The contribution of terminal complement components to acute and hyperacute allograft rejection in the rat

Acute rejection and antibody-mediated hyperacute allograft rejection are affected by activation of the complement cascade. Split products of early complement components influence the localization, activation, and effector functions of platelets, granulocytes, monocytes, and lymphocytes, while the formation of membrane attack complex (C5b-C9) can lead to rapid cell destruction. Therefore, we compared acute and Ab-mediated hyperacute allograft rejection in a recently described model of C6 deficient PVG (C-) (RT1c) rats and their normal counterpart PVG (C+) (RT1c) rats. Cardiac allografts from fully MHC disparate ACI donors were heterotopically grafted into naive and skin graft sensitized PVG (C-) and PVG (C+) rats. ACI cardiac allografts were rejected acutely (8.3 +/- 2 days; n = 7) by naive PVG (C+) recipients, but survived significantly longer in PVG (C-) recipients (22 +/- 10 days; n = 10). Presensitized PVG (C+) rats rejected ACI cardiac allografts hyperacutely in 6.1 +/- 2.4 hr (n = 5). In contrast, ACI cardiac allografts transplanted into presensitized (PVG (C-) rats had markedly longer survival of 91 +/- 14 hr (n = 5). The alloantibody responses of naive PVG (C+) and PVG (C-) recipients 7 days after cardiac allografting, and of presensitized PVG (C+) and PVG (C-) recipients at time of cardiac allografting were not significantly different as measured by flow cytometry against ACI lymphocytes. Immunofluorescence demonstrated deposition of IgM, IgG and C3 in ACI allografts in PVG (C-) as well as in PVG (C+) recipients. Deposition of C6 was only found in grafts rejected by PVG (C+). The significantly longer survival of ACI cardiac allografts in C6-deficient PVG (C-) rats indicates that the membrane attack complex contributes to acute as well as antibody-mediated hyperacute allograft rejection.     

Specific interaction between erythrocytes and a glucose-carrying polymer mediated by the type-1 glucose transporter (GLUT-1) on the cell membrane

A reducing glucose-carrying polymer, called poly [3-O-(4'-vinylbenzyl)-D-glucose] (PVG), interacted with erythrocytes carrying the type-1 glucose transporter (GLUT-1) on the cell membrane. The cooperative interaction between a number of GLUT-1s and a number of reducing 3-O-methyl-D-glucose moieties on a PVG polymer chain is responsible for the increase in the interaction with erythrocytes. In contrast to the PVG homopolymer, other sugar-carrying polymers showed lower interaction with erythrocytes. The affinity of erythrocytes and PVG was studied using FITC-labeled glycopolymers. The fluorescence intensity significantly changed, whereas a small change in fluorescence intensity was observed for other homopolymers. The specific interaction between GLUT-1 on erythrocytes and the PVG polymer carrying reducing glucose was suppressed by the inhibitors, phloretin, phloridzin, and cytochalasin B, and a monoclonal antibody to GLUT-1. Direct observation by confocal laser microscopy with the use of FITC-labeled PVG demonstrated that erythrocytes interacted with the soluble form of the PVG polymer via GLUT-1, while fluorescence labeling of the cell surface was prevented on pretreatment with the monoclonal antibody to GLUT-1.     

Application of flagella-based immunoassays for serologic detection of Salmonella pullorum infection in chickens

Behavior of two rat strains was analyzed with and without 1-week pretest handling. Male rats (150-200 g body weight) of the strains PVG/OlaHsd (PVG) and Hsd:Sprague-DawleySD (SPRD) were tested once in a standard open field and an enriched open field and twice in an elevated plus-maze. Behavioral analysis revealed significant differences between the two strains and differential effects of the pretest handling procedure. SPRD rats displayed higher levels of activity and exploratory behavior than the PVG rats, whereas PVG rats were obviously less anxious. One-week pretest handling had an "anxiolytic" effect and changed activity and exploration-related behavior of the animals in both strains. Activity-related parameters were mainly affected in SPRD rats and anxiety-related ones in PVG rats. The data give evidence that differences in behavior of rats are not only determined genetically but also by preceding handling procedures. Because the two rat strains responded differentially to the pretest handling, we recommend to use a well-defined handling procedure before starting a behavioral test, especially when drug applications are included.     

