Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers.

Protein tyrosine phosphorylation mediates signal transduction of cellular processes with protein tyrosine kinases (PTKs) regulating virtually all signalling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into cellular functions: extracellular response kinases (ERK), c-Jun N-terminal kinases (JNK) and p38. Previously conducted studies using two chicken lines (A and B) show line A heterophils are functionally more responsive and produce a differential cytokine/chemokine profile compared with line B, which also translates to increased resistance to bacterial challenges. Therefore, we hypothesize the differences between the lines result from distinctive signalling cascades that mediate heterophil function. Heterophils from lines A and B were isolated from 1-day-old chickens and total phosphorylated PTK and p38, JNK, ERK, and transcription factor (activator protein 1 (AP-1) and nuclear factor kappa B (NF-κB)) protein levels quantified following interaction with Salmonella Enteritidis (SE). Control and SE-treated heterophils from line A had greater (P≤0.05) PTK phosphorylation compared to line B with increased (P≤0.05) activation of p38. Conversely, line B heterophils activated JNK (P≤0.05). There were no differences in ERK between control and activated heterophils for either line. Defined signalling inhibitors were used to show specificity. The AP-1 and NF-κB transcription factor families were also examined, and c-Jun and p50, respectively, were the only members different between the lines and both were up-regulated in line A compared with line B. These data indicate that increased responsiveness of line A heterophils is mediated, largely, by an increased ability to activate PTKs, the p38 MAPK pathway and specific transcription factors, all of which directly affect the innate immune response.

Engagement of CD31 delivers an activating signal that contributes to the survival of chronic lymphocytic leukaemia cells.

The present study showed that engagement of CD31 delivers a survival signal in chronic lymphocytic leukaemia (CLL) cells. We describe two groups of CLL, showing different kinetics of apoptosis in vitro and distinct ratios between anti-apoptotic and pro-apoptotic proteins: CLL-I displayed low Bcl-x(L) /Bax and Bcl-2/Bax ratio and underwent rapid apoptosis in vitro; CLL-II had high Bcl-x(L) /Bax and Bcl-2/Bax ratio and were resistant to apoptosis for several days. Nurse-like cells, expressing vimentin, CD68 and CD31 were detected mainly in CLL-II cultures. Of note, CD31 cross-linking, obtained with a specific monoclonal antibody (mAb), induced phosphatidylinositol-3-kinase-dependent Akt phosphorylation and nuclear translocation of the nuclear factor-kBp65 and p52 subunits in both CLL groups, leading to upregulation of Bcl-2 and Bcl-x(L) transcription and increased cell survival. Binding to CD31(+) stable transfectants, could also deliver an anti-apoptotic signal in B cells of both CLL-I and CLL-II, increasing the Bcl-2 and Bcl-x(L) protein content, regardless the expression of CD38. On the other hand, the addition of the F(ab')2 (that is unable to oligomerize the target molecule) of the anti-CD31 mAb prevented these effects. These data suggest that the CD31 adhesion system may play a role also in vivo in maintaining CLL survival.

Antibody-mediated activation of the classical complement pathway in xenograft rejection.

Transplant rejection is a multifactorial process involving complex interactions between components of the innate and the acquired immune system. In view of the shortage of donor organs available for transplantation, xenotransplantation of pig organs into man has been considered as a potential solution. However, in comparison to allografts, xenografts are subject to extremely potent rejection processes that are currently incompletely defined. Consequently, an appropriate and safe treatment protocol ensuring long-term graft survival is not yet available. The first barrier that has to be taken for a xenograft is hyperacute rejection, a rapid process induced by the binding of pre-formed antibodies from the host to the graft endothelium, followed by activation of the classical complement pathway. The present review concentrates on the role of antibodies and complement in xenograft rejection as well as on the approaches for treatment that target these components. The first part focuses on porcine xenoantigens that are recognized by human xenoreactive antibodies and the different treatment strategies that aim on interference in antibody binding. The second part of the review deals with complement activation by xenoreactive antibodies, and summarizes the role of complement in the induction of endothelial cell damage and cell activation. Finally, various options that are currently under development for complement inhibition are discussed, with special reference to the specific inhibition of the classical complement pathway by soluble complement inhibitors.

Natural killer cell- and macrophage mediated discordant guinea pig-->rat xenograft rejection in the absence of complement, xenoantibody and T cell immunity.

