Antigen persistence and time of T-cell tolerization determine the efficacy of tolerization protocols for prevention of skin graft rejection.

We studied antigen-specific T-cell tolerization therapy using skin transplantation across a defined minor histocompatibility antigen difference. Specific tolerization protocols using short-lived peptide or long-lived spleen cells presenting the peptide as antigen prevented graft rejection without immunosuppression when started before or as long as 10 days after transplantation. Peptide-induced T-cell tolerance was transient, and antigen presentation by the graft was not sufficient to maintain tolerance. In contrast, transfer of antigen-expressing lymphoid cells induced long-lasting tolerance correlating with donor cell chimerism. These findings show that antigen-specific tolerization can induce graft acceptance even when begun after transplantation and that long-term graft survival depends on persistence of the tolerizing antigen.

Delayed developmental immunotoxicity of prenatal benzodiazepines.

Treatment of pregnant rats with low doses of classical benzodiazepines (BDZ, e.g. 1.25 mg diazepam/kg body weight) or a peripheral type BDZ receptor (PBR) agonist between gestational days 14 and 20 has been shown to result in a long-lasting depression of cellular and humoral immune responses in the offspring. Considerable alterations in mitogen-stimulated cytokine production in rats exposed to diazepam prenatally have now been observed: TNF-alpha liberation by splenocytes of diazepam-exposed rats was reduced at 2 wk of age and increased above control values at 8 wk, and interleukin (IL)-6 was depressed in the offspring at 2 and 8 wk of age. IL-1 was diminished during post-weaning and adult periods in male offspring but only in adult life in female offspring. In contrast, T-cell derived IL-2 was decreased during the postnatal period and normalized in adulthood. Prostaglandin E(2) (PGE(2)), which is known to down-regulate tumour necrosis factor-alpha (TNF-alpha) was increased and interferon-gamma (IFN-gamma), which stimulates TNF-alpha release, was depressed in 2-wk-old offspring that had been treated prenatally. Release of PGE(2) and IFN-gamma was still altered in young adulthood. While the initial action on the foetal immune system remains unknown, an interaction of the drugs with the PBR is suggested by the effectiveness of the PBR agonist and by altered characteristics of PBR (i.e. a decreased B(max) of [(3)H]PK 11195 binding to macrophage membranes of 8-wk-old offspring and an increased Kd of spleen cell membranes of 2-wk-old offspring).

Diazepam treatment of pregnant rats differentially affects interleukin-1 and interleukin-2 secretion in their offspring during different phases of postnatal development.

Treatment of pregnant Long Evans rats with benzodiazepines was found to cause alterations in cellular immune responses in their offspring. We now report on changes in interleukin-1 (IL-1) and IL-2 secretion which were analyzed in rats from birth until 12 weeks. Time-pregnant rats were treated with diazepam (1.25 mg/kg/day subcutaneously) from gestational day 14 to 20. Lipopolysaccharide-stimulated release of macrophage-derived IL-1 by spleen cells, determined on D10.G4.1 cells, remained in the control range during the preweaning period (postnatal day 6-28), then decreased in prenatally diazepam-exposed offspring, significantly in males during the postweaning period (postnatal day 34-61) and in both sexes in adults (postnatal day 62-83). Concanavalin A-stimulated release of T lymphocyte-derived IL-2 from spleen cells, determined on CTLL-2 cells, was reduced in male and female offspring during preweaning (postnatal day 3-28) and postweaning (postnatal day 33-55) periods and normalized in adulthood (postnatal day 60-84). The percentage of IL-2 receptor expressing (CD25+) cells was unaffected. From these and our earlier data it is evident that prenatal exposure to low doses of benzodiazepines can result in long-lasting alterations of the cytokine network, as indicated by reduced release of TNF-alpha, IL-1, IL-6, IL-2 and interferon-gamma. The concomitant reduction of peripheral type benzodiazepine receptors on macrophages is discussed as a possible link between prenatal treatment and disturbed function.

Release of IL-1 and IL-2 and expression of IL-2 receptors as a function of postnatal age and sex.

