Evidence of CNIH3 involvement in opioid dependence.

Opioid dependence, a severe addictive disorder and major societal problem, has been demonstrated to be moderately heritable. We conducted a genome-wide association study in Comorbidity and Trauma Study data comparing opioid-dependent daily injectors (N=1167) with opioid misusers who never progressed to daily injection (N=161). The strongest associations, observed for CNIH3 single-nucleotide polymorphisms (SNPs), were confirmed in two independent samples, the Yale-Penn genetic studies of opioid, cocaine and alcohol dependence and the Study of Addiction: Genetics and Environment, which both contain non-dependent opioid misusers and opioid-dependent individuals. Meta-analyses found five genome-wide significant CNIH3 SNPs. The A allele of rs10799590, the most highly associated SNP, was robustly protective (P=4.30E-9; odds ratio 0.64 (95% confidence interval 0.55-0.74)). Epigenetic annotation predicts that this SNP is functional in fetal brain. Neuroimaging data from the Duke Neurogenetics Study (N=312) provide evidence of this SNP's in vivo functionality; rs10799590 A allele carriers displayed significantly greater right amygdala habituation to threat-related facial expressions, a phenotype associated with resilience to psychopathology. Computational genetic analyses of physical dependence on morphine across 23 mouse strains yielded significant correlations for haplotypes in CNIH3 and functionally related genes. These convergent findings support CNIH3 involvement in the pathophysiology of opioid dependence, complementing prior studies implicating the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate system.

Concanavalin A-induced alteration of surface marker expression on murine T cells.

The activation of suppressor T cells by Concanavalin A (Con A) was characterized by changes in expression of T cell surface markers. Treatment of normal mouse spleen cells with monoclonal antibody to Ly-1 prior to activation with Con A consistently reduced the resulting suppressor cell activity as assayed by mixed culture with primed spleen cells responding to sheep erythrocytes. Addition of indomethacin to anti-Ly-1 depleted spleen cells together with Con A had no effect on the resulting suppressor cell activity, whereas its addition to normal spleen cells together with Con A significantly abrogated suppressor T cell induction. In contrast, treatment of normal spleen cells with anti-Ly-1 following 24h culture with Con A had no effect on resulting suppressor cell activity. Treatment of spleen cells either prior to or following culture with Con A with monoclonal antibody to Ly-2 completely abrogated suppressor T cell activity. Suppressor T cells activated by Con A were depleted significantly by fractionation on columns containing insolubilized histamine. In contrast, fractionation of normal spleen cells on histamine columns followed by culture with Con A had no effect on subsequent suppressor T cell induction. Furthermore, direct addition of histamine (5 x 10(-4) M - 5 x 10(-8) M) to Con A preculture had no effect on resultant suppressor T cell generation, suggesting that possible immunodulatory actions of histamine may be expressed at the effector rather than during induction stages of Con A-induced suppressor T cell activation.

Abrogation of suppressor cell function by inhibitors of prostaglandin synthesis.

The weekly intraperitoneal injection of rat erythrocytes into mice induces both a stable autoimmune state, as judged by the appearance of anti-mouse erythrocyte autoantibody and suppressor T cells capable of regulating this response; the latter being demonstrable only in a subsequent transfer system. This autoimmune response and the parallel anti-rat erythrocyte response were both insensitive to exogenous prostaglandin E2 (PGE2). The administration of prostaglandin synthetase inhibitors (indomethacin or aspirin) to mice undergoing immunization with rat erythrocytes had no effect on the anti-rat response, yet mildly exacerbated the onset of the autoimmune state and potently inhibited the generation of suppressor cells. Furthermore the administration of these drugs to recipients of suppressor cells virtually abrogated suppressor cell activity. These observations imply that both the generation and effector function of these suppressor cells may be modulated by prostaglandin synthetase inhibitors while at the same time T helper and B cell functions remain unimpaired.

Attenuation of murine graft-versus-host reactivity by azathioprine.

