels in transplant recipients, as well as baseline HCT donor follistatin levels, and allogeneic HCT outcomes. Higher recipient baseline follistatin levels were predictive of development of aGvHD (P=0.04). High donor follistatin levels were also associated with the incidence of aGvHD (P<0.01). Elevated follistatin levels on day 28 were associated with the onset of grade II-IV aGvHD before day 28, higher 1-year non-relapse mortality (NRM) and lower overall survival. In multivariate analyses, individuals with follistatin levels >1088 pg/mL at day 28 had a 4-fold increased risk for NRM (relative risk (RR)=4.3, 95% confidence interval (CI) 1.9-9.9, P<0.01) and a nearly three-fold increased overall risk for mortality (RR=2.8, 95% CI 1.5-5.2, P<0.01). Given the multiple roles of follistatin in tissue inflammation and repair, and the confirmation that this biomarker is predictive of important HCT outcomes, the pathobiology of these relationships need further study.

Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin.

Human neutrophils (PMN), when stimulated with such chemotaxins as phorbol myristate acetate (PMA), destroy erythrocytes and other targets. Cytotoxicity depends on PMN-generated reactive oxygen metabolites, yet the exact toxic specie and its mode of production is a matter of some dispute. Using 51Cr-labeled erythrocytes as targets, we compared various reactive-O2 generating systems for their abilities to lyse erythrocytes as well as to oxidize hemoglobin to methemoglobin. PMA-activated PMNs or xanthine oxidase plus acetaldehyde were added to target erythrocytes in amounts that provided similar levels of superoxide. PMNs lysed 68.3 +/- 2.9% (SEM) of targets, whereas the xanthine oxidase system was virtually impotent (2.3 +/- 0.8%). In contrast, methemoglobin formation by xanthine oxidase plus acetaldehyde was significantly greater than that caused by stimulated PMNs (P less than 0.001). A similar dichotomy was noted with added reagent H2O2 or the H2O2-generating system, glucose plus glucose oxidase; neither of these caused 51Cr release, but induced 10-70% methemoglobin formation. Thus, although O2- and H2O2 can cross the erythrocyte membrane and rapidly oxidize hemoglobin, they do so evidently without damaging the cell membrane. That a granule constituent of PMNs is required to promote target cell lysis was suggested by the fact that agranular PMN cytoplasts (neutroplasts), although added to generate equal amounts of O2- as intact PMNs, were significantly less lytic to target erythrocytes (P less than 0.01). Iron was shown to be directly involved in lytic efficiency by supplementation studies with 2 microM iron citrate; such supplementation increased PMN cytotoxicity by approximately 30%, but had much less effect on erythrocyte lysis by neutroplasts (approximately 3% increase), and no effect on lysis in the enzymatic oxygen radical-generating systems. These results suggest a critical role for an iron-liganding moiety that is abundantly present in PMN, marginally so in neutroplasts, and not at all in purified enzymatic systems--a moiety that we presume catalyzes very toxic O2 specie generation in the vicinity of juxtaposed erythrocyte targets. The obvious candidate is lactoferrin (LF), and indeed, antilactoferrin IgG, but not nonspecific IgG, reduced PMN cytotoxicity by greater than 85%. Re-adding 10(-8) M pure LF to neutroplasts increased their ability to promote hemolysis by 48.4 +/- 0.9%--to a level near that of intact PMNs. We conclude that O-2 and H2O2 are not sufficient to mediate target cell lysis, but require iron bound to LF, which, in turn, probably generates and focuses toxic O2 radicals, such as OH, to target membrane sites.

Molecular mimicry in Candida albicans. Role of an integrin analogue in adhesion of the yeast to human endothelium.

