In the face of chronic aspiration, prolonged ischemic time exacerbates obliterative bronchiolitis in rat pulmonary allografts.

Aspiration of gastric fluid into the lung mediates the development of obliterative bronchiolitis (OB) in orthotopic WKY-to-F344 rat pulmonary transplants that have been subjected to immunosuppression with cyclosporine. However, the contribution of ischemic time to this process remains unknown. In this study, the effect of long (n = 16) and short (n = 12) ischemic times (average of 6 h and of 73 min, respectively) on rat lung transplants receiving aspiration of gastric fluid was assessed. Long ischemic times (LIT) led to significantly (p < 0.05) greater development of OB (ratio of OB lesions/total airways = 0.45 ± 0.07, mean ± standard error) compared to short ischemic times (ratio = 0.19 ± 0.05). However, the development of OB was dependent on aspiration, as controls receiving aspiration with normal saline showed little development of OB, regardless of ischemic time (p < 0.05). These data suggest that LIT, while insufficient by itself to lead to OB, works synergistically with aspiration of gastric fluid to exacerbate the development of OB.

Chronic aspiration of gastric fluid induces the development of obliterative bronchiolitis in rat lung transplants.

Long-term survival of a pulmonary allograft is currently hampered by obliterative bronchiolitis (OB), a form of chronic rejection that is unique to lung transplantation. While tracheobronchial aspiration from gastroesophageal reflux disease (GERD) has clinically been associated with OB, no experimental model exists to investigate this problem. Using a WKY-to-F344 rat orthotopic left lung transplant model, the effects of chronic aspiration on pulmonary allograft were evaluated. Recipients received cyclosporine with or without 8 weekly aspirations of gastric fluid into the allograft. Six (66.7%) of 9 allografts with aspiration demonstrated bronchioles with surrounding monocytic infiltrates, fibrosis and loss of normal lumen anatomy, consistent with the development of OB. In contrast, none of the allografts without aspiration (n = 10) demonstrated these findings (p = 0.002). Of the grafts examined grossly, 83% of the allografts with chronic aspiration but only 20% without aspiration appeared consolidated (p = 0.013). Aspiration was associated with increased levels of IL-1 alpha, IL-1 beta, IL-6, IL-10, TNF-alpha and TGF-beta in BAL and of IL-1 alpha, IL-4 and GM-CSF in serum. This study provides experimental evidence linking chronic aspiration to the development of OB and suggests that strategies aimed at preventing aspiration-related injuries might improve outcomes in clinical lung transplantation.

Chronic aspiration shifts the immune response from Th1 to Th2 in a murine model of asthma.

BACKGROUND: Chronic aspiration associated with gastro oesophageal reflux disease (GERD) is thought to play a substantial role in the development of asthma, the incidence of which is dramatically increasing in industrially developed countries. The majority of data examining the association between aspiration and asthma has been obtained from epidemiological studies, which show that between 50 and 90% of individuals with asthma experience some element of GERD. This study describes the effect of chronic aspiration on a model of experimentally induced airway hypersensitivity in Balb/c mice. MATERIALS AND METHODS: Four experimental groups were utilized: Aspiration/Asthma, Sham/Asthma, Aspiration/Sham and Sham/Sham. Mice were sensitized with aerosolized 1% ovalbumin on days 1 to 10 (sensitization phase), followed by repeated exposure on days 31 to 40 (challenge phase). Aspiration events occurred on days 1, 8,15, 22, 29, 36, 43 and 50. Animals were sacrificed on days 56 and 57. RESULTS: Chronic aspiration of 10 microL of murine gastric fluid per week for eight weeks produced an injury pattern distinct from that of acute aspiration, with lung injury characterized by hyperplasia, neutrophil infiltration of the bronchioles and relative parenchymal sparing. Aspiration during induction of ovalbumin-induced airway hypersensitivity was associated with a trend toward decreased production of antiovalbumin IgG, antiovalbumin IgE, and total IgE. Further, aspiration induced a substantial and significant increase in antiovalbumin IgG1/IgG2a ratios, consistent with a shift toward a predominantly Th2 response. CONCLUSION: These findings indicate that chronic aspiration has a profound effect on the nature of the immune response to aerosolized allergens in a model of experimentally induced airway hypersensitivity.