The therapeutic effects of azathioprine were evaluated in a murine graft-versus-host (GVH) model. A single high dose (200 mg/kg) of azathioprine, administered to F1 recipients 2 to 3 days after the injection of parental spleen cells, abrogated the ensuing GVH reaction. Lower doses of the drug, even when injected over an extended period of time (14 days), were found to be ineffective. However, high doses of azathioprine failed to protect when F1 recipients were sublethally irradiated or injected with cyclophosphamide before GVH induction, and even the transfer of syngeneic F1 spleen cells immediately after irradiation failed to alter this outcome. Further analysis of the changes that occurred in sublethally irradiated recipients revealed that the protective effect of azathioprine on CBA leads to (CBA x C57BL)F1 GVH reaction was totally abrogated by doses of irradiation as low as 200 rad. Further experiments in which death was used as an indicator of GVH disease showed that lethality could not be reversed in sublethally irradiated F1 recipients by the transfer of syngeneic F1 spleen cells unless approximately 10 days were allowed to elapse between syngeneic reconstitution and GVH induction. Since syngeneic F1 cells from spleen, lymph node, or bone marrow all behaved similarly, it seems likely that the increased severity of GVH reaction induced by prior immunosuppression may not be attributable to a simple cell deletion event.

The induction of suppressor T cells by concanavalin A is independent of cellular proliferation and protein synthesis.

Factors that govern the induction of suppressor T cells after stimulation with concanavalin A (Con A) were investigated in a two-stage culture system. Normal mouse spleen cells were incubated with Con A in the presence of a variety of drugs and then assayed for suppressive activity by means of a secondary anti-sheep erythrocyte response in vitro. The inclusion of inhibitors of mitosis (vinblastine sulphate or mytomycin C) or protein synthesis (cycloheximide or pactamycin) into normal spleen cell cultures containing Con A failed to inhibit the subsequent development of suppressor cells. Furthermore, spleen cells from mice previously irradiated with 900 rad or injected with cyclophosphamide expressed a level of suppressor activity after Con A stimulation which was equivalent to that of normal spleen cells. However, the inclusion of drugs that inhibit microtubule or microfilament function (colchicine or cytocholasin B) did prevent suppressor cell induction. Kinetic studies also revealed that significant suppressor activity was detectable in normal spleen cells after only 3 h exposure to Con A. These results indicate that the induction of suppressor T cells in this system is a maturation event involving changes in the cell membrane and is entirely independent of protein synthesis and cellular proliferation.

The capacity of microsomally-activated cyclophosphamide to induce immunosuppression in vitro.

Cyclophosphamide (CY) was activated in vitro with washed rat liver microsomes and cofactors. Pretreatment of mouse spleen cells in vitro with the activated drug abolished their capacity to give a primary antibody response to SRBC and levan on transfer to irradiated syngeneic recipients. However, responsiveness returned if challenge was delayed for 7 or more days after transfer. Part of this was shown to be of donor origin by an allotype marker. The treatment of normal spleen cells with activated CY in vitro also prevented B cells from regenerating their immunoglobulin receptors after capping with anti-immunoglobulin serum. The induction of suppression required contact between lymphocytes and activated CY for at least 30 min at 37 degrees and did not appear following incubation for 1 h at 0 degrees. Since the antibody response of drug-treated spleen cells to SRBC could not be restored with purified normal B or T cells, it is probable that B and T lymphocytes are both susceptible to suppression by activated CY in vitro. Similar pretreatment abrogated the graft-versus-host (GVH) reactivity of spleen cells as measured by survival and in a popliteal lymph node assay. B cell chimerism in F1 recipients of drug-treated parental spleen cells was demonstrated by the presence of congenic allotype markers. This suggests a possible approach for the attenuation of GVH disease which is associated with bone marrow transplantation in man.

Ly and Ia phenotype of suppressor T cells induced by graft-vs.-host reaction.

Suppressor T cells were generated in (CBA X C57BL)F1 mice undergoing graft-vs.-host (GvH) reaction and treated with anti-Ly and anti-Ia antisera to determine their membrane antigen phenotype. Pretreatment of CBA-induced GvH suppressor T cells with anti-Ly-1.1, anti-Ly-2.1 or anti-Iak antisera plus complement abrogated their suppressive properties when tested against primed F1 spleen cells in vitro. In contrast, concanavalin A (Con A)-induced suppressor T cells were insensitive to anti-Ly-1.1 serum. It is concluded that GvH-induced suppressor T cells are Ly-1+,2+,3+,Ia+, and thus distinct from Con A or antigen-induced suppressor T cells.