Combination of open field and elevated plus-maze: a suitable test battery to assess strain as well as treatment differences in rat behavior

1. A test battery consisting of a standard open field, an enriched open field and an elevated plus maze was used to study behavior in rats. 2. Male rats of the strains PVG/OlaHsd (PVG) and Sprague-Dawley-Hsd (SPRD) (150-200 g body wt) were used to assess interstrain differences as well as handling effects. In a subsequent experiment an other set of male PVG rats (150-200 g body wt) treated either with diazepam or zolpidem was used to evaluate the test battery for pharmacological purposes. 3. SPRD rats displayed higher motor activity levels and also higher levels of exploratory behavior than the PVG rats. In contrast plus-maze activity indicated more anxiety of SPRD than PVG rats. One week pre-test handling increased the activity of both strains but it increased explorative behavior in the enriched open field only in SPRD rats. Diazepam had a substantial anxiolytic effect. Zolpidem enhanced the explorative activity in a differently to diazepam and exerted only minor anxiolytic properties. 4. We concluded that the test battery used here enables to reveal differentially strain, and treatment effects in rats.     

Complement C6 and C2 biosynthesis in syngeneic PVG/c- and PVG/c+ rat strains

A PVG rat with total deficiency of C6 and partial deficiency of C2 (PVG/c-), and a syngeneic control strain (PVG/c+), were used to study the production of extrahepatically synthesized complement. Livers of complement deficient rats were transplanted in sufficient rats (Tx-L). The C6 and C2 levels in Tx-L rats declined within 2 days to 25% and 30%, respectively, and remained stable for more than 6 weeks. To investigate the contribution of C6 synthesis by the liver, C6 sufficient livers were grafted in deficient rats (Tx + 1). After an initial increase, with maximum C6 levels of 119% at 10 days following transplantation, the C6 levels decreased gradually and C6 was no longer detectable 28 days after transplantation. This decline in C6 levels was dependent on antibody production against C6. No significant change in the C3, C4, factor H and factor B levels was observed. Expression of C6 mRNA in the grafted PVG/c+ sufficient liver was comparable to the expression of C6 mRNA in control PVG/c+ livers while C6 mRNA expression in the transplanted PVG/ c- liver and the control PVG/c- liver was lower. In conclusion, it was demonstrated in vivo that not only C6 but also C2 is synthesized extrahepatically in PVG/c rats.     

Tolerance to rat liver allografts: IV. Acceptance depends on the quantity of donor tissue and on donor leukocytes

Liver allografts in some rat strains are often spontaneously accepted across a complete major histocompatibility barrier without the requirement for immunosuppression while other nonliver allografts are rejected. In previous studies, we have shown that spontaneous acceptance is dependent on liver passenger leukocytes. Depletion of passenger leukocytes by donor irradiation allows rejection, with DA recipients of irradiated PVG livers having a median survival time (MST) of 16 days. Here we show that, in this model, spontaneous acceptance is reconstituted by intravenous injection of donor leukocytes. Intravenous injection of 3-5x10(7) PVG liver leukocytes significantly prolonged DA survival time (MST=96 days, P=0.026), as did 5x10(7) spleen leukocytes (MST>100 days, P=0.002). Deletion of T cells from the reconstituting inoculum reduced survival time (MST=78 days, P=0.039), whereas deletion of B cells or monocytes/macrophages had no effect on survival time. In contrast, PVG hearts are regularly rejected by DA recipients, and PVG liver or spleen leukocytes, even at doses of greater than 3x10(8) cells/recipient, were unable to induce heart acceptance. To investigate the possibility that acceptance of the irradiated liver but not the heart might be due to the large mass of the liver, two kidneys and two hearts of PVG origin were transplanted to each DA recipient together with 1.5x10(8) PVG leukocytes. These organs survived for greater than 200 days, thereby showing that a large mass of donor tissue, in association with donor leukocytes, leads to acceptance of organs that are rejected if transplanted singly. It appears likely that spontaneous liver transplant tolerance is a high-dose or activation-associated immune phenomenon.     