Release patterns of the mitogen-induced cytokines IL-1 and IL2 by Long Evans rat unfractionated spleen cells, as well as expression of IL-2R on such cells at different postnatal days (neonatal to adult) were studied for female and male rats. IL-1 release was found to be low but in a more or less steady fashion in cells of both sexes over the entire period. However, IL-2 synthesis increased from an almost undetectable level up to a significant secretion at the time of weaning and continued to increase in female but not male spleen cells. IL-2 receptors were found to be expressed in moderate amounts already at early postnatal days and up to weaning. Thereafter, the synthesis of IL-2R followed a course essentially similar to that of IL-2, i.e. higher for female than for male cells. In the female rats, a hormonal influence was observed on the formation of IL-1 and IL-2 but not on the expression of IL-2R.

In vitro inhibition of cellular immune responses by benzodiazepines and PK 11195. Effects on mitogen- and alloantigen-driven lymphocyte proliferation and on IL-1, IL-2 synthesis and IL-2 receptor expression.

In vitro mitogen-driven lymphocyte proliferation tests (Con A, LPS) on murine lymph node and spleen cells revealed inhibition of T and B cell stimulation by different benzodiazepines and by PK 11195, with IC50 values in the low micromolar range. T cell responses as a consequence of recognition of alloantigens, as measured in mixed lymphocyte cultures (MLC), were affected in an analogous way. In all systems, agonists at peripheral type benzodiazepine receptors (Ro 5-4864 and the non-benzodiazepine compound PK 11195) and diazepam which acts on both, central and peripheral type benzodiazepine receptors, were most potent; clonazepam, a central type agonist, proved about half as active. The central type antagonist Ro 15-1788 failed to antagonize the action of diazepam and clonazepam. Variations among cells from several congenic strains of mice were modest. Cytotoxicity could not be made responsible for drug effects. The most susceptible stage of mitogen-triggered T and B lymphocyte proliferation was found to be at incipience. Radioresistant, adherent spleen cells, upon LPS-stimulation formed only small amounts of the cytokine IL-1. Its release was affected only at very high drug concentrations. Similar small amounts of IL-1 were generated during MLC; in this case, the drugs were about 10 times less potent than in mitogen-induced proliferation assays. Peripheral agonists were more active on IL-1 synthesis. Spleen cells stimulated with Con A and cultivated with the highest concentration of diazepam and clonazepam formed markedly greater amounts of IL-2 than those cultivated in medium, while at this concentration PK 11195 allowed no formation of the lymphokine.(ABSTRACT TRUNCATED AT 250 WORDS)

Nervous and immune systems as targets for developmental effects of benzodiazepines. A review of recent studies.

Prenatal exposure to benzodiazepines (BDZ) can cause behavioral dysfunctions both in humans and in experimental animals. In addition, prolonged impairment of cellular immune functions is found in rats after low dose BDZ exposure (e.g., diazepam 1.25 mg/kg/day) during part of fetal life [gestational days (GD) 14-20]. Analysis of diazepam and its metabolites in maternal and fetal tissues revealed that in this rat model the drug is no longer present at birth, which excludes direct effects of diazepam during the postnatal period. The main target of BDZ in brain, the GABAA receptor complex, is structurally and functionally heterogeneous. Besides alpha- and beta-subunits, gamma 2- or gamma 3-subunit should be coexpressed for a fully functional BDZ response. Signals of mRNAs encoding for alpha 1, beta 2 and gamma 2 are detected in fetal rat spinal cord and lower brainstem by GD 14 and reach telencephalic regions in later fetal life, reminiscent of BDZ receptor ontogeny. Regional subunit distribution differs from the adult brain, one interesting feature being a preponderance of gamma 2 mRNA throughout fetal life. Since subunit composition influences the sensitivity to BDZ, these data suggest that prenatal effects of BDZ depend upon regional subunit compositions present at different developmental stages. The delayed depression of cellular immune responses in prenatally BDZ-exposed rat offspring during the first 2 postnatal months is accompanied by various changes in immune cell biology. Binding characteristics of the peripheral (omega 3) type BDZ receptor are altered until adulthood (8 weeks). Membranes of spleen cell preparations containing mainly lymphocytes exhibit a decrease of affinity for the peripheral ligand [3H]PK11195, splenic macrophage preparations a decrease of maximal binding capacity. Various defects in cytokine production by macrophages and T lymphocytes were observed: Mitogen-stimulated release of macrophage-derived tumor necrosis factor-alpha (TNF-alpha) and of the T cell-derived interleukin-2 (IL-2) was drastically reduced at 2 and 4 weeks of life and recovered in young adulthood, exhibiting the same time course of depression as lymphocyte proliferation in response to immune stimuli. Interleukin-6 (IL-6) release remained diminished until adulthood. In female offspring, additional alterations were found in splenic noradrenaline turnover after immune stimulation. The mechanisms underlying the breakdown of the cytokine network in prenatally diazepam-exposed offspring, and the long-term consequences are as yet unknown.