The adoptive secondary response to human serum albumin under conditions of high antigen pressure. The response of high and low avidity B cell subsets.

Thoracic duct lymphocytes (TDL) from (AS2 x AS)F1 rats previously injected with human serum albumin (HSA) were transferred to 900 r irradiated syngeneic recipients which were challenged with various doses of soluble HSA (s-HSA). TDL from partially tolerant rats, which were deficient in high avidity B cells, produced a maximum PFC response to the largest challenge dose (1 g s-HSA). In contrast, high avidity B cells from primed donors were optimally stimulated by 1 microgram and maximally inhibited by 1 mg s-HSA (day 7 PFC). An additional increase in antigen concentration by 1000 fold failed to diminish the PFC numbers further. Plaque inhibition profiles indicated that these antibody forming cells resisting inhibition were of the same high avidity as those triggered by low doses of antigen. The inability of s-HSA to completely inhibit antibody synthesis is discussed with regard to current views on B cell inactivation. Evidence is also presented which indicates that the standard haemolysis-in-gel test may fail to detect many low avidity antibody forming cells to proteins.

Cellular events in protein-tolerant inbred rats. IV. The mechanism of immunogen-maintained tolerance.

The ability of immunogens to maintain or extend a state of unresponsiveness was investigated in unbred rats using a human serum albumin (HSA) model of tolerance. Rats initially challenged with immunogen within two weeks of high or low dose tolerance induction by tolerogen (soluble HSA) remained hyporesponsive even a year and a half later and in some cases became less responsive following a subsequent challenge. An inhibitory effect of immunogen on escape from tolerance was formally demonstrated: in comparison with an unchallenged group, tolerant rats which received a second immunogen challenge 6 months after the first, synthesized less antibody; this antibody underwent a gradual decline in affinity after each challenge which suggested that higher avidity B cells were progressively lost. In addition, immunogen-maintained tolerant rats (a) had demonstrable helper T cell activity among their thoracic duct lymphocytes on adoptive transfer, (b) did not produce a significant increase in antibody synthesis after receiving peripheral T cells and (c) provided no evidence that suppressor cells were playing a role. The results suggested that the mechanisms of tolerance induction by tolerogen and tolerance maintenance by immunogen are fundamentally different.

Analysis of immunosuppression generated by the graft-versus-host reaction. II. Characterization of the suppression cell and its mechanism of action.

Spleen cells from (CBA X C57/BL) F1 mice undergoing graft-versus-host (GVH) reaction induced by injection of parental cells 7-14 days previously are capable of suppressing an immune response by normal or primed F1 spleen cells to chicken erythrocytes and levan in vivo and sheep erythrocytes in vitro. The cells in these GVH spleens which were responsible for the suppression were sensitive to treatment with anti-0 serum, resistant to 900 rad irradiation in vivo and not retained by anti-immunoglobulin columns. Suppressor activity in vitro was present only in the non-adherent fraction of these GVH cell suspensions. Furthermore, the T-cell fraction, purified by affinity chromatography, suppressed the in vitro response of macrophage-depleted normal F1 cells to DNP-levan. Collectively, these observations imply that suppressor T cells generated by GVH reaction can affect B-cell functions directly without intermediary macrophage participation. Spleen cells from (CBA X C57/BL) F1 mice undergoing GVH reaction induced by C57/BL cells were depleted of their F1 content by treatment with anti-CBA alloantiserum. The suppressive activity of the residual donor component was still expressed against other F1 cells (AKR X C57/BL) which were H-2 compatible with the original host, but not against H-2-incompatible cells (DBA/1 X C57/BL) F1. However, the latter were suppressed in the presence of (CBA X C57/BL) F1 cells. Thus, interaction of donor T cells with F1 target cells containing those H-2 antigens towards which they are sensitized is mandatory for the subsequent manifestation of immunosuppressive activity. GVH cells suppressed the response of primed F1 cells in double Marbrook chambers when the two populations were separated were by a cell-impermeable membrane, provided the GVH suspension contained F1 cells to which donor T cells were sensitized. This suggests that soluble factors are involved in the mechanism of GVH-induced immunosuppression.