Antibody- and cell-mediated immune responses of Actinobacillus pleuropneumoniae-infected and bacterin-vaccinated pigs

The number involved in and the rate of migration of donor leucocytes into the following recipient organs (spleen, thymus, bone marrow, lung and mesenteric lymph nodes) were measured in two rat models of orthotopic liver transplantation (OLT) using donor-specific MHC class I antibodies. The first OLT model is one that does not require immunosuppression in order to achieve tolerance and involved the transplantation of DA (MHC haplotype, RT1a) livers into PVG (RT1c) recipients. The second model was one that required a 7-day (10 mg/kg) treatment with cyclosporin A (CsA) to achieve tolerance and used DA donors into Lewis (RT1(1)) recipients. Recipient organs were biopsied on days 3, 20 and 87 following OLT and donor leucocyte migration was quantified by immunohistochemistry and computer densitometry of immunoblots of detergent-solublized tissues in order to resolve both membrane-bound and soluble donor MHC class I antigen. In a separate experiment, spleen biopsies were taken following OLT on days 3 and 15 from the naturally tolerizing OLT model (DA into PVG), treated with and without CsA for 7 days and compared with the (DA into Lewis) model. The initial rate of leucocyte migration between days 3 and 21 following OLT was found to be the most rapid into the spleen, followed by the bone marrow and mesenteric lymph nodes in the naturally tolerant (DA into PVG) model when compared with the (DA into Lewis) model. The number of donor leucocytes in the spleen and mesenteric lymph nodes in both models was, however, approximately the same by 87 days. No real difference in the rate of leucocyte migration was seen in the thymus or the lung for both transplant models over the time course assayed. CsA was found to lower the rate of donor leucocyte migration only over the period it was administered. The rate of donor leucocyte migration into the spleen was still much lower 15 days after OLT in the (DA into Lewis) model compared with the (DA into PVG) model treated with and without CsA. Thus the differences in the rate of donor leucocyte migration into the spleen, bone marrow and mesenteric lymph nodes immediately following OLT may offer an explanation as to why the (DA into PVG) combination is able to accept a transplanted liver without immunosuppressive therapy.     

Contribution of abdomen and legs to central blood volume expansion in humans during immersion

Collagen-induced arthritis (CIA) is a T cell-dependent disease in which susceptibility is controlled by genes both within and outside the major histocompatibility complex (MHC). In the present study, we compared the humoral responses and kinetics of cytokine secretion patterns in the draining lymph nodes of arthritis-susceptible DA rats and arthritis-resistant F344 and DA MHC congenic PVG.1AV1 rats immunized with rat type II collagen (RCII) in incomplete Freund's adjuvant. The results demonstrate a marked humoral RCII response and a Th1 cytokine profile, with expression of interferon-gamma and interleukin (IL)-2 mRNA in DA rats; a limited humoral RCII response and a Th2 cytokine profile, with expression of IL-4 mRNA in arthritis-resistant F344 rats; and a marked humoral RCII response in arthritis-resistant PVG.1AV1 rats. However, in contrast to DA rats, PVG.1AV1 rats produce IgG1 autoantibodies which, together with strong expression of IL-4 mRNA, indicates the involvement of Th2 subsets. From these data, we conclude that non-MHC gene(s) determines the direction of the anti-RCII response towards a Th1 disease-promoting, or a Th2 disease-limiting response.     