Partial sequence of human platelet heparitinase and evidence of its ability to polymerize.

Heparitinase cleaves heparan sulfate, a glycosaminoglycan associated with all nucleated mammalian cells and extracellular matrices. Despite the important physiologic role heparitinase is postulated to play in such processes as tumor metastasis and inflammation, the identity of the enzyme remains a matter of controversy and there is a question of whether heparitinase is CTAP III. We report a 900,000-fold purification of heparitinase from human platelets. A multi-step procedure utilizing chromatography on heparin, DEAE, hydroxyapatite and size exclusion matrices was employed and yielded a single protein as judged by Coomassie staining of protein separated by SDS-PAGE. The purified protein had an apparent molecular mass of 35 kDa by size exclusion chromatography and 55 kDa by SDS-PAGE. During purification, heparitinase activity co-eluted from the hydroxyapatite and size exclusion columns with the 35-55 kDa protein, confirming that the purified protein was indeed heparitinase. The 35-55 kDa protein reacted strongly with concanavalin A, a lectin known to bind to heparitinase, further confirming that the protein was heparitinase. Platelet heparitinase formed dimers and tetramers upon storage in a purified form, possibly accounting for the various molecular weights previously reported for the enzyme. A partial amino acid sequence of the protein revealed that heparitinase has not been previously sequenced.

Porcine platelet antigens recognized by human xenoreactive natural antibodies.

Hyperacute xenograft rejection of porcine organs by primates is initiated by the binding of recipient natural antibodies to endothelium in the donor organ. We showed previously that human natural antibodies recognize 3 porcine endothelial cell glycoproteins with molecular masses of 115 kDa, 125 kDa, and 135 kDa; we called the glycoproteins gp115/135. We also showed that porcine platelets contain glycoprotein antigens that are the same or very similar to gp115/135 from endothelial cells. The studies reported here were aimed at identifying these porcine platelet antigens and evaluating their potential relevance for the pathogenesis of xenograft injury. The importance of gp115/135 as targets of human natural antibodies was supported by the demonstration that (a) antibodies against porcine platelet gp115/135 are absorbed from the blood of nonhuman primates during the perfusion of a porcine kidney; (b) purified gp115/135 glycoproteins at concentrations < 500 pM, block the binding of human natural antibodies to cultured porcine endothelial cells; (c) the binding of human antibodies to porcine platelet extracts is significantly decreased by removal of gp115 or gp135 from the extracts; (d) antibody binding to gp115/135 initiates the activation of complement. Amino-terminal sequencing of gp115/135 revealed that gp115 has significant homology to human integrin beta 3 chains, gp125 to human alpha IIb chains, and gp135 to human alpha 2 chains. Ligand binding properties of the porcine glycoproteins were consistent with the identity of the antigens revealed by amino acid sequencing and molecular weight. Human natural antibodies also recognized a 250 kDa porcine platelet glycoprotein which was found to be homologous to human von Willebrand factor (vWF). Antibodies against vWF in the blood of nonhuman primates are absorbed during ex vivo perfusion of a porcine organ. The identification of gp115/135 as integrins, the functions of which in endothelium might include cell signaling and maintenance of barrier function, provides potential insight into the pathogenesis of the rejection reaction in which these processes are manifestly aberrant. The identification of vWF as a potential target antigen raises several questions, including whether vWF provides one basis for antibody binding to xenogeneic endothelium or whether upon secretion from an endothelial cell vWF might actually block antibody binding.

Isohemagglutinins and xenoreactive antibodies: members of a distinct family of natural antibodies.