Hematogenous infection with the yeast Candida albicans now occurs with increasing frequency in the neonate, the immunocompromised patient, and the hyperglycemic or hyperalimented host. Yeast-phase C. albicans expresses a protein that is antigenically and structurally related to CD11b/CD18, a member of the beta 2 integrins and a well-characterized adhesin for mammalian neutrophils. Both the neutrophil protein and its analogue in C. albicans have an identical affinity for the C3 ligand iC3b, and both proteins are significantly increased in expression at 37 degrees C. Given these several similarities, we therefore studied the role of the integrin analogue on C. albicans in the adhesion of the yeast to human umbilical vein endothelium (HUVE). After growth of C. albicans in 20 mM D-glucose, as opposed to 20 mM L-glutamate, flow cytometric analysis with monoclonal antibodies recognizing the alpha-subunit of CD11b/CD18 demonstrated a 25.0% increase in mean channel fluorescence (range 18.4-31.8%), as well as an increased percentage of yeasts fluorescing (P less than 0.02). This increased intensity of fluorescence, which corresponds to increased expression of the integrin analogue, also correlated with a significant increase of 30-80% in adhesion of glucose-grown C. albicans to HUVE (P less than 0.02). Blockade of the integrin analogue on C. albicans by monoclonal antibodies recognizing adhesive epitopes on neutrophil CD11b/CD18 inhibited glucose-enhanced adhesion of C. albicans to HUVE. Incubation of glucose-grown C. albicans with saturating concentrations of purified human iC3b, the ligand for CD11b/CD18, reduced adhesion of the yeast to HUVE by 49.7%, whereas BSA in equimolar concentration had no effect (P less than 0.001). These results identify a glucose-responsive integrin analogue on C. albicans as one of possibly several cellular structures that mediate adhesion of the yeast to human endothelium.

Eosinophil cationic granule proteins impair thrombomodulin function. A potential mechanism for thromboembolism in hypereosinophilic heart disease.

Thromboembolism is a prominent but poorly understood feature of eosinophilic, or Loeffler's endocarditis. Eosinophil (EO) specific granule proteins, in particular major basic protein (MBP), accumulate on endocardial surfaces in the course of this disease. We hypothesized that these unusually cationic proteins promote thrombosis by binding to the anionic endothelial protein thrombomodulin (TM) and impairing its anticoagulant activities. We find that MBP potently (IC50 of 1-2 microM) inhibits the capacity of endothelial cell surface TM to generate the natural anticoagulant activated protein C (APC). MBP also inhibits APC generation by purified soluble rabbit TM with an IC50 of 100 nM without altering its apparent Kd for thrombin or Km for protein C. This inhibition is reversed by polyanions such as chondroitin sulfate E and heparin. A TM polypeptide fragment comprising the extracellular domain that includes its naturally occurring anionic glycosaminoglycan (GAG) moiety (TMD-105) is strongly inhibited by MBP, whereas its counterpart lacking the GAG moiety (TMD-75) is not. MBP also curtails the capacity of TMD-105 but not TMD-75 to prolong the thrombin clotting time. Thus, EO cationic proteins potently inhibit anticoagulant activities of the glycosylated form of TM, thereby suggesting a potential mechanism for thromboembolism in hypereosinophilic heart disease.

Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat.

Heme proteins such as myoglobin or hemoglobin, when released into the extracellular space, can instigate tissue toxicity. Myoglobin is directly implicated in the pathogenesis of renal failure in rhabdomyolysis. In the glycerol model of this syndrome, we demonstrate that the kidney responds to such inordinate amounts of heme proteins by inducing the heme-degradative enzyme, heme oxygenase, as well as increasing the synthesis of ferritin, the major cellular repository for iron. Prior recruitment of this response with a single preinfusion of hemoglobin prevents kidney failure and drastically reduces mortality (from 100% to 14%). Conversely, ablating this response with a competitive inhibitor of heme oxygenase exacerbates kidney dysfunction. We provide the first in vivo evidence that induction of heme oxygenase coupled to ferritin synthesis is a rapid, protective antioxidant response. Our findings suggest a therapeutic strategy for populations at a high risk for rhabdomyolysis.

Potentiated adherence of sickle erythrocytes to endothelium infected by virus.