Nuclear myxovirus-resistance protein Mx is a minor histocompatibility antigen.

Minor histocompatibility antigens (MiHAgs) cause slow-to-rapid organ transplant rejection by immunocompetent hosts and mild-to-severe graft-versus-host reactions in immunosuppressed hosts. MiHAgs are allelic forms of major histocompatibility complex (MHC) class I-restricted self-antigens recognized by cytotoxic T cells and usually are defined immunogenetically. Although structurally not identified as yet, it is assumed that MiHAgs are internal cell antigens that are processed and then presented by MHC class I proteins similar to viral antigens. To define a MiHAg both molecularly and functionally, we took advantage of the allelic difference of the structurally characterized intracellular myxovirus-resistance protein (Mx) and investigated its antigenicity. Skin grafts from congenic Mx+ mice carrying the functional Mx1 gene were rejected by mice lacking a functional Mx1 gene (Mx- mice). In parallel, cytotoxic MHC class I-restricted effector T cells specific for Mx protein and the H-2Kk antigen (but not for several other allelic H-2 antigens) were strongly induced in Mx- mice immunized with spleen cells from interferon-treated Mx+ mice. These data show that allelic forms of cell internal proteins presented by MHC class I may act as MiHAgs.

Prenatal benzodiazepine immunosuppression: possible involvement of peripheral benzodiazepine site.

Treatment of pregnant Long Evans rats with a low dose of diazepam (1.25 mg/kg from gestational day 14-20) produced offspring suffering from suppression of cellular immune responses. Analogous effects were produced by clonazepam, a benzodiazepine (BDZ) with high affinity for the central-type, and Ro 5-4864, a BDZ with selective affinity for the peripheral-type BDZ receptor. Peripheral-type BDZ receptors are found to develop early in fetal life in peripheral organs including primary (thymus) and secondary (spleen) lymphoid organs, in the central nervous system and on immune cells (lymphocytes). In prenatally diazepam-exposed offspring the affinity constant is significantly changed. BDZ and PK 11195 also inhibit mitogen and alloantigen-induced T and B cell proliferation in vitro in adult murine lymphocytes. Diazepam, Ro 5-4864 and PK 11195 were found to be the most active compounds.

Diazepam effects on the fetus.

Treatment of time-pregnant Long Evans rats with 1.25 mg/kg s.c. diazepam (2.5 mg/kg in Sprague Dawley rats) from gestational day 14 to 20 produced transient depression of an olfactory guided behavior (nest odor behavior) in suckling offspring. Enhanced drug sensitivity to diazepam was seen in adult male and female off-spring as indicated by increased temperature depression. In addition, increased sensitivity to an opiate (morphine) was noted for the female offspring in the tail flick test. Treatment of the pregnant dam with diazepam or clonazepam, a benzodiazepine with selective affinity for the central benzodiazepine receptor, resulted in a marked depression of cellular immune responses in the offspring of both sexes up to 2 months of age. Drug treatment during early fetal period (GD 12-16), at a time central benzodiazepine receptors are not present in all brain regions of the fetal brain, did not affect the quality of cellular immune responses, whereas treatment from GD 16 to 20 was effective. Prenatal diazepam effects are discussed in view of presence and functionality of both central and peripheral benzodiazepine binding sites in the fetus.

Prenatal diazepam induced persisting depression of cellular immune responses.

