Development of an ex vivo model of pig kidney perfused with human lymphocytes. Analysis of xenogeneic cellular reactions.

BACKGROUND: Because of the explosive nature and the extremely rapid process of hyperacute rejection (HAR), significant infiltration of the xenograft by immunocompetent cells is not observed, and the role and the mechanism of action of cell-mediated rejection in discordant xenografts are therefore still under discussion. METHOD: We developed an experimental approach using pig kidneys perfused with human peripheral blood lymphocytes (PBL) in which the immunologic barrier of hyperacute rejection was excluded and which mimics the in vivo situation. RESULTS: PBL retention in the kidney was evaluated at 20-minute intervals for 3 hours. Retention increased from 30% to 80% with the time of perfusion and was specific because significantly fewer syngeneic lymphocytes were retained. Phenotype analysis of recovered PBL showed a significant decrease in natural killer (NK) cells. Immunohistochemical studies revealed the presence of NK cells and T lymphocytes in the glomerular and interstitial tubular structures of the kidney. Functional studies showed a progressive cessation of diuresis and augmentation of renal vascular resistance when the kidney was perfused with PBL. Electron microscopy examinations of kidney sections perfused with PBL showed swollen endothelial zones, suggesting alterations to and damage of the endothelium. CONCLUSIONS: This system provides a valuable model for the study of early discordant xenogeneic cellular rejection and demonstrates the predominance of xenograft infiltration by NK cells.

Docosahexaenoic and eicosapentaenoic acids inhibit in vitro human endothelial cell production of interleukin-6.

The interaction between lymphocytes, cytokines, and endothelial cells (EC) is a key step in the inflammatory process. Interleukin-6 (IL-6) a pleiotropic cytokine in its effects, seems to be an early indicator of acute systemic inflammation. In this study, we have examined the effects of polyunsaturated fatty acids (PUFAs) on the production of IL-6 by human unstimulated EC or EC stimulated with TNF-alpha (100 U/ml); IL-4 (100 U/ml); LPS (1 ug/ml); or allogeneic peripheral blood lymphocytes (PBL). Twenty-four hour culture supernatants of immunoreactive IL-6 were measured by Sandwich ELISA. We have shown that the production of IL-6 was potentiated when EC were stimulated with TNF-alpha; IL-4; LPS; or monocyte-depleted PBL in comparison to unstimulated EC. The addition of n-3 PUFAs in culture medium (100 ug/ml DHA or EPA) significantly reduces the production of IL-6 by unstimulated EC; or stimulated with TNF-alpha; IL-4 pg/ml); LPS or depleted PBL respectively for DHA and EPA, whereas the n-6 PUFAs (Arachidonic acid), even used at the highest concentration, was ineffective. This inhibitory effect is PUFA dose dependent but is more potent with EPA than DHA. Regardless of the mode of action, since IL-6 is known to be involved in hematopoiesis, in the regulation of the immune response and in the inflammatory reaction, these results suggest that n-3 PUFAs may play a role in suppressing inflammation. Further studies are needed to elucidate the mechanism involved and the choice between the two fatty acids for clinical and therapeutic purposes.

Docosahexaenoic and eicosapentaenoic acids inhibit in vitro human lymphocyte-endothelial cell adhesion.

Dietary supplementation with fish oil, which contains high amounts of long chain omega 3 ((n-3)) polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), has recently been shown to have protective and ameliorative effects on diseases characterized by chronic inflammatory reactions. Interactions between vascular endothelium, mononuclear cells, and cytokines are crucial steps in the course of inflammatory processes such as chronic graft rejection. We therefore studied the effects of DHA and EPA on both the adhesion of peripheral blood lymphocytes (PBL) to human endothelial cells (EC) in culture and the expression of EC-adhesion molecules and their counterreceptors on PBL. The addition of DHA or EPA to the adhesion assay significantly decreased the adhesion of PBL to untreated EC and tumor necrosis factor-alpha (TNF alpha)-, interleukin (IL) 4-, and lipopolysaccharide-stimulated EC. When EC were pretreated with (n-3) PUFAs for 18 hr, washed, and then stimulated by TNF alpha, IL-4, or lipopolysaccharide, PBL adhesion was also significantly reduced compared with controls. We also showed that PBL preincubated with DHA or EPA, and then washed and chromium radiolabeled, still exhibited an adhesion inhibition to TNF alpha- and IL-4-treated EC as well as untreated EC. Cytofluorometry and immunoenzymatic analyses indicated that pretreatment of EC with (n-3) PUFAs before their activation significantly reduced the EC-induced expression of vascular cell adhesion molecule 1, whereas the level of expression of intercellular adhesion molecule 1 and E-selectin was not modified. Furthermore, we showed that incubation of PBL with DHA or EPA moderately reduced the level of cell surface expression of L-selectin and leukocyte function-associated antigen 1, but not of very late antigen 4. In all cases, the inhibitory effect of (n-3) PUFAs was specific and dose dependent. In addition, DHA seems to be a more potent inhibitor than EPA, but the two compounds in association had an additive effect. Regardless of the mode of action, this inhibitory effect may explain the protective and ameliorative effects of (n-3) PUFAs on diseases involving chronic inflammatory reaction.