BACKGROUND: The role of natural killer (NK) cells and macrophages (Møs) in the absence of T cell immunity in discordant xenograft (xg) rejection was investigated. METHODS: Guinea pig hearts were transplanted into athymic nude rats receiving no treatment or antixenoantibody (xAb), anticomplement (C), antinatural killer (NK) cell therapy (antiasialo GM1 [anti-ASGM1]) or their combinations. For anti-xAb therapy, natural xAbs were absorbed/neutralized by pretransplant guinea pig blood transfusion (pGPBT), followed by administration of the malononitriloamide MNA715. Cobra venom factor (CVF) was administered as anti-C therapy. FACScan analysis and a standard cytotoxicity assay determined NK cell number and cytotoxicity, respectively. ELISA and the CH50 assay measured titers of xAb and C activity, respectively. Rejected Xgs were examined by light microscopy and by immunohistochemistry. RESULTS: All hyperacutely (15+/-4 min) rejected Xgs from untreated rats showed deposits of C3 and IgM without cellular infiltrates. Combined anti-xAb and anti-C (pGPBT/MNA715/CVF) treatment significantly prolonged the survival of Xgs (3.7+/-0.6 days, P<0.001 vs. control group) showing NK cell and Mø infiltration without deposition of xAbs or C3. NK cell depletion (day -12) followed by exposure of recovering NK cells to the guinea pig antigens failed to induce specific NK cell nonresponsiveness and to further prolong xg survival in combined anti-xAb/anti-C group. In contrast, adding of continuous and repetitive depletion of NK cells significantly further prolonged xg survival to 7.4+/-0.5 days (P<0,001 vs. the anti-xAb/anti-C group) with rejected Xgs densely infiltrated by activated Møs without involvement of NK cells, C or xAbs. CONCLUSIONS: When xAb and C are suppressed in the discordant guinea pig-into-nude rat model, NK cells play an important role in xg rejection. When also NK cells are suppressed, activated Møs seem to reject discordant Xgs. The induction of specific NK cell nonresponsiveness fails in the guinea pig-into-rat combination.

Accommodated xenografts survive in the presence of anti-donor antibodies and complement that precipitate rejection of naive xenografts.

Hamster hearts transplanted into transiently complement-depleted and continuously cyclosporin A (CyA)-immunosuppressed rats survive long-term despite deposition of anti-donor IgM Abs and complement on the graft vascular endothelium. This phenomenon is referred to as "accommodation." The hypothesis tested here is that accommodated xenografts are resistant to IgM Abs and complement that could result in rejection of naive xenografts. After first hamster hearts had been surviving in cobra venom factor (CVF) + CyA-treated rats for 10 days, a time when the anti-donor IgM Ab level was maximal and complement activity had returned to approximately 50% of pretreatment levels, naive hamster hearts or hamster hearts that had been accommodating in another rat for 14 days were transplanted into those rats carrying the surviving first graft. The naive hearts were all hyperacutely rejected. In contrast, a majority of regrafted accommodating hearts survived long-term. There was widespread Ab and activated complement deposition on the vascular endothelium of accommodating first hearts, second accommodating hearts, and rejected second naive hearts. However, only the rejected naive hearts showed extensive endothelial cell damage, myocardial necrosis, fibrin deposition, and other signs of inflammation. Accommodating first and second hearts but not rejected second naive hearts expressed high levels of the protective genes A20, heme oxygenase-1 (HO-1), bcl-2, and bcl-xL. These data demonstrate that accommodated xenografts become resistant to effects of anti-donor IgM Abs and complement that normally mediate rejection of xenografts. We hypothesize that this resistance involves expression by accommodated xenografts of protective genes.

Decreased fibrinolytic activity in porcine-to-primate cardiac xenotransplantation.