Just as anti-blood group A and anti-blood group B antibodies pose a strong humoral barrier to the transplantation of allogeneic organs or blood, xenoreactive natural antibodies directed against Gal alpha 1-3Gal pose a barrier to the transplantation of xenogeneic organs or blood. We tested the idea that, although "natural" iso-hemagglutinins and xenoreactive natural antibodies recognize distinct structures, they have a similar origin and function. Anti-A antibodies, anti-B antibodies, and xenoreactive natural antibodies were present in serum at similar concentrations and varied with age, gender, and the concentration of total IgM in serum in a similar manner. Anti-A antibodies, anti-B antibodies, and xenoreactive natural antibodies, unlike some elicited antibodies, had a high degree of thermal lability and bound more avidly at lower temperatures. The natural antibodies manifest remarkable homogeneity and high functional avidity for determinants on a cell surface but only a weak affinity for monovalent ligands. These findings suggest that anti-A antibodies, anti-B antibodies, and xenoreactive natural antibodies specific for Gal alpha 1-3Gal have a common origin and function and, given similar antigen density on target cells, provide similar humoral barriers to transplantation or transfusion and that these antibodies may be members of a common "family" of antibodies.

Humoral responses to pig-to-baboon cardiac transplantation: implications for the pathogenesis and treatment of acute vascular rejection and for accommodation.

Organs transplanted between phylogenetically-disparate species, such as from the pig into the primate, are subject to hyperacute and acute vascular rejection. Hyperacute rejection of a porcine organ by a primate is thought to be initiated by the binding of xenoreactive natural antibodies to Galalpha1-3Gal expressed on the endothelial lining of blood vessels in the xenograft. The factor(s) which initiates acute vascular rejection is uncertain; however, there is some evidence implicating xenoreactive antibodies. The nature of the humoral response which might contribute to acute vascular rejection of a porcine organ was investigated in baboons which received a porcine cardiac xenograft plus immunosuppression with methylprednisolone, azathioprine, and cyclosporine. Following rejection and surgical removal of the xenografts, the serum concentration of xenoreactive antibodies increased in untreated animals but in immunosuppressed animals was similar to the concentration in preimmune serum. The antibodies in the sensitized recipients were specific for Galalpha1-3Gal (70-95%) and other determinants (5-30%). However, cross-blocking studies showed that, following xenotransplantation, the immunosuppressed baboons had no detectable IgM or IgG directed against "new" endothelial antigens. These results indicate that antibodies made by immunosuppressed individuals in response to xenotransplantation are much like xenoreactive natural antibodies and suggest that acute vascular rejection might in some cases be addressed by therapeutic strategies aimed at those antibodies.

Identification of porcine endothelial cell membrane antigens recognized by human xenoreactive natural antibodies.

Rejection of an organ transplanted into a phylogenetically disparate recipient may be initiated by the binding of xenoreactive natural Abs of the recipient to Ags expressed on donor endothelium leading to the activation of the C system. Having previously shown that human xenoreactive Abs bind predominantly to N-linked oligosaccharides on porcine endothelial cell glycoproteins, we sought to identify those glycoproteins to gain insight into the pathogenesis of the rejection reaction and to develop improved methods for depleting those Abs. Based on amino-terminal and internal sequencing of the glycoproteins, reactivity with monospecific rabbit Abs, and functional properties, the porcine endothelial cell targets of human natural Abs were shown to be the von Willebrand factor, integrin alpha 1, alpha v, alpha 3/alpha 5, beta 1, and beta 3 chains and a 95-kDa glycoprotein homologous with chick DM-GRASP. Human and baboon Abs directed against those glycoproteins were shown to be absorbed during perfusion of porcine organs and the binding of human IgM to the isolated glycoproteins to initiate the activation of C leading to formation of iC3b neoantigen. The determinants recognized by human xenoreactive Abs had immunodominant alpha-galactosyl residues based on affinity chromatography, reactivity with anti-Gal alpha(1-3)Gal Abs and sensitivity to alpha-galactosidase. The findings show that members of the integrin family and the von Willebrand factor are major targets of xenoreactive Abs and suggest potential mechanisms by which the binding of Abs to endothelial cells might perturb the physiology of those cells and thus cause aberrant functioning of a tissue or organ.

