Induction of human complement activation without cytolysis by mouse monoclonal antibodies to human leukocyte antigens

Ten monoclonal antibodies to human leukocyte subsets that had previously been shown to lyse their respective target cells in the presence of rabbit serum as complement source were evaluated for their cytolytic capacity with human complement. Four of the ten were lytic with human complement. All were of IgM type. Antibodies were also evaluated for their capacity to induce C3 binding to target cells. With this method we could demonstrate that, indeed, 3 of the 6 noncytolytic antibodies had the capacity to initiate the human complement activation process and to induce C3 binding. Two of these 3 antibodies were of IgM class (VIT3 and VIM13), one of IgG3 (562). From the practical point of view the most interesting of these 3 antibodies is the nonmitogenic anti-CD3 pan-T cell antibody VIT3. Therefore, this antibody was analyzed in more detail. VIT3 antibody concentrations needed to induce detectable C3 binding to human T cells are very low (down to 1 ng VIT3/ml). Human serum as complement source can also be considerably (100X) diluted before C3 binding becomes undetectable. Activation of C3 is a prerequesite for VIT3-induced C3 binding, and bound C3 seems to lack the C3a fragment. Bound C3, in contrast to the quickly occuring antigenic modulation of the CD3 complex and the simultaneous disappearance of the antibody coat, remains expressed also after prolonged incubation at 37 degrees C. C3 fragments bound to T cells after activation with VIT3 are also recognized by cells bearing C3 receptors of types CR1 and CR2.     

Variability of anti-αGal antibodies in human serum and their relation to serum cytotoxicity against pig cells

Abstract: One of the major obstacles in pig-to-human xenografting is hyperacute rejection (HAR) of pig cells caused by preformed anti-pig antibodies and complement. In 1991 we suggested that anti-αGal antibodies play a major role in the HAR of pig cells. Anti-αGal antibodies recognize terminal α-galactose-containing epitopes on glycoproteins and glycolipids. They are present in humans, apes, and Old World monkeys, but not in lower mammals such as pigs. However, pigs, unlike humans, express terminal α-galactose epitopes on vascular endothelium which represent targets for human anti-αGal antibodies. Despite increasing recognition that anti-αGal antibodies are an important factor, many questions related to their precise role in HAR remain to be answered.
In this study, we analyzed 75 human AB sera in terms of (i) cytotoxic activity against cultured pig (PK-15) cells, (ii) anti-αGal titers, (iii) immunoglobulin-binding to pig cells, and (iv) immunoglobulin concentrations. The results demonstrated considerable variability in cytotoxicity, anti-αGal titers, and immunoglobulin binding to pig cells, whereas the serum immunoglobulin concentrations were less variable. Positive correlations were found between cytotoxicity and binding of IgG and IgM to the surface of pig cells. The surface-bound IgG and IgM also correlated with the serum anti-αGal IgG and IgM titers. Anti-αGal IgA, however, did not show any relation with cytotoxicity or cell binding. Concentrations of serum total immunoglobulins correlated with neither I cytotoxic activity nor cell binding.     

Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.     

Microcarrier enhanced survival of human and rat fetal ventral mesencephalon cells implanted in the rat striatum

The transplantation of tissue containing dopamine-producing cells into the mammalian central nervous system is an emerging treatment for Parkinson's disease, despite relatively poor survival of implanted tissue. Recent evidence has suggested that Cytodex microcarriers enhance the survival of dopaminergic rat chromaffin cells transplanted into the rat striatum in the absence of immunosuppression. The current study was undertaken to evaluate the survival of rat and human fetal ventral mesencephalic neurons (VM) implanted alone or after attachment to microcarriers in the striatum of rats without immuno suppression. Rat fetal VM neurons demonstrated enhanced survival in the rat striatum when transplanted on microcarriers, compared to their transplantation alone during the 3-mo period examined in the present study. Transplants of human fetal VM neurons on microcarriers also survived remarkably well in the rat striatum without systemic immunosuppression. In contrast, human fetal VM cells transplanted alone into the rat striatum did not survive without systemic immunosuppression. There was no evidence of TH fiber sprouting in the vicinity of any transplant site. These data indicated that Cytodex microcarriers provide enhanced survival of both rat allograft and human xenograft fetal mesencephalic cells in the rat striatum without the necessity of systemic immunosuppression, perhaps by inducing a unique neuron-glia environment.     