BACKGROUND: One major barrier to successful xenotransplantation is acute vascular rejection, a process pathologically characterized by microvascular thrombosis and diffuse fibrin deposition in transplant blood vessels. This pathologic picture may result from a disturbance in the coagulant or fibrinolytic pathways that regulate normal vascular patency. This study evaluated the regulation of fibrinolytic activity defined by tissue plasminogen activator and plasminogen activator inhibitor-1 as it may exist in the setting of acute vascular rejection. MATERIALS AND METHODS, RESULTS: Serial biopsies from cardiac xenotransplants evaluated by immunofluorescence microscopy demonstrated progressive decreases in tissue plasminogen activator and increases in plasminogen activator inhibitor-1. In vitro studies measuring fibrinolytic activity of cell culture medium from porcine aortic endothelial cells stimulated with human serum or autologous porcine serum revealed that human serum triggered as much as 93% increase in antifibrinolytic activity. CONCLUSIONS: These findings demonstrate that porcine vascular endothelial cells change toward an antifibrinolytic state following stimulation with human xenoreactive antibodies and complement. The shift is at least partly explained by an increased ratio of plasminogen activator inhibitor-1 to tissue plasminogen activator, and is at least in part mediated by the activation of complement. This increased antifibrinolytic activity may contribute to the thrombotic diathesis seen in acute vascular rejection in pig-to-primate xenografts.

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.

Human IgG xenoreactive antibodies mediate damage to porcine endothelial cells in vitro by both humoral and cellular mechanisms.

A major barrier to the transplantation of a porcine organ into a human recipient is the hyperacute rejection response which has been shown to be mediated by xenoreactive antibodies (XAb) and complement proteins. Here, evidence is presented that normal human sera contain IgG XAb that are able to mediate increased adhesion of polymorphonuclear leucocytes (PMN) to porcine aortic endothelial cells (PAEC) in vitro, to initiate damage to endothelial cells via antibody-dependent cellular cytotoxicity mechanisms (ADCC) along with peripheral blood mononuclear cells (PBMC) and to activate the classical complement cascade. PMN exhibit a 2.8-fold increase in adhesion to PAEC from 19.9 +/- 4% to 56 +/- 1.7% at an IgG concentration of 16 mg/ml. This increase is independent of treatment of the PMN with interferon-gamma. PAEC are lysed in the presence of complement: 42.8 +/- 0.7% lysis occurs with a 1/8 dilution of human serum as a source of immunoglobulin of all classes, while 39.3 +/- 3% lysis occurs with purified IgG at 13 mg/ml in the presence of baby rabbit complement. PAEC are also lysed by PBMC in the presence of human IgG XAb, a maximum of 52.0 +/- 5% being observed at effector-to-target (E:T) ratios of 30:1. PBMC bearing Fc gamma RIII receptors for the Fc portion of the IgG molecule mediate the endothelial cell damage since the anti-Fc gamma RIII monoclonal antibody, 3G8, can inhibit lysis by up to 77 +/- 5%. We conclude that human IgG are able to damage porcine endothelial cells using cellular and humoral mechanisms and that IgG XAb can efficiently activate the classical complement cascade in this model system.

Evaluación de la actividad fagocitaria y bactericida de los monocitos y heterófilos aviares contra Samonella gallinarum / Evaluation of phagocytic and bactericidal activities of avian monocytes and heterophils against Salmonella gallinarum in vitro

Se determinó, mediante ensayos in vitro, la capacidad fagocítica y bactericida de los deterófilos y de los monocitos aviares contra Salmonella gallinarum, en presencia y en ausencia de 10 por ciento de suero hiperinmune. En los ensayos de fagocitosis se observó que los hetrófilos fagocitaron al 28 ñ 4.7 por ciento de las bacterias sin opsonizar y al 45 ñ 9.9 por ciento de las bacterias opsonizadas, obteniéndose diferencias significativas (P< 0.05). En contraste, los monocitos sólo fagocitaron un 10.3 ñ 4.2 por ciento y un 11.7 ñ 3.8 por ciento de bacterias opsonizadas y sin opsonizar respectivamente (P> 0.05). En los ensayos bactericidas se observó que los heterófilos destruyeron al 90.46 ñ 3.3 por ciento de la bacteria sin opsonizar y 90 ñ 2.3 por ciento de la bacteria opsonizada (P> 0.05); sin embargo, en los monocitos se obtuvo un 10.5 ñ 6.6 por ciento y un 84.74 ñ 5 por ciento respectivamante, obteniéndose diferencias significativas (P< 0.05). Los resultados del presente estudio indican que la fagocitosis de los heterófilos fue significativamente incrementada por la opsonización; en el caso de los monocitos, no hubo un efecto significativamente mayor. Aproximadamente el 90 por ciento de las bacterias fagocitadas por los heterófilos fueron destruidos, como se determinó en el ensayo. La opsonización no incrementó significativamante el porcentaje de bacteria destruida por parte de los heterófilos, sin embargo, la psonización de Salmonella gallinarum sí favoreció la capacidad bactericida de los monocitos

Biochemical and functional characterization of xenoreactive natural antibodies in hu-PBL-SCID mice.