Modulation of natural IgM binding and complement activation by natural IgG antibodies: a role for IgG anti-Gal alpha1-3Gal antibodies.

The most abundant natural IgG Abs in human serum are thought to be Abs specific for Gal alpha1-3Gal, a carbohydrate expressed in lower mammals. IgG Abs specific for Gal alpha1-3Gal have been postulated to contribute to host defense and to participate in the rejection of interspecies organ grafts. Our previous studies indicated, however, that IgM and not IgG anti-Gal alpha1-3Gal Abs activate complement on foreign surfaces, and thus the physiologic role of IgG anti-Gal alpha1-3Gal remains uncertain. We tested whether the IgG anti-Gal alpha1-3Gal in a human serum might in fact compete with IgM for binding and thus modulate complement fixation by IgM. Several lines of evidence suggested such competition might occur. First, the functional avidity of IgG and IgM for Gal alpha1-3Gal on cell surfaces were nearly within the same order of magnitude, and in some sera the molar concentrations of IgG and IgM anti-Gal alpha1-3Gal were comparable. Second, binding of human IgM to Gal alpha1-3Gal on cell surfaces was inversely correlated with the concentration of IgG anti-Gal alpha1-3Gal in serum. Third, combination of IgG and IgM Abs specific for Gal alpha1-3Gal demonstrated direct competition for binding. The presence of IgG anti-Gal alpha1-3Gal, which was predominantly IgG2, attenuated by up to 80% the fixation of C1q mediated by IgM, presumably by competing for antigenic sites recognized by IgM Abs that fix complement. Thus, IgG Abs specific for Gal alpha1-3Gal modulate complement activation by IgM specific for that structure and might in this way modulate the consequences that ensue when human blood is brought into contact with foreign organisms or xenogenic cells.

Characterization and affinity isolation of xenoreactive human natural antibodies.

Natural Abs, which are thought to provide an initial defense against invasive microorganisms, include isohemagglutinins, anti-phosphatidylcholine Abs, and anti-alpha-galactose Abs. We have evaluated the physiologic properties of the fraction of human natural Abs that bind to porcine endothelial cells and that would, as a result, initiate the rejection of a porcine organ transplanted into a human. The concentration of xenoreactive IgM in the serum varied widely in the population (5 to 105 micrograms/ml), but was highly dependent on the concentration of IgM in the serum (r = 0.85). Despite this variation and the potential diversity of epitopes recognized, human xenoreactive natural Abs exhibited surprisingly homogeneous binding characteristics, both in one individual and in the population. The apparent avidity determined by using a direct ELISA yielded a functional dissociation constant of 10(-8) M to 10(-10) M, depending on the temperature used. This high functional Kd apparently results from polyvalent interactions between the IgM and the porcine cell surface. Although the xenoreactive IgMs were absorbed by structurally diverse molecules such as ssDNA and thyroglobulin, about 80% of the xenoreactive Abs were specific for the terminal alpha-galactose determinant. A method was developed for affinity isolation of xenoreactive natural Abs by using a thermal extraction procedure. The method quantitatively accounts for all xenoreactive IgM, yielding functional IgM as evidenced by Ag binding and complement activation. Given the overlapping specificity of xenoreactive Abs in the population and the homogeneity of the functional Kd, the natural humoral immunologic barrier to xenotransplantation may be far less formidable than previously thought.

Specificity and function of "natural" antibodies in immunodeficient subjects: clues to B cell lineage and development.