Systemic viral infection is a known precipitant of vasocclusive crisis in sickle patients, but the mechanism underlying this clinical observation is unknown. In the present studies, human umbilical vein endothelial cells were infected with Herpes simplex virus type 1 (HSV) to model systemic viral disease. The already abnormal adherence of sickle erythrocytes to control endothelium is enhanced 1.8 +/- 0.4-fold to HSV-infected endothelium (P less than 0.001). This component of potentiated adherence is eliminated by maneuvers that block Fc receptors, it is prevented by tunicamycin, and it is not seen using a mutant HSV that is unable to express the Fc receptor glycoprotein. Thus, the incremental adherence seen here occurs due to expression of Fc receptor activity on HSV-infected endothelium and the consequent recognition of abnormal amounts of IgG on sickle erythrocytes. We conclude that systemic viral infection potentially can induce a novel mechanism for enhancement of erythrocyte adherence to endothelium and that this may increase the likelihood of vasocclusion during viral infection.

Tumor cell heme uptake induces ferritin synthesis resulting in altered oxidant sensitivity: possible role in chemotherapy efficacy.

Neovascularization and hemorrhage are common features of malignant tumors. We wondered whether hemoglobin derived from extravasated RBC deposits heme-derived iron into the tumor, which could modulate the sensitivity of cancer cells to oxidant-mediated injury. A brief exposure (1 h) of 51Cr-radiolabeled breast cancer cells (BT-20) but not colon cancer cells (Caco-2) to hemin (10 microM) or FeSO4 (10 microM) significantly enhances cytotoxicity mediated by 0.5 mM hydrogen peroxide (H2O2). Associated with Caco-2 resistance, these cells were found to be enriched in the endogenous iron chelator, ferritin. If cellular ferritin is even further increased through 1 h incubation (24 h prior to H2O2 exposure) of both cell types with hemin, FeSO4, or exogenous spleen apoferritin itself (24 h), marked resistance to H2O2-mediated cytotoxicity is manifest. Under several conditions, the sensitivity of tumor cells to oxidant-mediated lysis is inversely proportional to their ferritin content. Pretreatment of BT-20 and Caco-2 cells with hemin or FeSO4 rapidly increases H-ferritin mRNA but only slightly increases L-ferritin mRNA; nevertheless, large increases in overall ferritin content of iron-exposed cells result. Data analogous to those with H2O2-mediated cytotoxicity were obtained in studies of bleomycin-engendered DNA strand breakage and cell damage, i.e., brief treatment of BT-20 cells with both hemin or FeSO4 significantly increases their sensitivity to bleomycin (100 micrograms/ml), whereas treatment followed by 24 h incubation with media alone significantly protects against bleomycin toxicity. We speculate that acute exposure of tumors to iron (e.g., derived from heme-proteins in hemorrhagic cancerous lesions) may increase sensitivity of some cancer cells, particularly those relatively low in endogenous ferritin, to oxidant-mediated lysis. In contrast, repeated, more chronic, exposure effector cells or chemotherapeutic agents, an effect derived from their increased synthesis and accumulation of the intracellular iron scavenger, ferritin.

Mechanism of cultured endothelial injury induced by lymphokine-activated killer cells.

A new form of therapy of experimental tumors, utilizing lymphokine-activated killer (LAK) cells and high doses of interleukin 2, has recently been applied in the treatment of human neoplasms. Severe side effects, suggestive of a diffuse vascular injury of unknown etiology, have prevented a more widespread application of this form of therapy. We have investigated the etiology of this clinical capillary leak syndrome, using an in vitro model of endothelial injury. LAK cells, but not interleukin 2 itself, are cytotoxic to cultured human endothelial cells, and this cytotoxicity is time and dose dependent. This human endothelial cell cytotoxicity can be inhibited by depletion of extracellular Ca2+, inhibition of the effector cell microtubular system, and inhibitors of serine proteases, but is not inhibited in the presence of toxic oxygen radical scavengers. LAK cell-mediated endothelial cytotoxicity is far more potent than that exhibited by maximally stimulated polymorphonucleocytes. LAK cell-mediated injury of human endothelium may possibly be responsible for the capillary leak syndrome observed in patients treated with high doses of interleukin 2 and LAK cells.

Cytomegalovirus inhibits p53 nuclear localization signal function.