Reactivity to human islets and fetal pig proislets by peripheral blood mononuclear cells from subjects with preclinical and clinical insulin-dependent diabetes

A simple, direct assay for T-lymphocyte reactivity to islet antigen(s) in human insulin-dependent diabetes mellitus (IDDM) should facilitate preclinical diagnosis and the evaluation of intervention therapy to avert autoimmune-mediated beta-cell destruction. In subjects with preclinical or clinical IDDM, we measured the reactivity of peripheral blood mononuclear cells (PBMCs) incubated over 6 days with either adult human islets or fetal pig proislets, or other fetal pig tissues, and with human insulin. With islets, the stimulation index (SI) of [3H]thymidine uptake by PBMCs exceeded the mean + 2SD of control subjects in 6 of 6 preclinical subjects (SI 8.7 +/- 3.7), 7 of 11 clinical subjects (SI 5.2 +/- 3.4), and 1 of 12 control subjects (SI 2.7 +/- 1.7); with insulin, the responses were less in frequency and magnitude, being 4 of 6 (2.7 +/- 1.6), 3 of 11 (2.2 +/- 1.1), and 0 of 12 (1.20 +/- 0.55), respectively. The mean responses to islets of PBMCs from preclinical and clinical subjects differed significantly from control subjects (P less than 0.02 by 2-tailed Kruskal-Wallis test). Secretion of granulocyte macrophage colony-stimulating factor by PBMCs over 6 days was assayed in the preclinical group and generally paralleled the uptake of [3H]thymidine. PBMC reactivity to islets appeared to be at least as sensitive a marker of preclinical IDDM as autoantibodies to a 64,000-Mr protein, presumably the enzyme glutamic acid decarboxylase, in fetal pig proislets. In conclusion, islet-reactive T lymphocytes in subjects with preclinical and clinical IDDM can be identified in bulk culture of PBMCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sensitivity to human serum of gammaretroviruses produced from pig endothelial cells transduced with glycosyltransferase genes

Abstract: Reduction of pig cell-surface α-galactosyl (Gal) epitope, Galα1, 3Galβ1, 4GlcNAc-R, by the introduction of glycosyltransferase genes is effective in suppressing hyperacute rejection (HAR) in pig- to-human xenotransplantation. The transmission of porcine endogenous retroviruses (PERVs) has been recognized as a potential risk factor associated with xenotransplantation. In this study, effects of the introduction of glycosyltransferase genes to pig cells on the sensitivity of gammaretroviruses to human serum were investigated. Pig endothelial cells (PEC), PEC transduced with α1,2 fucosyltransferase (FT), α2,3 sialyltransferase (ST) or N -acetylglucosaminyltransferase III (GnT-III), and human embryonic kidney (HEK) 293 cells were transduced with the LacZ gene with the packaging signal of murine leukemia virus (MuLV) under the control of the long terminal repeat of MuLV by a pseudotype infection. Then, the cells were further infected with PERV subtype B (PERV-B) or feline leukemia virus subgroup B (FeLV-B). Culture supernatants of the infected cells were mixed with human serum (HS) and then inoculated to HEK293 cells. The inoculated cells were histochemically stained and lacZ -positive blue foci were counted. Glycosyltransferase activity, xenoantigenicity, and α-Gal epitope density in the cells were measured at the time of the infection experiments. PERV-B or FeLV-B particles from the parental PEC were efficiently neutralized by HS, while those from PEC transduced with α1,2FT, α2,3ST or GnT-III were less sensitive to HS. The transduced PEC exhibited high levels of activity of the introduced glycosyltransferases, and expressed fewer xenoantigens and cell-surface α-Gal epitopes. Our results suggest that gammaretroviruses including PERVs produced by transgenic pigs, that are genetically modified to reduce the cell-surface α-Gal epitope to overcome the HAR in xenotransplantation, are less sensitive to HS.     