An in vivo model system to understand the mechanism of xenograft rejection was established using human peripheral blood leukocyte-reconstituted SCID (hu-PBL-SCID) mice. Human xenoreactive natural antibodies (XNA), of IgM and IgG subtypes, capable of binding to pig aortic endothelial cells (PAEC) were detected in the sera of hu-PBL-SCID by ELISA and flowcytometric methods. Western blot analysis of PAEC lysates showed that IgM and IgG XNA from hu-PBL-SCID recognized xenoantigens with similar molecular mass as those recognized by XNA from normal human serum (NHS). This result demonstrated that hu-PBL-SCID contained XNA representing the same repertoire as that of the NHS. XNA from NHS and hu-PBL-SCID were also able to induce intracellular Ca2+ signals in cultured PAEC several fold above the basal level. This result revealed their functional similarity and demonstrated for the first time that XNA in the absence of C can activate PAEC, which may lead to the pathology of xenograft rejection. In vivo, PAEC transplanted under the kidney capsule of hu-PBL-SCID mice showed deposition of human IgM and mouse C. In summary, the present study demonstrates that hu-PBL-SCID can serve as a useful model to characterize innate immunity against xenograft.

Heteroagglutinins and their significance in baboon hepatic xenotransplantation.

The role of preformed xenoreactive antibodies in xenograft recipients is unknown. Humans and baboons possess red cell agglutinating activity associated with isohemagglutinins and heteroagglutinins (HA). We examined the role of HA in two patients who received ABO-identical baboon livers. Human antibaboon HA were assessed by correlating serial titers with studies for rejection. Serial direct antiglobulin testing (DAT) was used to detect baboon antihuman HA, potentially produced by transplanted passenger lymphocytes. Patient 1 survived 70 days. The human antibaboon HA titers remained essentially unchanged from preoperative values. Although hyperacute rejection did not occur, and there was only mild cellular rejection, liver function was suboptimal. Postreperfusion immunoglobulin and complement deposition and histologic changes suggested complement-mediated injury. DAT testing was negative except for passively acquired anti-A from transfusion. At autopsy there was marked bile stasis, but no rejection. Patient 2 survived 26 days with essentially unchanged HA titers until preterminal. Although there was no hyperacute rejection and only mild humoral rejection (without cellular rejection), suboptimal liver function and bile stasis were again noted. Postreperfusion immunoglobulin and complement deposition again suggested complement-mediated injury. DAT testing was negative. At autopsy there was no rejection. Human antibaboon HA do not appear to be associated with hyperacute or cellular rejection, but their role in the complement-mediated injury, suspected in both cases, cannot be definitively excluded. Baboon antihuman HA were not detected in either patient.

Xenogeneic antibodies against hepatocyte plasma membranes suppress bile secretion in rats.

The action of xenogeneic antibodies against rat hepatocyte plasma membranes which were injected into the portal vein in the dose of 40 mg of protein per kg of body mass on the bile secretion and on the ultrastructure of the liver were studied. It was shown that these antibodies suppressed the bile flow, the secretion of bile acids and cholesterol and decreased the bile salt-independent bile flow. The action of antibodies was accompanied by ultrastructural damage of sinusoidal and lateral plasma membranes of some hepatocytes and by a decrease in the Na+, K(+)-ATPase activity. It was concluded that the decrease of Na+, K(+)-ATPase activity in the plasma membranes was the most important mechanism responsible for a decrease in the bile flow.

Evolutionary and immunological aspects of xenotransplantation.

Immunological aspects in xenotransplantation are directly linked to evolutionary aspects. The immunological network with its high antigen specificity and species-specific interactions was developed to fight against the host's outside world including xenografts. The tremendous amount of reaction and mechanisms triggered by the performed natural antibody-endothelial cell reaction clearly indicates that only basic changes might overcome these problems. Even the introduction of transgenic donors might not be sufficient to modify more than one parameter, for example, complement activity. Total depletion of preformed natural antibodies is not compatible with normal life and the regulation of hormones and enzymes under xenogeneic conditions has just been discovered. Cellular mechanisms depend, as far as we know, on the delicate system of receptors, interleukins, and MHC antigens. From today's very early results we know that major changes exist already between closely related species. The investigation of xenogenic mechanisms has opened a totally new field of immunological interactions. Up to now there are no signs of solution to bridge clinically relevant times with xenogenic organs from widely divergent suitable donors with promising quality of life.