Treatment of pregnant Long Evans rats with a low dose of diazepam (1.25 mg/kg per day s.c.) from gestational day (GD) 14 to 20 resulted in severe and long lasting depression of cellular immune responses in male and female offspring. T lymphocyte proliferation, induced by allogeneic stimulation in mixed lymphocyte culture (MLC) or geneic stimulation in mixed lymphocyte culture (MLC) or mitogenic stimulation (concanavalin A), decreased by 50 % or more over a postnatal period of about 2 months. Treatment of the pregnant dam during the early fetal period, from GD 12 to GD 16, did not significantly affect these immune parameters, whereas treatment during later gestation, from GD 16 to 20, significantly affected T lymphocyte function. Clonazepam, a benzodiazepine with high affinity for the central type benzodiazepine site, also affected cellular immune response in offspring. Our data indicate that benzodiazepine treatment during the fetal period may result in persistent postnatal deficiency of cellular immune responses. The relative role of central and peripheral type benzodiazepine receptor and possible interactions with maternal and fetal pituitary - adrenocortical systems are discussed.

Unimpaired first-set and second-set skin graft rejection in agammaglobulinemic mice.

B cell and antibody-deprived B10.BR chronically suppressed by rabbit antimouse IgM serum rejected first-set allogeneic skin grafts as rapidly as control mice. We confirmed this finding using BALB/c mice born from B cell-deprived mothers and chronically treated with anti-IgM antibodies. Such mice had previously been shown to be agammaglobulinemic except for the suppressing monoclonal rat antimouse IgM antibody in their serum. These results indicate that in mice neither first nor accelerated second-set skin graft rejection reactions depend upon preexisting natural or specifically induced antibodies.

Characterization of a product of histoincompatible spleen cells restoring class I-inhibited mixed-lymphocyte culture reactions.

Conditions leading to the formation of a recognition product (SEPIR) capable of restoring MLC responses inhibited by treating stimulator cells with mAb to Class I antigens have been investigated. SEPIR has been found to be present in supernatants of immunological one-way interactions of histoincompatible spleen lymphocytes from naive mice following cultivation for a few hours. It is active in relatively high dilutions. Similarities with cytokines of IL-1, IL-2, or interferon character could not be revealed. Formation of the heat-labile principle is governed by immunologically specific reactions and involves recognition of Class I (but not Class II) HMC antigens by T cells of Lyt-2 phenotype. B cells and Lyt-1+ cells failed to induce formation of the product. SEPIR appears to be a complex of T-cell receptors for Class I antigens with these antigens. The data indicate that in conventionally induced fully incompatible MLC responses a similar product might be formed.

Reconstitution of primary mixed-lymphocyte culture reactions inhibited with class I antibodies by a product of histoincompatible spleen cells.

Primary MLC reactions with spleen cells from mice of conventional inbred or congenic origin and incompatible at the entire H-2 or at K- or D-regions could be inhibited by pretreating stimulator but not responder cells with mAb to Class I MHC antigens. Inhibited MLC reactions, initiated in the presence of a product (SEPIR) present in supernatants after short-term cultivations of one-way histoincompatible spleen cells of the same haplotype as MLC cells, could be restored specifically to normal activities. It is suggested that the reconstituting factor represents a product of Class I antigen recognition capable of compensating the prevented recognition of these antigens by MLC responder cells. SEPIR was ineffective in restoring Class II antigen-inhibited MLC responses. Because the product might be formed spontaneously under normal MLC conditions, it had no effect when added to uninhibited MLC cells. Conceivably due to neutralization, SEPIR was also incapable of restoring MLC responses conducted in the continuous presence of Class I mAb. Inhibition of MLC responses and reconstitution with SEPIR depended on the effectiveness of Class I antigen inhibition.

Early recognition of transplantation antigens by T lymphocytes. I. Test system and cellular parameters.

Within 2-4 h of interaction of parental spleen cells from naive mice or of their supernates with alloantigen-bearing F1 hybrid spleen cells, a factor called soluble early product of immune recognition (SEPIR) is secreted. SEPIR could be revealed by its ability to enhance mixed leukocyte cultures (MLC) set up in suboptimal conditions. The factor appears to be generated by parental strain T but not B lymphocytes, is active at low concentration and acts in a pulse-like fashion. Its formation is triggered by unstimulated T cells reacting with H-2 antigens; no cytokine activity of IL 1, IL 2 or interferon character could be detected. It is suggested that the formation of SEPIR within the first few hours of MLC interaction is critically related to the further development of alloantigen-driven T cell proliferation. SEPIR might thus be the earliest discernible product of alloimmune recognition.

Early recognition of transplantation antigens by T lymphocytes. II. Specificity and characterization of the recognition process.

