Human T lymphocyte proliferative response to resting porcine endothelial cells results from an HLA-restricted, IL-10-sensitive, indirect presentation pathway but also depends on endothelial-specific costimulatory factors.

To investigate the mechanisms of cellular rejection in pig-to-human xenotransplantation, the proliferation of different human purified lymphocyte subpopulations in response to swine leukocyte Ag class II-negative porcine aortic endothelial cells (PAEC) was measured in the presence or absence of human autologous adherent cells (huAPC). CD8+ lymphocytes proliferated moderately in the absence of huAPC, and the immune response was slightly increased when huAPC were added. CD56+ lymphocytes failed to proliferate in response to PAEC whether huAPC were present or not. CD4+ lymphocytes alone did not proliferate in response to PAEC, but a strong proliferative response was observed in the presence of metabolically active huAPC. This response was totally abolished by mAbs directed against HLA class II molecules or by pretreatment of huAPC by human IL-10. Even in the presence of huAPC, CD4+ lymphocytes failed to respond to fixed PAEC or to PAEC-lysates, suggesting that PAEC must be viable to support lymphocyte proliferation. Finally, none of the nonendothelial porcine adherent cells tested was able to induce human lymphocyte proliferation, despite the fact that they also provided a large set of xenogeneic peptides. Our results show that the indirect presentation pathway of xenoantigens by huAPC to CD4+ lymphocytes is crucial in the response to porcine endothelial cells, and that IL-10 could be of therapeutic interest to prevent human lymphocyte activation by this pathway. Furthermore, we demonstrated that stimulatory signals specifically provided by endothelial cells are also necessary for this huAPC-restricted proliferative response.

Human NK cell-mediated direct and IgG-dependent cytotoxicity against xenogeneic porcine endothelial cells.

Once hyperacute rejection has been prevented, the pig-to-human xenograft might be exposed to vascular cell-mediated rejection directed against vascular structures. In order to evaluate the relative importance of direct and antibody-dependent anti-endothelial cell-mediated cytotoxicity in different individuals, freshly isolated human blood leukocytes were incubated with confluent porcine aortic endothelial cells (PAEC) in a 4 h Cr-release cytotoxicity assay. Peripheral blood mononuclear cells (PBMC) and lymphocytes (PBL) of all subjects tested (but not monocytes or neutrophils) directly killed PAEC, with wide interindividual variations (from 2.8% to 32%). The addition of heat-inactivated autologous serum to PBMC and PBL (but not to myeloid cells) always enhanced cytotoxicity. This antibody-dependent cell-mediated cytotoxicity (ADCC) was also observed in the presence of adult pooled serum and cord blood pooled serum and was eliminated after adsorption of adult pooled serum to insoluble protein A, demonstrating that IgG is the only class of immunoglobulin involved in this phenomenon. Moreover, blocking Fc gamma RIII with an anti-CD16 mAb eliminated ADCC without affecting direct cytotoxicity. When the ADCC exerted by the PBL of all subjects was assessed with the same preparation of purified IgG, wide interindividual variations were again observed. Surprisingly, there was no correlation between direct cytotoxicity and ADCC although, as depletion experiments demonstrated, both were due to CD16+ natural killer (NK) cells. These results argue that CD16+ NK cells could play an important role in early vascular rejection of porcine discordant xenografts, by both a direct and an IgG xenoreactive natural antibody-dependent cell-mediated cytotoxicity.

Removal of terminal alpha-galactosyl residues from xenogeneic porcine endothelial cells. Decrease in complement-mediated cytotoxicity but persistence of IgG1-mediated antibody-dependent cell-mediated cytotoxicity.

To determine the role of the terminal alpha-galactosyl residue in the endothelial damage mediated by human xenoreactive natural antibodies (IgM and IgG), we treated porcine endothelial cells in culture with green coffee bean alpha-galactosidase. A practically complete removal of terminal alpha-Gal residues (as evaluated by flow cytometry with Bandeiraea simplicifolia isolectin B4) and concomitant exposure of N-acetyllactosamine were obtained without altering cell viability. A dramatic decrease in IgM and IgG binding (from a pool of human sera) was observed, confirming the key role of the alpha-galactosyl residues. The enzyme treatment did not induce any nonspecific immunoglobulin binding sites, but led to the exposure of new epitopes for a minor fraction of IgM. The main residual IgM and IgG binding could be due to xenoantigens other than the alpha-galactosyl residues. When alpha-galactosidase-treated endothelial cells were used as targets in cytotoxicity experiments, they were less susceptible than untreated cells to complement-mediated cytotoxicity induced by fresh human serum. In contrast, they did not acquire resistance to human IgG-dependent cellular cytotoxicity, despite the decrease in IgG binding. Because it is known that antibody-dependent cytotoxicity mediated by CD16+ NK cells is dependent on IgG1 and IgG3, and not on IgG2 or IgG4, which was confirmed by blocking experiments, we studied the binding of all four subclasses to intact and alpha-galactosidase-treated endothelial cells. Two major subclasses, IgG1 and IgG2, bound to untreated endothelial cells, whereas IgG3 binding was low and IgG4 binding was negligible. A decrease in IgG1, IgG2, and IgG3 binding was observed upon alpha-galactosidase treatment, indicating that antibodies belonging to these three subclasses recognize alpha-galactosyl residues. The decrease in IgG2 binding was more pronounced than the decrease in IgG1 binding. Collectively, these data indicate that IgG1 xenoreactive natural antibodies, including those which are not directed at the alpha-galactosyl residues, could play a major role in the early delayed vascular rejection of pig xenografts.