The effect of complement regulatory protein expression on pig endothelial cells to porcine endogenous retrovirus lyses by human sera

INTRODUCTION: Expression of human complement regulatory proteins (CRP) on pig endothelial cells (PEC) has been useful to avoid hyperacute rejection by human sera. On the other hand, porcine endogenous retrovirus (PERV) from PEC transfectants with CRP may acquire resistance to human sera. In this study, we investigated the effects of the transfected CRP on PERV neutralization and/or lysis by human sera. METHODS: cDNA of membrane cofactor protein (MCP: CD46), decay accelerating factor (DAF: CD55), and CD59 were transfected to PEC lines by lipofection. The expressions of these CRPs were verified by FACS analysis. The PEC lines with human CRPs were then transfected with the LacZ gene and PERV subtype B (PERV-B) to investigate PERV infectivity by LacZ pseudotype assay. Culture supernates of PEC were inoculated to HEK293 cells with or without 10% human sera. The inoculated 293 cells were then histochemically stained to count the LacZ-positive blue foci and calculated the rate of reduction of LacZ-positive cells by serum. RESULTS: PERV from the PEC with DAF or CD59 showed a resistance to human sera compared with those of control PEC (DAF: 59.6% +/- 5.3%, CD59: 61.1% +/- 3.9% vs control: 31.3% +/- 3.6%; P < .01). However, PEC with MCP did not cause such an effect (28.8% +/- 2.5%). CONCLUSIONS: While expression of DAF and CD59 on PEC changed its PERV responsiveness to human sera, MCP did not improve it.     

The effect of pH and nucleophiles on complement activation by human proximal tubular epithelial cells.

BACKGROUND: Activation of urinary complement proteins in situ by proximal tubular epithelial cells (PTEC) may contribute to the mediation of tubulointerstitial injury in patients with significant proteinuria. However, the mechanism involved is unclear, and the role of changes in urinary pH and in the concentrations of urea or ammonia requires further clarification. METHODS: The protein fraction of urine samples from nine patients with proteinuria >1.5 g/day was purified. A cell ELISA involving cultured HK-2 PTEC was used to investigate the capacity of urinary protein to promote the deposition of both C3 and C9 on the cell surface. The effect of variations in pH (5.5-8.0) and in the concentration of urea and ammonia was also examined. C3 was purified and used to further investigate the mechanism of complement deposition. RESULTS: Urine samples from the majority of patients induced deposition of C3 and C9 on the surface of HK-2 cells via the alternative pathway. This process was maximal at acidic pH values. Preincubation of urinary complement or serum with urea or ammonia inhibited C3 deposition. Purified C3 incubated with HK-2 cells showed no evidence of activation in the absence of other complement components. CONCLUSIONS: These data suggest that bicarbonate protects against complement-mediated damage in the lumen by increasing the local pH, rather than by inhibiting the generation of ammonia. PTEC appear to activate complement through provision of a 'protected site' on their surface, rather than by the activation of C3 by convertase-like protease(s).     

Protection of hDAF-transgenic porcine endothelial cells against activation by human complement: role of the membrane attack complex

Xenograft rejection in the discordant pig-to-primate model is dependent on binding of natural antibodies to gal-α [ 1 –3 ]-gal epitopes on the porcine endothelial cell (EC). This leads to complement activation and deposition of activation products onto the membrane and results in perturbation of EC function and thrombus formation. Here we investigated the ability of human complement activation products to directly induce activation of porcine EC, with subsequent upregulation of adhesion and pro-coagulant molecules. Porcine aortic EC were isolated from wild-type and hDAF-transgenic pigs and incubated with human serum, either in the presence or absence of the soluble complement inhibitor TP10 (sCR1). Recombinant C5a, C1q-IgG immune complexes, C6-deficient human serum and serum containing anti-C9 Ab were used to identify EC activating complement products. Heat-inactivated human serum was used as a negative control. Cells were stained with antibodies against human C3, the MAC or with antibodies cross-reactive for porcine E-Selectin, VCAM-1 or Tissue Factor, and analyzed by flow cytometry. We found upregulation of E-Selectin and Tissue Factor on wild-type EC after incubation with human serum. This effect coincided with the deposition of C3 and MAC on the membrane of these cells. The addition of TP10 inhibited EC activation by up to 95%. In contrast, greatly reduced C3 and MAC deposition was detected on hDAF transgenic cells, and no complement-mediated EC activation was seen. Experiments with C6-deficient serum and incubation with anti-C9 Ab indicate a major role of the MAC in serum-mediated EC activation, whereas neither C5a nor C1q-IgG caused activation of EC. These data provide further explanation of the protective role of human DAF in the pig-to-primate xenotransplantation model.     