In crude supernates of interactions of parental strain T spleen cells with H-2 antigen-bearing F1 hybrid spleen cells, a product (soluble early product of immune recognition, SEPIR) can be found which enhances mixed leukocyte culture (MLC) reactions set up with suboptimal cell concentrations (SMLC). Supernates of parental T cells reacting with F1 spleen cell also produce this factor. The product ameliorates SMLC proliferative responses specifically since identical antigenic specificities have to be recognized in both SEPIR formation and in test SMLC. Further evidence for SEPIR being an immunologically specific product is documented by the finding that spleen cells from mice made specifically tolerant at birth cannot respond to the tolerogen with SEPIR formation, but are fully reactive towards unrelated H-2 antigens. No responses to syngeneic or autologous antigens could be discerned. Characterization of allorecognition resulting in SEPIR formation has revealed that spleen cells of Lyt 23 phenotype recognize H-2K/D-encoded antigens. Lyt 1 cells appear to be incapable of forming the early product and H-2I-determined antigens fail to incite a response. Reactivity to class I antigens was amenable to specific inhibition by monoclonal alloantibodies.

Anti-idiotypic antibodies induce formation of anti-H-2 sera and of idiotypes.

Alloantisera could be induced in parental strain recipients either by means of alloantigen (skin allografting) or by injecting anti-idiotypic (anti-T cell receptor) sera. The resulting sera displayed activity against H-2 antigens; they also contained soluble isiotypic structures. With these sera anti-idiotypic sera could be provoked in F1 hybrid hosts. If antibody cycles initiated with alloantigen were carried through four runs, specificity appeared to be preserved and titers obtained corresponded to those found in conventionally raised anti-idiotypic sera and in alloantisera.

Studies on the product of antigenic recognition. I. Formation of the product in recognition of alloantigens.

A product of antigenic recognition (PAR) was produced whenever receptors for alloantigens from T lymphocytes or a principle present in T-cell dependent alloantisera interacted with alloantigen. With two forms of the PAR assay (direct and indirect) the mechanisms underlying these interactions have been analyzed. For the interaction of T-lymphocyte receptors with alloantigen measured with direct PAR assays, the following conclusion emerged: upon confrontation with alloantigen, receptors (if not already present in secreted form) had first to be released from T-cell membranes. Shed T-cell receptors interacted with alloantigens by solubilizing them. Both processes could be prevented by fixing cells with formaldehyde. Release of T-cell receptors was temperature-dependent, solubilization of alloantigens was not. Because in mixed cell cultures receptors had first to be shed, this process was considerably slower and, in concordance with temperature dependence of receptor release, took place only at 37 degrees C. Titration of T lymphocytes with 'bound' receptors by the direct PAR test revealed that in the presence of excess alloantigen 10(2) T cells sufficed to give measurable responses. Supernates of parental strain lymphocytes containing numerous T-cell receptor specificities could be depleted of one of them. Alloantisera raised in presence of T helper cells ('T alloantisera') contained a principle capable of recognizing alloantigens, alloantisera incited in the absence of T helpers ('B alloantisera') did not. The recognizing principle appeared to be IgG. Like T-cell receptors, it was capable of solubilizing alloantigens form target cell membranes. B alloantisera lacked this capacity and their alloantigen-recognizing moiety was found to be monomeric IgM. The mode of interaction of this IgM with alloantigen most likely consisted in fixation to and shielding of antigen.

Studies on the product of antigenic recognition. III. Nature of PAR.

The product of antigenic recognition (PAR) is an antigen-antibody complex with granulotactic activity. The following results support his conclusion. PAR generated by cell-bound or shed T-cell RS or the recognizing structure of T alloantisera and alloantigen can be destroyed by heating at 56 degrees C for 30 min, because of inactivation of a heat-labile component supplied by alloantigen. Recognizing partners of these complexes are heat-stable. They form PAR anew when alloantigen is added or upon confrontation with anti-RS serum. They are also capable of inducing anti-RS sera. PAR preparations composed of these recognizing structures and antibodies to them are heat-inactivated at much higher temperatures, because both partners of the complex are relatively heat-stable. Heat-labile PAR complexes (recognizing structures and alloantigen) pass anti-mouse immunoglobulin immunosorbent, but are retained specifically by alloantiserum (via alloantigen of the complex) or by anti-RS serum (via RS of the complex) bound to the column.