Inhibition of induced lymphocyte proliferation by lipid and protein components of the syncytiotrophoblast plasma membrane.

PROBLEM: The aim of this work was to define the respective responsibilities of the lipid and protein components of syncytiotrophoblast plasma membranes on the inhibition of lymphocyte proliferation induced in vitro. METHOD: A fractionation method using octyl-beta-D-glucopyranoside enabled lipoprotein, lipid, and protein fractions to be isolated from the membrane. RESULTS: The lipid fraction was shown nonspecifically to inhibit lymphocyte proliferation, to a lower extent compared with the native membrane. Alternatively, the protein fraction used as a proteoliposome contained the totality of the cytostatic effect of the native fraction. CONCLUSION: These results are discussed generally in the context of the immunoregulatory role of membrane lipids and proteins and in relation to the local properties of syncytiotrophoblast plasma membrane components in fetal graft tolerance.

Human Sertoli cells in vitro. Lactate, estradiol-17 beta and transferrin production.

Human Sertoli cell parameters, namely lactate, estradiol-17 beta, and transferrin production, were determined after a 24-hour incubation with either human follicle stimulating hormone (FSH) or dbcAMP in the presence or absence of testosterone plus a phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine; MIX). Testicular tissues were obtained from 10 young patients (mean age, 29 years); using a 3-step enzymatic treatment, Sertoli cell enriched preparations (> 92%) were studied after 4 days as primary cultures. No significant changes in lactate, estradiol-17 beta, and transferrin outputs have been observed according to age in patients ranging in age from 16 years to 47 years. Sertoli cell production of the compounds is controlled by testosterone plus MIX; FSH (or dbcAMP) treatment only slightly improves their synthesis. It is suggested that human Sertoli cell function, as far as the parameters measured in this study are concerned, is likely regulated by cAMP-dependent and independent pathways.

Germ cell-Sertoli cell interactions and production of testosterone by purified Leydig cells from mature rat.

The addition of seminiferous tubule (ST) culture medium (STM) prepared from testes of either busulfan-treated (Bus) or cryptorchid (Cryp) or genetically sterile (hd) rats, to Percoll purified Leydig cells leads to a further increase of LH-stimulated testosterone (T) output (26, 43 and 14%, respectively). Taking into account that the Sertoli cell number per cm of ST is 2.6, 1.8 and 1.4-fold greater in Bus, Cryp and hd rats than in controls, the above STM effects on T output, expressed per 10(6) Sertoli cells are in fact lower (63, 44 and 43%, respectively) that those of control STM. Similar results have been obtained for the STM transferrin levels which are decreased, 74, 67 and 45%, respectively in Bus, Cryp and hd animals. So, it is likely that the Sertoli cell secretion of both the paracrine factor involved on Leydig cell T production and the transferrin is influenced mainly by spermatids and to a lesser extent by spermatocytes of mature rat testis.

Inhibitory effect of human syncytiotrophoblast plasma membrane vesicles on Jurkat cells activated by phorbol ester and calcium ionophore.

The effects of syncytiotrophoblast plasma membrane vesicles (STPM) on stimulated Jurkat leukemic T cells have been investigated. STPM inhibited IL-2 production and the expression of protein P55 of the IL-2 receptor (IL-2R P55), when Jurkat cells were stimulated by a combination of calcium ionophore A23187 (CaI) + phorbol 12-myristate 13-acetate (PMA). STPM also inhibited IL-2R P55 when cells were stimulated by PMA alone, a situation in which IL-2 production is negligible. On the other hand, STPM had no effect on the sustained mobilization of intracellular Ca2+ induced by CaI nor on the PKC-dependent CD3 down regulation induced by PMA. Finally STPM had no effect on intracellular cAMP levels. These results show that (i) the inhibitory effect of STPM on IL-2R P55 expression is independent of the inhibition of IL-2 production, and (ii) the inhibitory effects of STPM are at least partially independent of phosphatidylinositol 4,5-bisphosphate hydrolysis. They suggest that STPM affect a signaling pathway activated by PMA but possibly PKC independent.