Endothelial cells (EC) infected with the VHL strain of cytomegalovirus (CMV) are resistant to p53-mediated apoptosis, which may be relevant to EC dysfunction and atherogenesis. This resistance to apoptosis may be mediated by cytoplasmic sequestration of p53, which functions only in the nucleus. We explored the hypothesis that CMV sequesters p53 in the cytoplasm by blocking p53 nuclear localization signal (NLS) function. We transfected VHL CMV infected EC with recombinant p53 NLSI conjugated with chicken muscle pyruvate kinase (PK) plasmid. NLSI is responsible for 90% of p53 nuclear localization, and PK is not normally translocated to the nucleus after cytoplasmic production. Thus it cannot be localized in the nucleus without the assistance of the artificial NLSI. A double-labeling immunofluorescence staining method was used to identify the localization of p53 NLSI-conjugated PK in CMV-infected EC. We found that CMV infection sequesters PK and p53 in the cytoplasm by blocking NLSI function. This inactivation of NLSI function is dependent upon infection stage; it occurs only in the early and late phases and not the immediate early phase of infection. These findings may be relevant to endothelial dysfunction and initiation of atherogenesis. Our study also suggests a novel mechanism of the p53 inactivation by virus, which may be important for atherogenesis and tumorgenesis.

Oxidation of hemoglobin by lipid hydroperoxide associated with low-density lipoprotein (LDL) and increased cytotoxic effect by LDL oxidation in heme oxygenase-1 (HO-1) deficiency.

Heme-catalyzed oxidation of low-density lipoprotein (LDL) is one of the relevant mechanisms involved in LDL modification. We previously revealed a substantial oxidation of plasma hemoglobin to methemoglobin and a subsequent heme-catalyzed LDL oxidation generating moieties toxic to endothelium in heme oxygenase-1 (HO-1)-deficiency in human. Drawing upon our previous observation we posited a pathway for oxidation of plasma hemoglobin in the HO-1-deficient child involving LDL-associated lipid hydroperoxide. In support, LDL-associated lipid hydroperoxide oxidized ferrohemoglobin to methemoglobin--known to readily release its heme moieties--in a dose-dependent manner. Repeated heme exposure of the child s LDL further increased its lipid hydroperoxide content within min leading to additional cytotoxic effect on endothelium. Both cytotoxicity and HO-1 inducing ability of the oxidized LDL were strongly dependent on its lipid hydroperoxide content. We wondered if cells of the HO-1-deficient patient were prone to oxidative damage arising from heme-mediated oxidation of LDL. Indeed, we found elevated cytotoxicity induced by heme-catalyzed oxidation of LDL in lymphoblastoid cells derived from the HO-1-deficient patient. We conclude that oxidation of hemoglobin to methemoglobin by LDL-associated lipid hydroperoxide and increased sensitivity of cells of the HO-1-deficient child to stress of oxidized LDL might contribute to the vascular disorders reported earlier.

Thrombin-treated endothelium primes neutrophil functions: inhibition by platelet-activating factor receptor antagonists.

We have shown previously that fluid phase platelet-activating factor (PAF) can enhance or "prime" polymorphonuclear (PMN) responses to subsequent stimulation with agonists such as formyl-methionine-leucine-phenylalanine (FMLP). Since thrombin induces PAF production in endothelial cells, we tested whether this thrombin-provoked endothelial PAF primes responses of marginated PMNs. Monolayers of human umbilical vein endothelial cells were exposed to either thrombin (0.5-5.0 units/ml) or buffer for up to 5 min and then PMNs were layered on top of the endothelial cells. After a further 5 min incubation, the PMNs were stimulated with a suboptimal concentration of FMLP (10(-7) M), and their superoxide production, elastase release, adhesion to endothelium, and capacity to cause endothelial cell lysis and detachment were assessed. Thrombin pretreatment significantly enhanced each of these FMLP-stimulated neutrophil responses. The extent of this enhancement correlated with both the dose and duration of thrombin treatment of endothelial cells and also the duration of PMN incubation with thrombin-exposed endothelium. Evidence that the augmentation was due to endothelial-derived PAF was obtained as follows: (1) thrombin induced [3H]acetate incorporation into endothelial PAF (assayed in lipid extracts); (2) antithrombin III conjointly inhibited this [3H]acetate uptake and prevented the priming effect of thrombin-treated endothelium on PMN responses; and (3) the PAF receptor antagonist BN52021, when preincubated with PMNs, also effectively blocked the enhancement of PMN responses. We conclude that thrombin stimulation of endothelial cells initiates a sequence of events culminating in the production of PAF--a membrane phospholipid capable of priming marginated PMNs. We suggest that this coagulation-fostered endothelial/PMN interaction may underlie a paracrine response that may potentiate PMN-mediated endothelial injury during sepsis and other thrombin-generating disorders.