Ouabain binds with high affinity to the Na,K-ATPase in human polycystic kidney cells and induces extracellular signal-regulated kinase activation and cell proliferation

In autosomal dominant polycystic kidney disease (ADPKD), cyst formation and enlargement require proliferation of mural renal epithelial cells and the transepithelial secretion of fluid into the cyst cavity. Na,K-ATPase is essential for solute and water transport in ADPKD cells, and ouabain blocks fluid secretion in these cells. By binding to the Na,K-ATPase, ouabain also induces proliferation in some cell types. Surprisingly, it was found that nanomolar concentrations of ouabain, similar to those circulating in blood, induced ADPKD cell proliferation but had no statistically significant effect on normal human kidney (NHK) cells. Ouabain, acting from the basolateral side of the cells, also caused an increase in the level of phosphorylated extracellular signal-regulated kinases (ERK). Mitogen-activated protein kinase kinase (MEK) inhibitor U0126 blocked ouabain-induced ERK activation and cell proliferation, suggesting that ouabain effect is mediated through the MEK-ERK pathway. In contrast to NHK cells, the dose-response curve for ouabain inhibition of Na,K-ATPase activity indicated that approximately 20% of the enzyme in ADPKD cells exhibits a higher affinity for ouabain. The increased ouabain affinity of ADPKD cells was not due to differences in Na,K-ATPase isoform expression because these cells, like NHK cells, possess only the alpha1 and beta1 subunits. The gamma variants of the Na,K-ATPase also are expressed in the cells but are elevated in ADPKD cells. Currently, the basis for the differences in ouabain sensitivity of NHK and ADPKD cells is unknown. It is concluded that ouabain stimulates proliferation in ADPKD cells by binding to the Na,K-ATPase with high affinity and via activation of the MEK-ERK pathway.     

Different oxygenators for cardiopulmonary bypass lead to varying degrees of human complement activation in vitro

Complement activation was studied in vitro with six different membrane and bubble oxygenators for cardiopulmonary bypass. There was a similar increase in terminal (C5 to C9) activation with all oxygenators (p less than 0.001), ranging from 281% (117% to 444%) to 453% (225% to 680%) after 60 minutes (median and 95% confidence intervals). C3 activation was not observed with a hollow fiber membrane and a soft shell bubble oxygenator. On the other hand, a capillary membrane, a sheet membrane, a nonporous membrane, and a hard shell bubble oxygenator all induced a similar increase in C3 activation (p less than 0.01), ranging from 107% (23% to 346%) to 272% (88% to 395%) after 60 minutes. The differences in C3 activation could not be explained by the blood contact materials or any other single factor known to induce activation, which suggests that overall complement activation during cardiopulmonary bypass is a multifactorial effect. The tubing set per se induced only minor C3 activation but contributed to the overall formation of terminal complement complex. The study further indicates that an arterial line blood filter prevents activated neutrophils from being reinfused to the patient and should be used regardless of type of oxygenator.     

Antisense inhibition of pig α1,3galactosyl-transferase leads to a reduction in expression of the major target for human natural antibodies on pig vascular endothelial cells

Abstract: The expression of epitopes on pig cells for human natural antibodies (NAb) currently constitutes a major barrier to the use of pig organs and tissues in clinical transplantation. This is of particular significance to vascularized organs where the presence of carbohydrate antigens invariably leads to a hyperacute rejection when exposed to human blood or serum. It has been strongly suggested that the major antigen in this context consists of a terminal Galα1,3Galβ1,4GlcNAc trisaccharide. We have previously proposed that decreased expression of this epitope for human NAb may lead to elimination of hyperacute rejection. We have now used mRNA targeted antisense oligonucleotides to decrease the expression of the α1,3galactosyltransferase directing the terminal synthesis of this epitope on pig vascular endothelial cells. This mRNA antisense targeting leads to a decreased expression of the Galα1,3Galβ1,4GlcNAc structure as detected by epitope specific lectin. Moreover, a very similar reduction in mean fluorescence was seen when staining the same cells with human Galα1,3Galα1,4GlcNAc-specific IgM NAb. We feel these findings constitute an important step in the process of providing conclusive evidence that reduction of or complete elimination of this epitope will overcome the hyperacute response in this species combination. In addition, such pig tissues may be less susceptible to further antibody rejection once the hyperacute phase has been overcome.     