The inhibitory effect of human syncytiotrophoblast plasma membrane vesicles on in vitro lymphocyte proliferation is associated with reduced interleukin 2 receptor expression.

The mechanisms by which vesicles of syncytiotrophoblast plasma membranes (STPM) prepared from full-term human placentas inhibit lymphocyte proliferation have been investigated. In the presence of STPM, IL-2 secretion and the expression of protein P55 (IL-2R P55) from its receptor were examined in two models of PBMC proliferation: induced by PHA in 3-day-old cultures, and induced by IL-2 in 6-day-old cultures. In the case of PHA stimulation, STPM strongly inhibited IL-2 (but not IL-1) secretion and IL-2R P55 expression at a concentration where lymphocyte proliferation was also blocked. In these conditions, the addition of excess recombinant IL-2 (rIL-2) only partially restored proliferation and IL-2R P55 expression. In addition, STPM inhibited proliferation and IL-2R P55 expression when resting PBMC were stimulated by a high concentration of rIL-2. These results suggest that STPM inhibit lymphocyte proliferation by affecting one or several events occurring in the synthesis and/or expression of IL-2R P55 by a mechanism which is at least partially independent of its inhibitory effect on IL-2 secretion. The significance of these results is discussed in the context of the survival of the fetal allograft.

Syncytiotrophoblast plasma membrane inhibits membrane expression of activation markers on PHA-stimulated human lymphocytes.

Studies have been carried out on the effects of full-term human syncytiotrophoblast plasma membrane (STPM) preparations on the membrane expression of the lymphocyte activation markers HLA-DR, IL-2R, TfR and CD69 during PHA-induced lymphoproliferation. STPM considerably decreases the expression of both late (HLA-DR) and early (IL-2R, TfR, CD69) activation markers at doses which inhibit PHA-induced lymphoproliferation. These results favour the hypothesis that STPM inhibits a very early phase of PHA-induced lymphocyte activation.

Sodium diethyldithiocarbamate protects against the MPTP-induced inhibition of immune responses in mice.

The effects of 1-methyl-4-phenyl - 1,2,3,6-tetrahydropyridine (MPTP) on immune parameters, and the restorative influence of sodium diethyldithiocarbamate (DTC) or deprenyl were evaluated in mice. The concentrations of dopamine (DA), 3-methoxytyramine (3-MT), 3-4-dihydroxyphenyl acetic acid (DOPAC), and homovanillic acid (HVA), were concomitantly measured in the striatum. MPTP depressed T-cell responses. DTC restored these responses as well as the concentration of striatal DA. Deprenyl had no effect on the concentrations of DA and its metabolites, yet it modified the immune responses alike MPTP. The findings suggest a dopamine pathway could be involved in the brain-controlled immunostimulation afforded by DTC.

Immunopharmacology and immunotoxicity of zinc diethyldithiocarbamate.

Zinc is essential for the functions of the immune system, and sodium diethyldithiocarbamate (imuthiol) restores and regulates the numbers and activities of cells of the T-cell lineage. The combination of both elements was therefore tested for immune enhancement and immunotoxicity. The data presented herein show that the administration of zinc diethyldithiocarbamate was devoid of immunoenhancing influence on the responses to T-cell mitogens, and exerted a cytocidal effect on spleen lymphocytes.

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.

Effect of continuous low-dose gamma-irradiation on rat Sertoli cell function.

Continuous low-dose gamma-irradiation of mature rats induced a progressive degeneration of the germ cells. Blood FSH increased by 127, 176 and 214%, respectively, after 55, 70 and 85 days of treatment when compared to FSH levels in control rats (8.50 +/- 0.60 ng/ml); conversely, serum LH and testosterone levels were unchanged. The Sertoli cell function was affected by the treatment from 70 days on, as attested by androgen binding protein (ABP) and transferrin secretions which diminished 35-40%. Serum ABP levels were not altered, whatever the duration of irradiation, even though epididymal ABP contents (as well as concentrations) diminished 34-60% when compared to those of the controls. Moreover, in purified Leydig cells, LH-stimulated intracellular cAMP levels, which were decreased by seminiferous tubule medium (STM) from control rats, were enhanced in presence of STM from treated animals. Testosterone output was stimulated 9-fold in presence of oLH and further increased (46-76%) from stages XIV-V by STM prepared from control and irradiated rats, respectively. After 85 days the STM effects on both cAMP and testosterone syntheses were zero. These results demonstrate a probable alteration of Sertoli cell function after irradiation, but also a role of the germ cells in the regulation of the synthesis of ABP, transferrin and Sertoli cell paracrine factors.