Potential role of viruses in thrombosis and atherosclerosis.

Viruses have been implicated as triggers of endothelial injury initiating the process of atherosclerosis. Because patients with atherosclerosis suffer acute events associated with arterial thrombi obstructing a vital vessel, the role of viruses in promoting a procoagulant endothelium has been explored. This review targets the evidence that viruses contribute to atherogenesis, and discusses mechanisms by which specific herpes viruses promote inflammation and thrombosis.

Nitric oxide donors modulate ferritin and protect endothelium from oxidative injury.

Ferritin protects endothelial cells from the damaging effects of iron-catalyzed oxidative injury. Regulation of ferritin occurs through the formation of an iron-sulfur cluster within a cytoplasmic protein, the iron regulatory protein (IRP) that controls ferritin mRNA translation. Nitric oxide has been shown to inhibit iron-sulfur proteins and is present at vascular sites of inflammation; therefore, we undertook a study to examine the influence of nitric oxide on changes in endothelial cell ferritin content in response to iron exposure, and the subsequent effects on susceptibility to oxidative injury. Iron-loaded endothelial cells (EC) exposed to nitric oxide donors synthesize markedly less ferritin. Treatment of EC with a nitric oxide donor increases IRP affinity for ferritin mRNA concomitant with a loss of cytoplasmic aconitase activity in iron-laden EC. Iron-treated EC exposed to NO donors were resistant to oxidative injury despite their low ferritin content when examined 1 h after the treatment period. In contrast, 24 h later, these same cells become sensitive to oxidants, whereas iron-treated EC that are ferritin-rich continue to be resistant. In conclusion, NO inhibits the increase of EC ferritin after exposure to iron but provides short-term protection against oxidants; ferritin, in turn, provides durable cytoprotection by inactivating reactive iron.

Human cytomegalovirus immediate early proteins upregulate endothelial p53 function.

Infected endothelial cells are found to be resistant to apoptosis possibly mediated by p53 cytoplasmic sequestration. We explored whether the immediate early 84 kDa protein (IE84) of cytomegalovirus (CMV) is responsible for p53 cytoplasmic sequestration. The endothelial cells were transfected with plasmids containing IE1 and 2 coding regions which are known to synthesize IE84 and 72 proteins. Our study found that p53 expression was significantly elevated in endothelial cells transfected with IE1 and 2 plasmids. However, p53 was only found in the nucleus rather than sequestered in the cytoplasm. We have demonstrated that IE84 and 72 are not responsible for p53 dysfunction caused by CMV infection, rather they upregulate p53 function and promote endothelial apoptosis.

Inhibition of complement-mediated endothelial cell cytotoxicity by decay-accelerating factor. Potential for prevention of xenograft hyperacute rejection.

Complement plays a major role in hyperacute rejection of discordant xenografts. In immediately vascularized xenografts, such as porcine organs to humans, C activation contributes to triggering of endothelial cell activation and adhesion of leukocytes and platelets to the endothelial cells, which is followed by thrombosis and tissue necrosis. We investigated the potential utility of the membrane-associated inhibitor of C, decay accelerating factor (DAF), in the prevention of C-mediated tissue injury. We used an in vitro model of xenotransplantation consisting of porcine aortic endothelial cells incubated with human serum as the source of xenogeneic natural antibodies and C. Because C inhibitors such as DAF may be relatively species-specific, we tested whether human DAF would incorporate into porcine endothelial cells and function to inhibit cytotoxicity of such cells by human C. We found that purified radiolabeled human DAF incorporated into porcine endothelial cells in a dose-dependent manner and that human DAF very significantly protected the endothelial cells from the cytotoxic effect of human C.