Docosahexaenoic and eicosapentaenoic acids inhibit human lymphoproliferative responses in vitro but not the expression of T cell surface activation markers.

The effects of polyunsaturated fatty acids (PUFAs: docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids) on induced lymphocyte proliferation and expression of CD25alpha chain of interleukin-2 receptor, CD71 and HLA-DR were investigated. PUFAs had no effect on phytohaemagglutinin (PHA)-induced lymphocyte agglutination, but they strongly inhibited the lymphoproliferative response to PHA. This inhibitory effect is PUFA dose-dependent and seems to be more potent with DHA than EPA, Pre-incubation experiments showed that lymphocytes cultured with PUFAs for 6 h then washed and exposed to PHA, still inhibited lymphocyte proliferation. The authors also showed that this inhibitory activity was time dependent but became nonsignificant when PUFAs were added after 48 h lymphocyte culture. The addition of excess exogenous human recombinant rIL-2 partly restored PHA-lymphocyte proliferation inhibited by EPA but not by DHA. On the other hand, the authors showed that PUFAS did not inhibit IL-2 stimulated lymphocyte proliferation. The addition of PUFAs to cell culture medium had no inhibitory action on the PHA-induced lymphocyte expression of CD25, CD71 and HLA-DR. Furthermore, this effect appeared independent of eicosanoid synthesis or peroxide formation. Indeed, the inclusion of aspirin and vitamin E in the culture medium did not prevent the inhibitory effects of PUFAs on lymphocyte proliferation. Regardless of the mechanism of action, the inhibitory effect of PUFAs on activated lymphocytes may explain why some clinical trials of fish oil supplemented diets containing high amounts of DHA and EPA have been successful in improving the health status of patients suffering from inflammatory and autoimmune disorders.

Tissue factor activity of syncytiotrophoblast plasma membranes and tumoral trophoblast cells in culture.

During pregnancy, important modifications of hemostasis occur resulting in mothers in hypercoagulability and the role of placental cells such as trophoblast cells has been hypothesized. In this study, we first showed that syncytiotrophoblast plasma membranes, isolated from normal human placenta, expressed a strong tissue factor (TF) activity. We then studied TF activity of two continuous trophoblast cell lines (JEG-3 and BeWo) in comparison to human umbilical vein endothelial cells (HUVEC) and transformed human endothelial cells (ECV-304). TF assays were performed on intact detached confluent cells. Unstimulated JEG-3 and BeWo cells exhibited a very high TF activity which slightly increased after 2 to 4 h TNF-alpha stimulation. In contrast, HUVEC and ECV-304 had a lower basal TF activity which was mainly inducible by TNF-alpha, with a maximum effect after 4 to 6 h stimulation. For both cell types, TF activity was decreased to basal value after 16-hour TNF-alpha stimulation. These results support that trophoblast cells are able to express TF but the involvement of this property in the hemostatic physiological changes observed during pregnancy, remains to be demonstrated.

Long-term study of serum glycosyl-transferase levels in pregnant women and their newborn infants.

1. The authors have studied changes of glucosyl-, galactosyl- and sialyl-transferase activities in the sera of women during pregnancy and the early post-partum period, and in neonates from birth to 1 yr of age. 2. Galactosyl- and glucosyl-transferase activities throughout pregnancy were high in comparison to control values, and increased with advancing gestation. In the early post-partum period, the activities of these enzymes decreased markedly, reaching values similar to those of control subjects. 3. There were no variations, however, in serum sialyl-transferase activity at any stage of pregnancy or the post-partum period. In newborn infants, serum glucosyl- and sialyl-transferase activities were identical to control values. 4. Galactosyl-transferase activity was high at birth, decreased progressively and returned to the normal range only after 1 yr. 5. The physiological significance of these glycosyl-transferases in the sera of pregnant women and newborn infants is discussed.

Seminal fluid transferrin as an index of gonadal function in men.

Seminal fluid transferrin concentrations of proven fertile donors and normozoospermic patients were significantly higher (P less than 0.001) than those in other groups examined. There were no significant differences in the transferrin values among vasectomized, azoospermic and very severe oligozoospermic subjects. Values were also similar in patients affected by secretory or excretory azoospermia. Regression analysis showed a positive correlation (P less than 0.001, r = 0.72) between seminal fluid transferrin concentrations and sperm density. A negative correlation (P less than 0.02, r = 0.28) existed between circulating FSH and seminal fluid transferrin concentrations. There were no significant differences between seminal fluid transferrin and the percentages of abnormal sperm cells or immature seminal line elements. These results indicate that the nature of seminiferous tubule dysfunction can be precisely defined by examining seminal fluid transferrin in combination with other biological values usually used to explore testicular function.

In vitro inhibitory effect of human syncytiotrophoblast plasma membranes on the cytolytic activities of CTL and NK cells.