Early changes in immune parameters induced by an acute nonantigenic inflammation in mouse: influence of imuthiol.

Calcium pyrophosphate (CaPP)-induced pleurisy, may represent one of the simplest expressions of inflammation in that the irritant is a non-diffusible, non-antigenic and non-pyrogenic agent. Spleen or lymph node T or B cell numbers and activities, as well as NK activity, were modified at distance by CaPP-pleurisy. An intense increase in blood polymorphonuclear cells was also triggered by the inflammatory process. Treatment with imuthiol (sodium diethyldithiocarbamate), an agent known to be active on the T-cell lineage, restored towards control values the inflammatory response and tended to normalize white blood cell percentages altered by the inflammatory process. The findings suggest imuthiol could be employed as a virtually nontoxic and non-steroidal anti-inflammatory agent.

Fate and distribution of radioactive sodium diethyldithiocarbamate (Imuthiol) in the mouse.

The distribution of 35S in mouse tissues has been investigated by radioactivity counts and by autoradiography after the intravenous injection of 35S-labeled imuthiol (sodium diethyldithiocarbamate). Radioactive Imuthiol is selectively localized on liver, thymus and brain neocortex, most likely as the methyl ester, within minutes after dosing. Lung or white brain matter did not fix the labeled thiol. Blood, kidney and guts show rapid elimination patterns, and can be considered as passage organs which did not fix Imuthiol. The findings are consistent with the immunopharmacological data which demonstrate that Imuthiol exerts its T-cell recruiting and activating influence through a multi-step pathway involving the brain neocortex, the thymus and the liver.

Favorable influences of imuthiol on mouse reproduction and immune system of offspring.

A physiological immature immune system in newborns is a common feature frequently associated with increased susceptibility to infections. The properties of imuthiol (purified sodium diethyldithiocarbamate), an agent specifically active on the T-cell lineage, and virtually devoid of toxicity for man or animals, encouraged us to determine whether imuthiol administered to the dams could increase the immune capability of offspring without altering fecundability and birth rate. Experiments performed either in histocompatible or histoincompatible mating systems, show that chronic administration of imuthiol prior to mating and/or during pregnancy stimulated newborn mice to increased T-cell-dependent responses, without altering birth rates and growth curves in progenies. The data suggest that imuthiol has no teratogenicity or deleterious influences on mouse gametes, and might be useful to prevent immunodepression-associated infections in newborns.

Sodium diethyldithiocarbamate influences the early immunological changes evoked by an acute non-antigenic inflammation process.

Calcium pyrophosphate (CaPp) produces an acute local inflammatory process when administered intra-pleurally. It is the simplest model of an inflammation uncomplicated by pharmacological properties of the agent or the presence of an antigen. Spleen or lymph-node T- or B-cell numbers and activities, including NK activity, are modified within 2 h under these conditions. A treatment with sodium diethyldithiocarbamate (lmuthiol), already known as a T-cell augmenting agent, restores towards normal values both the inflammatory reaction and the modified immune parameters induced in mice by CaPp pleurisy. The use of lmuthiol as a non-toxic, non-steroidal antiinflammatory agent is therefore suggested.

Involvement of brain neocortex and liver in the regulation of T cells: the mode of action of sodium diethyldithiocarbamate (imuthiol).

Sodium diethyldithiocarbamate ( imuthiol ), a non-antigenic and non-carcinogenic compound, devoid of toxic effects at immunostimulant doses, shows distinctive properties in recruitment and activation of T cells. Studies on its mode of action disclosed unsuspected links between the immune system, the endocrine liver and the central nervous system. Evidence was obtained indicating that the brain neocortex modulates T-cell mediated events, most likely via control of specific hormonal synthesis by the liver. The influence of imuthiol is determined at the level of the brain neocortex.

Kinetics of the histological changes in lymphoid organs and of the T-cell inducing capacity of serum in mice treated with Imuthiol (sodium diethyldithiocarbamate).

Sodium diethyldithiocarbamate ( Imuthiol ) triggers in mice an increased production of T-cell recruiting factors and an early and prolonged hyperplasia in the T-cell dependent areas of peripheral lymphoid organs, partially independent from thymus modification. The data confirm previous findings on the role of an extrathymic factor and are consonant with the notion of a feedback regulation to control T-cell differentiation.