Anti-vitronectin antibodies enhance anti-Thy-1-induced proteinuria in PVG/c, but not in Wistar rats

Injection of rats with mouse monoclonal IgG2a anti-Thy1.1 antibodies (ER4G) results in rapid development of proteinuria in Wistar rats, reaching average values of 160 mg/24 h on day 3 after antibody administration. In contrast, no overt proteinuria was observed in PVG/c+ rats (maximum, 40 mg/24 h on day 3). This study investigates whether differences in the inactivation of C5b-9 complexes in the glomerulus by complement inhibitors are responsible for the differences in proteinuria between the two rat strains. Regardless of the presence of proteinuria, an increased expression of Crry by mesangial cells (MC) was observed within 24 h after injection of ER4G in both Wistar and PVG/c+ rats. Double-label immunofluorescence using goat anti-mouse Ig antibodies demonstrated an expression of Crry exclusively on MC. Furthermore, Crry colocalized with C5b-9 complexes on MC, as detected by a monoclonal antibody against the rat C5b-9 neo-antigen. In PVG/c+ rats, C5b-9 complexes persisted in the mesangial area for at least 7 d and colocalized immediately (within 1 h) and homogeneously with vitronectin. However, in proteinuric Wistar rats, C5b-9 complexes disappeared from the glomerular mesangium within 6 d. In these rats, mesangial colocalization of C5b-9 with vitronectin could only occasionally be detected. Pretreatment of PVG/c+ rats with antibodies against vitronectin, followed by administration of ER4G, resulted in the immediate development of proteinuria (maximum, 119 mg/24 h on day 3; P < 0.05), whereas Wistar rats did not become more proteinuric. This study provides evidence that differences in susceptibility of PVG/c+ and Wistar rats to complement-mediated damage of the glomerulus may be related to the degree of inactivation of C5b-9 complexes by complement regulatory factors.     

Regulatory T cells in the control of autoimmunity: the essential role of transforming growth factor beta and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4(+)CD45RC- cells and CD4(+)CD8(-) thymocytes

Previous studies have shown that induction of autoimmune diabetes by adult thymectomy and split dose irradiation of PVG.RT1(u) rats can be prevented by their reconstitution with peripheral CD4(+)CD45RC-TCR-alpha/beta+RT6(+) cells and CD4(+)CD8(-) thymocytes from normal syngeneic donors. These data provide evidence for the role of regulatory T cells in the prevention of a tissue-specific autoimmune disease but the mode of action of these cells has not been reported previously. In this study, autoimmune thyroiditis was induced in PVG.RT1(c) rats using a similar protocol of thymectomy and irradiation. Although a cell-mediated mechanism has been implicated in the pathogenesis of diabetes in PVG.RT1(u) rats, development of thyroiditis is independent of CD8(+) T cells and is characterized by high titers of immunoglobulin (Ig)G1 antithyroglobulin antibodies, indicating a major humoral component in the pathogenesis of disease. As with autoimmune diabetes in PVG. RT1(u) rats, development of thyroiditis was prevented by the transfer of CD4(+)CD45RC- and CD4(+)CD8(-) thymocytes from normal donors but not by CD4(+)CD45RC+ peripheral T cells. We now show that transforming growth factor (TGF)-beta and interleukin (IL)-4 both play essential roles in the mechanism of this protection since administration of monoclonal antibodies that block the biological activity of either of these cytokines abrogates the protective effect of the donor cells in the recipient rats. The prevention of both diabetes and thyroiditis by CD4(+)CD45RC- peripheral cells and CD4(+)CD8(-) thymocytes therefore does not support the view that the mechanism of regulation involves a switch from a T helper cell type 1 (Th1) to a Th2-like response, but rather relies upon a specific suppression of the autoimmune responses involving TGF-beta and IL-4. The observation that the same two cytokines were implicated in the protective mechanism, whether thymocytes or peripheral cells were used to prevent autoimmunity, strongly suggests that the regulatory cells from both sources act in the same way and that the thymocytes are programmed in the periphery for their protective role. The implications of this result with respect to immunological homeostasis are discussed.     