The origin of natural antibodies has long been a subject of controversy. Polyreactive natural antibodies recognize multiple ligands and are thought to arise from B1 B cells. Natural antibodies against carbohydrate antigens such as Gal alpha 1-3Gal or against blood groups A and B are thought to be "elicited" by gut bacteria, but their origin is uncertain. To explore the origin of naturally occurring anticarbohydrate antibodies, the specificity and function of the xenoreactive antibodies and isohemagglutinins were investigated in immunodeficient subjects. Subjects with defects in T cell-dependent antibody synthesis had normal levels of xenoreactive natural antibodies, most of which, like xenoreactive antibodies from normal individuals, were specific for Gal alpha 1-3Gal. On the other hand, some subjects with hyper-IgM syndrome who were able to synthesize abundant quantities of xenoreactive antibodies and polyreactive antibodies were devoid of anti-Gal alpha 1-3Gal antibodies. These results suggest that the lineages of B cells giving rise to anti-Gal alpha 1-3Gal antibodies and isohemagglutinins are distinct from B1 B cells or at least exist at a more "advanced" stage of development than those B1 B cells that give rise to polyreactive antibodies. The findings also suggest that B cells which synthesize anti-Gal alpha 1-3Gal antibodies and isohemagglutinins may be distinct from B2 B cells or exist at a more "primitive" stage of development than B2 B cells that synthesize elicited antibodies in normal individuals.

Transplantation of discordant xenografts: a challenge revisited.

Six years ago, Jeffrey Platt and colleagues reviewed the biological hurdles to transplanting organs between species. The ensuing years have allowed the concepts advanced at that time to be tested leading to significant progress in understanding the immunology of xenotransplantation and in developing strategies for potential clinical application. Here, William Parker and colleagues review that progress.

Identification of antigens on porcine pulmonary microvascular endothelial cells recognized by human xenoreactive natural antibodies.

Transplantation of organs between species is prevented in part by humoral immune responses triggered by xenoreactive natural antibodies. Although the immune barrier to xenotransplantation of the lung is thought to be qualitatively and quantitatively different than the immune barrier to xenotransplantation of the kidney or heart, the antibody-antigen reactions responsible for rejection of pulmonary xenografts have not been characterized. To begin to address this issue for porcine lungs transplanted into humans, we analyzed the porcine pulmonary endothelial antigens recognized by human xenoreactive natural antibodies. Human and baboon natural antibodies recognized glycoprotein and glycolipid antigens isolated from the membranes of porcine pulmonary microvascular endothelial cells. The antigens included the integrin chains alpha1, alpha2, alpha3, alpha5, alpha(v), beta1, beta 3, the von Willebrand Factor, and fibronectin. These glycoproteins seemed to be recognized by the same antibodies that bind to porcine kidney or cardiac xenografts. Natural antibodies also recognized at least four glycolipids containing from one to five sugar residues, although at a lower level per unit number of cells than glycoprotein antigens. The epitope recognized by natural antibodies was predominantly Gal alpha1-3Gal, a structure expressed by lower mammals but not by humans and baboons. The antigens recognized by human antibodies in the porcine lung may provide insight into the pathogenesis of the rejection reaction. Moreover, the similarity of porcine lung antigens to porcine kidney and heart antigens suggests that differences in the rejection reactions between these organs reflects the distinct responses of the organs to humoral immunity.

Immune complex formation after xenotransplantation : evidence of type III as well as type II immune reactions provide clues to pathophysiology.

Rejection of renal and cardiac xenografts is initiated when natural antibodies of the recipient bind to donor endothelium, activating complement on the surface of endothelial cells. Pulmonary xenotransplants, however, reveal less evidence of antibody binding and complement activation and, in contrast to other xenografts, fare worse when the complement of the graft recipient is depleted. Accordingly, we asked whether distinct immunochemical reactions might occur after xenotransplantation of the lung and what implications such reactions might have for pulmonary pathophysiology. Analysis of serum from baboons after transplantation with porcine lungs revealed complexes containing baboon IgM and porcine von Willebrand factor. The baboon IgM in these complexes was specific for Galalpha1-3Gal. Immune complexes were also seen, albeit to a lesser extent, in the serum of kidney and heart xenotransplant recipients. Deposits of porcine von Willebrand factor and baboon C3 were detected in livers and spleens of transplanted baboons. These results indicate pulmonary xenotransplantation eventuates in formation of immune complexes and in the deposition of those complexes at distant sites. Immune complex formation could explain the peculiar fate of xenoreactive antibodies after pulmonary xenotransplantation and might contribute to the pathophysiology of the lung and systemic changes not previously considered a complication of xenotransplantation.