Effect of anti-Ig on rabbit B cell function and Ig expression.

We have examined the effect which xenogeneic anti-Ig has on rabbit B cell function and Ig expression in an effort to understand the phenomenon of antibody mediated suppression. Treatment of rabbit lymphocyte cultures with xenogeneic anti-rabbit Ig causes 2.5-3.8 fold decrease in the level of Ig secreting cells with little or no long-term effect on surface Ig. This suppression in B cell secretory function is not the result of suppression of Ig gene expression since xenogeneic anti-rabbit Ig treatment causes a 1.7-2.7 fold increase in Ig L and H chain mRNA levels. Collectively, these data are consistent with the hypothesis that antibody mediated suppression of B cell function occurs at a post-transcriptional level involving either the secretory pathway of Ig expression and/or blockage in B cell differentiation.

Heterogeneity in the specificity of the islet cell cytoplasmic antibody response in insulin-dependent diabetes mellitus.

We assessed the heterogeneity in the islet cell cytoplasmic antibody (ICA) response of insulin-dependent diabetes mellitus (IDDM) patients via indirect immunofluorescence on frozen sections of human, bovine, and porcine pancreas. The three substrates detected comparable frequencies of ICA positives among the IDDM sera tested, whereas control sera were ICA negative on all three substrates. However, individual IDDM serum samples showed heterogeneity in ICA binding on the three pancreata. Of 28 sera tested on all three substrates, 22 were ICA positive on human pancreas, three were ICA positive on bovine pancreas, and two were ICA positive on porcine pancreas. Sensitivity of ICA epitopes to neuraminidase treatment and periodate oxidation suggests that glycoconjugates are recognized by serum ICA. Cholera toxin blocked ICA binding. However, the functional cholera toxin receptor ganglioside Gm1 is resistant to neuraminidase treatment and periodate oxidation. Therefore, it is unlikely that Gm1 is the ICA determinant. These data suggest that not all ICA antigens are equivalently expressed on islets from different pancreata and/or that each individual responds to a hierarchy of islet antigens such that restricted patterns of specific ICA binding are found.

Fc gamma 2b receptor-mediated phagocytosis by a murine macrophage-like cell line (P388D1) and peritoneal resident macrophages. Up-regulation by the inhibitors of phospholipase A2 and cyclooxygenase.

The mechanisms of Fc gamma R-mediated phagocytosis of immune complexes were investigated by the use of a murine macrophage-like cell line (P388D1) and murine peritoneal resident macrophages. About 40 to 80% of P388D1 cells phagocytosed SRBC coated with IgG2a subclass anti-SRBC mAb (EA2a) within 60 min, whereas only 10 to 20% of the cells phagocytosed EA2b during the same period. The treatment of P388D1 cells with inhibitors of phospholipase A2 (p-bromophenacylbromide, EGTA, or dexamethasone) or of cyclooxygenase (indomethacin or aspirin) significantly promoted the Fc gamma 2bR-mediated phagocytosis of EA2b, but did not affect the Fc gamma 2aR-mediated phagocytosis of EA2a. These results suggest that the activation of phospholipase A2 activity associated with Fc gamma 2bR may lead to the inhibition of phagocytosis of EA2b. This inhibition appeared to be due to the blockade of the interaction of Fc gamma 2bR with various cytoskeletal components, because the association of Fc gamma 2bR and these cytoskeletal components, which could be eliminated by cytochalasin D, was found to be increased by the inhibition of phospholipase A2 activity.

Effect of antibodies against the gap junction protein on differentiation of induced embryonic cells.

Microinjections of antibodies against gap junction protein to early blastomeres of Xenopus laevis were succeeded by the treatments of mesodermal inductor. It was found that induction was not affected by the injection of the antibodies but the differentiation of notochord and muscle was impeded at different degrees. Cell interactions via gap junction communication seem to be involved in the differentiation of the induced cells.