Strain-specific expression of microglial keratan sulfate proteoglycans in the normal rat central nervous system: inverse correlation with constitutive expression of major histocompatibility complex class II antigens

We have recently demonstrated that a novel type of keratan sulfate proteoglycan (KSPG) identified by the monoclonal antibody (mAb) 5D4 is expressed on ramified microglia but downregulated coincident with T-cell-mediated autoimmune inflammation of the spinal cord in Lewis (LEW) rats. In this study we show by immunocytochemistry and Western blot analysis that various inbred rat strains differ significantly in their constitutive expression of KSPG on ramified microglia in the normal CNS. Microglial KSPG was high in LEW and Fischer 344 rats but low in DA, Brown Norway (BN), and PVG rats. The KSPG low-expressing strains exhibited constitutive expression of major histocompatibility complex (MHC) class II antigens on ramified microglia that was not detectable in the KSPG high-expressing strains. Thus, an inverse correlation between constitutive KSPG and MHC class II expression was present. The KSPG-low-/MHC class II-positive phenotype is associated with resistance to experimental autoimmune encephalomyelitis in BN and PVG, but not DA rats. These findings suggest a significant impact of genetic factors on the molecular differentiation of resident macrophages in the CNS.     

Pitx2 participates in the late phase of the pathway controlling left-right asymmetry

BACKGROUND: We have studied the role of the different MHC (RT1) subregions in acute natural killer (NK) cell-mediated bone marrow allograft rejection in lethally irradiated, bone marrow cell (BMC) reconstituted rats. METHODS: We employed a series of MHC congenic and intra-MHC recombinant rat strains so that effects of mismatches in defined RT1 subregions could be studied systematically. BMC allograft survival was measured as 125IUdR uptake in the spleen between day 5 and day 7 after irradiation and BMC reconstitution. RESULTS: We found that in certain RT1 haplotype combinations, nonclassical RT1.C disparities by themselves could determine graft rejection (i.e., in the u/av1 recombinant haplotypes), whereas in another combination (between the av1 and c haplotypes) a mismatch for an isolated classical RT1.A region was decisive for engraftment. Thus, PVG.R1 BMC failed to proliferate in PVG rats, differing in the RT1.A region only, whereas in PVG.1U rats rejection could be determined by isolated differences in the RT1.C region (LEW.1WR1). Also, RT1 homozygous rats (RT1.U) rejected semi-allogeneic F1 hybrid BMC. The acute rejection of BMC was mediated by NK cells, as athymic nude rats, lacking alloreactive T cells but with normal alloreactive NK cells, showed the same patterns of rejection as did normal rats. Nude rats also rejected allogeneic lymphocytes, a previously documented NK-mediated phenomenon, with identical requirements of MHC disparity. CONCLUSIONS: This investigation shows that rat effector NK cells are radioresistant, independent of the thymus, and capable of recognizing and rejecting MHC mismatched transplanted BMC on the basis of mismatches in both classical and nonclassical class I regions in vivo. The studies underline the importance also of NK cells in determining BMC allograft survival.     

Renal microvascular injury induced by antibody to glomerular endothelial cells is mediated by C5b-9

We have recently developed a model of thrombotic microangiopathy with injury to the glomerular endothelial cell (GEN) induced by heterologous antibody to rat GEN. In addition to GEN injury rats developed glomerular platelet aggregation and fibrin deposition, acute renal failure, and acute tubular necrosis with interstitial inflammation. To study the role of complement in mediating this lesion, we induced the disease in normal complement PVG rats and measured the effects of generalized complement depletion with cobra venom factor (CVF) and of selective C6 deficiency using genetically C6 deficient PVG animals. Complement sufficient rats developed severe endothelial injury accompanied by platelet aggregation, fibrin deposition, decrease in endothelial cells assessed by antibody staining in the glomerulus, and macrophage infiltration. These changes were associated with marked reduction in renal function. These features were either absent or markedly diminished in complement depleted or C6 deficient rats. This demonstrates that C5b-9, the terminal product of activation of the complement cascade, plays an important role in the pathogenesis of this immune renal microvascular endothelial injury model. Thus, the complement system may play a pathogenic role in renal microvascular diseases such as thrombotic microangiopathy.     