An ELISA technique for quantitation of human xenoantibodies binding to pig cells: Application in patients with pig kidneys extracorporeally connected to the circulation

ABSTRACT: A quantitative ELISA technique for determination of human anti-pig xenoantibody number in serum samples has been established using pig lymphocytes and pig/rabbit erythrocytes as target cells and a pool of serum from human blood group AB donors. The number of low affinity antibodies binding to the cells was determined by quantitation following the use of aqueous washing of the cells and separation of bound and unbound antibodies with the phthalate oil method. The efficiency of different soluble Galal-3Gal-terminating di- and tri-saccharides to inhibit antibody binding was tested and found to vary between 70–90% at a saccharide concentration of 10 mg/ml. The assay was used to evaluate the antibody changes in two patients who, after plasmapheresis treatments, had pig kidneys extracorporeally connected to their blood circulation. The number of anti-pig IgM/IgG antibodies bound to each pig lymphocyte were reduced from 5,600/13,200 to 1,300/3,100 in patient 1 and from 1,200/6,500 to 500/2,100 in patient 2 by three consecutive daily plasmapheresis treatments. Although the lymphocytotoxic titers were reduced to very low levels, the antibody numbers still present in the blood of patient 1 caused a hyperacute rejection of the pig kidney. However, the antibody levels in patient 2 did not cause rejection of this kidney during 15 min perfusion time. A strong anti-pig antibody response 3 weeks after the perfusion experiment was found in patient 1 as shown by 27,600/245,300 IgM/IgG molecules bound to pig lymphocytes corresponding to an increase of lymphocytotoxic titer from 8 to 512. The second patient showed a much weaker immune response with 1,400/19,800 IgM/IgG antibodies corresponding to a lymphocytotoxic titer increase from 8 to 32. The use of this quantitation technique enables more accurate investigation of antibody bindine to xenoeenic tareet cells than conventional titration techniaues.     

Aggregation of human platelets induced by porcine endothelial cells is dependent upon both activation of complement and thrombin generation

Abstract: The binding of human xenoreactive antibody (XNA) to porcine endothelium with complement (C) activation via the classical pathways is considered the major event triggering hyperacute rejection (HAR) with microvascular thrombosis in vivo. As C components are linked to key events in blood coagulation, we have examined pathways whereby activation of complement by endothelial cells results in xenogeneic platelet activation in vitro. Methods: Cultured porcine aortic endothelial cells (pEC), human aortic endothelial cells (HAEC) or human umbilical vein endothelial cells (HUVEC) were prepared in suspension (5 × 10⁶/ml) using EDTA-collagenase. Human platelet rich plasma (PRP) and washed platelets with platelet poor plasma (PPP) were prepared from control, drug free, volunteer donors. All aggregation tests used a two-sample, four-channel, model 560 Ca Lumi-Aggregometer (Chronolog Corporation, Havertown, PA). Selected assays for complement (C3a; C5b-C9; CH50; and AP50) were then performed on supernatant fluids. To test the effects of complement inhibition and thrombin antagonists, the following agents were pre-incubated with PRP (or PPP) in various titrations for 10 min at 37°C prior to combination with pEC in the aggregometer: soluble complement receptor typel (sCR1); Cobra Venom Factor (CVF), heparin, hirudin, and anti-CD31 (anti-PECAM). In addition, pEC, HAEC, and HUVEC were incubated with 10–20% human PPP; supernatant fluids were harvested at various time points and used for platelet activation assays and for functional tests of thrombin or levels of thrombin-antithrombin complexes (TAT). Results: pEC but not HAEC/HUVEC resulted in activation of PRP or washed platelets only in the presence of supplemental PPP. Platelet activation could be inhibited by pre-incubation of samples with CVF (2–5 IU/ml to deplete complement components) and to a variable extent with sCRl (1–2 mg/ml). Complement assays confirmed activation of C3 by the classical pathway with reduction of the CH50 while C6 deficient samples also supported platelet activation. Aggregation of platelets was inhibited by preincubation of PRP with concentrations of hirudin (1 unit/ml) and heparin (5–10 units/ml) sufficient to neutralize thrombin generated. Supernatant fluids containing PPP removed from pEC were found to have increased levels of thrombin and thrombin-antithrombin complexes and could also activate platelets in a hirudin sensitive manner. Discussion: Platelet and pEC combinations underwent aggregation only in the presence of complement components. This process appeared independent, at least in part, of the assembly of terminal complement components. Consequent pEC generation of thrombin appeared adequate to trigger platelet aggregation which could in turn be inhibited by hirudin or heparin in vitro.