We have previously described the purification procedure for syncytiotrophoblast plasma membranes (STPM) and have now studied their immunomodulatory properties on the in vitro cytotoxic assays of generated cytotoxic T-lymphocytes (CTL) and natural killer cells (NK). STPM inhibited the cytolytic activities of CTL and NK against their target cells, whereas RBC ghosts, even at the highest protein concentration used, were ineffective. This inhibitory effect was dose-dependent upon the STPM-protein concentration and appeared to be particularly distinct at low effector/target ratios. It is hypothesized that the inhibitory activity of STPM may be exerted by blocking the effector cells or by masking their targets. Regardless of the mode of action, since cytotoxic cell activities are known to play an important role in the allograft rejection process, this suppressive inhibitory effect of STPM might be a crucial mechanism in the tolerance of the semiallogenic fetal graft.

Effect of human syncytiotrophoblast plasma membrane-soluble extracts on in vitro mitogen-induced lymphocyte proliferation. A possible inhibition mechanism involving the transferrin receptor.

Transferrin (Tf) binding to lymphocytes and to syncytiotrophoblast plasma membranes (STPM) and their soluble extracts (STPM-SE) was examined, as well as the effect of the latter on mitogen-induced lymphoproliferation. Lymphocytes only express Tf receptors (Rtf) after mitogen (phytohemagglutinin or concanavalin A) stimulation, and the percentage of Tf bound by stimulated lymphocytes increased as a function of contact time with the mitogen, reaching a maximum at 72 hr. Studies of Tf binding to STPM-SE showed that the percentage of bound Tf increased proportionally to the protein concentration, but was additionally enhanced by a factor of three when STPM were pretreated with 3 M KCl. Scatchard analysis of Tf binding to lymphocytes cultured for 72 hr in the presence of mitogen, as well as to STPM and STPM-SE, revealed that this binding was specific and occurs via a single category of identical and independent receptors the numbers and affinity constants (Ka) of which have been determined. The results obtained by using STPM indicate that the Ka does not vary significantly from one preparation to another, but that the number of sites per milligram of protein increases by a factor of 10 when the STPM are pretreated with 3 M KCl (KCl-STPM). Finally, STPM-SE inhibited the mitogen-induced lymphoproliferative response whether or not they were treated with 3 M KCl. This inhibition was not due to lymphocytotoxicity, was dose dependent regardless of the preparation used, but was maximized with the KCl-STPM-SE fraction. The correlation between the inhibitory capacities of the soluble STPM extracts and the numbers of RTf sites present on their membranes leads to the hypothesis that the observed inhibition could involve the RTf. This effect may help in protecting the fetus from the maternal immune system at the time of the semi-allogenic fetal graft.

Characterization of the human syncytiotrophoblast plasma membrane associated components.

A purification procedure of synctiotrophoblast plasma membrane (STPM) has previously been described. We now report a further characterization of their associated components. By immunochemical analysis two groups of proteins were found. The first fraction solubilized by chaotrope treatment was made up with a complex of IgG, albumin, transferrin and alpha-fetoprotein. The second fraction, insolubilized by KCl-treatment but by detergents, did not react with antisera to human proteins; it contained human chorionic gonadotropin and placental lactogen. The amino acid, carbohydrate and lipid content of STPM were determined. High galactosyl- and sialyl-transferase activities were found in purified STPM. The role of these various components on the acceptance of the fetal allograft is discussed.

A simple and sensitive solid-phase radioimmunoassay for measuring the transferrin content of human biological fluids: its application to seminal plasma.

A highly sensitive and reproducible radioimmunoassay was established to detect transferrin in human fluids. By this technique, applied to seminal fluid, transferrin levels (micrograms/ml) were found in normozoospermic individuals (64.49 +/- 25.41) at level higher than in oligozoospermic (38.93 +/- 21.35), azoospermic (19.49 +/- 10.23), or vasectomized (19.61 +/- 8.95) subjects. A relationship between transferrin and spermatozoid concentration in sperm was shown. These results reinforce previous findings that seminal transferrin can be used as a reliable clinical marker of Sertoli Cell function.

Effect of human syncytiotrophoblast extract on in vitro proliferative responses.

The authors studied the effects of soluble syncytiotrophoblast extract (STE) on the proliferative responses of lymphocytes to different lectins (PHA and Con A) and to allogeneic cells in one-way mixed lymphocyte cultures. STE suppressed lymphocyte reactivity to lectins and to allogeneic cell cultures. The inhibitory effect was dose dependent. Increased concentrations of lectins failed to overcome the inhibitory effect of STE. Lymphocytes preincubated with STE for 18 hr, then washed and exposed to lectins still exhibited an inhibition of cellular proliferation. STE added to lymphocyte cultures at various times in the presence of both mitogens or of allogeneic cells continued to inhibit lymphoproliferative responses even when it was added after 43 hr of cell culture. Furthermore, STE was able to reduce the spontaneous proliferation of tumor cell lines K562 and LHN13 maintained in vitro culture prior to testing. In all cases, the inhibition observed was not due to lymphocytotoxicity or to tumor cell mortality. The inhibitory effect of STE on the proliferative responses of lymphocytes to stimulants may be due to a cytostatic effect that may represent a contributing factor in the nonrejection of the fetus by a competent immune system.