Minisatellite origins in yeast and humans

BACKGROUND: In the rat, orthotopic liver transplantation from a DA strain donor to a PVG recipient causes an early rejection response that spontaneously resolves over the following weeks to yield long-lasting, donor-specific tolerance. METHODS: Limiting dilution analysis was used to estimate the frequencies of host CD4+ cells able to proliferate in response to donor antigens in the grafted liver and spleen of recipients during and after tolerance induction. RESULTS: Compared with naive PVG rats, both the frequencies and absolute numbers of donor-reactive host CD4+ cells in the liver and spleen rose significantly during the first week after transplantation and remained elevated for at least 3 months. CONCLUSION: We conclude that the development of tolerance in this model is not associated with deletion of clonogenic donor-reactive CD4+ T cells by clonal exhaustion or any other mechanism.     

Molecular recognition with C-clamp porphyrins: synthesis, structural, and complexation studies

The cholesterolaemic effect of 2 hypercholesterolaemic diets was tested in 12 rat inbred strains. Diet I is a commercial diet supplemented with 2.0% (w/w) cholesterol and 5.0% (w/w) olive oil; diet II is identical to diet I with addition of 0.5% (w/w) sodium cholate. Strains with the highest plasma cholesterol response after diet I (BN and LEW) also had the highest cholesterol response after diet II (hyperresponders, mean response > 3.5 mmol/l). In the strains DA, SHR, BC, WAG, LOU, PVG and BUF the strain mean cholesterol response remained below 1.3 mmol/l after both diets (hyporesponders). Strains F344 and OM had an intermediate cholesterol response after both diets (normoresponders, mean response between 1.3 and 3.5 mmol/l). Only in the strains LOU, PVG and SHR there appeared to be a significant higher cholesterol response after diet II when compared with the cholesterol response after diet I. In the strain WKY this difference was of a borderline significance (P = 0.052) and this strain turned from a normoresponder after diet I into a hyperresponder after diet II. Liver cholesterol levels as measured after feeding diet II for two weeks also appeared to be strain-specific. No correlation was found between the plasma cholesterol response after diet II and the liver cholesterol levels. Changes in plasma phospholipid and triglyceride levels have been measured for both diet I and diet II. For group means a correlation between the cholesterol response and the change in phospholipid levels was found (r = 0.86 for diet I, P < 0.001 and r = 0.76 for diet II, P < 0.01). No such correlation was found for triglyceride levels.     

Preferential involvement of Go and Gz proteins in mediating rat natural killer cell lysis of allogeneic and tumor target cells

IL-2-activated NK cells from PVG rats potently lyse target cells expressing allo-MHC class I determinants. Here, we investigated the role that G proteins play in mediating this activity. Pretreatment of NK cells with pertussis toxin (PT) or cholera toxin (CT) inhibited NK cell killing of tumor (YAC-1 or P815), and allogeneic target cells. ADP ribosylation assay revealed that PT ADP ribosylates a 39-kDa G protein, whereas CT ADP ribosylates a 45 to 47-kDa G protein in PVG NK cell membranes. Membranes prepared from intoxicated NK cells with either PT or CT lost their ability to incorporate [32P]NAD. These membranes possess Gi, Go, Gs, and Gz as demonstrated by immunoblot analysis. However, Gq was not clearly detected by this method. IL-2-activated NK cells were permeabilized with streptolysin O. Permeabilized cells incorporated Abs to Gi, Go, Gz, Gs, and Gq as determined by flow cytometric analysis. When Abs to Go or Gz, but not to Gi, Gs, or Gq, were incorporated inside permeabilized NK cells, a significant reduction in the lysis of tumor or allo-MHC target cells was observed, suggesting that Go and Gz play important roles in transducing the signals necessary to lyse target cells. Our results show for the first time a role for G proteins in mediating NK cell killing of allo-MHC-encoded target cells, and provide evidence for Gz protein involvement in NK cell recognition of target cells. The effect of Gz is novel and has not been previously described in any other system or cell type.