Persisting T cells in rats tolerant of human serum albumin. The significance of tolerant and nonimmune T cells which preferentially restrict high affinity antibody synthesis.

The adoptive response of primed rat thoracic duct lymphocytes ('TDL) following specific antigen challenge (soluble human serum albumin, s-HSA) was restricted when cells were transferred into syngeneic, adult (AS2 X AS)F1 hybrid recipients in comparison with irradiated hosts. This adoptive memory response was also inhibited in irradiated recipients by transferring nonimmune TDL along with 'TDL. Recirculating B cells (B-TDL) did not inhibit the 'TDL response, indicating that the adoptive secondary response was regulated by T cells. Antibody synthesis was preferentially restricted in the high affinity memory cell precursor population, demonstrating a role for T cells in regulating the maturation of antibody affinity. The adoptive memory response was liberated from this T regulatory effect in adult recipients when hosts were challenged with the alum-precipitated adjuvant form (HSA-adj) rather than the soluble form of HSA. Since the adoptive memory response was sensitive to the presence or absence of T cells, this experimental model was used to determine whether or not T cells were eliminated from HSA-tolerant rats. Antibody synthesis by 'TDL was reduced approximately 10-fold compared with controls when transferred into tolerant recipients and challenged with either s-HSA or HSA-adj; a similar reduction was not observed by substituting bovine serum albumin (BSA) 'TDL and challenging with s-BSA. The tolerance-induced inhibition of HSA 'TDL was destroyed by irradiation and TDL from HSA-tolerant donors were more effective than normal nonimmune TDL in reducing the adoptive HSA 'TDL response. HSA-tolerant TDL did not inhibit the BSA 'TDL response significantly. The results indicate that T cells are not eliminated by tolerance induction in this model and after interaction with tolerogen may exert an active (or competitive) role in restricting antibody synthesis by high affinity B memory cell precursors. However, the fact that tolerant T cells are not able to prevent a primary response suggests that unresponsiveness to HSA in the T compartment represents a functional deficiency and not an active suppression at this level. Nevertheless, the presence of these tolerant cells probably accounts for the failure of antibody affinity to mature in partially tolerant rats.

Analysis of immunosuppression generated by the graft-versus-host reaction. I. A suppressor T-cell component studied in vivo.

The kinetics and cellular characteristics of immunosuppression in (CBA-p X C57/Bl)F1 mice during graft-versus-host (GVH) reaction induced with spleen cells from either parental strain have been investigated. Effects on PFC responses to thymus-dependent (chicken red blood cells) and independent (levan) antigens have been compared. The unresponsive state which developed in GVH mice was expressed to comparable extent by their spleen cells following transfer to irradiated recipients. Furthermore, GVH spleen cells suppressed normal spleen cells in a mixed transfer system, but this effect was lost completely by day 21 whereas the original donors (or their cells) were still totally refractory after 80 days. This active suppressor effect was found to be mediated by T cells of perental origin based on cell fractionation analysis and selective deletion of donor or host cells by alloimmune attack. The delayed transfer of GVH cells to irradiated repopulated recipients challenged with antigen, indicated that suppressor T cells exert an anti-mitotic influence on antigen-stimulated B-cell proliferation. Supplementation of macrophage-depleted, anti-theta-treated GVH spleen cells with purified normal T cells demonstrated that B cells in GVH mice are normally reactive even when active suppression by T cells is no longer demonstrable. The likelihood that this later phase of immunosuppression is attributable to another mechanism is discussed.

Changes in lymphocyte recirculation and liberation of the adoptive memory response from cellular regulation in irradiated recipients.

Thoracic duct lymphocytes from previously immunized (AS2 x AS)F1 rats ('TDL) were adoptively transferred to syngeneic recipients and triggered by soluble HSA (s-HSA) the following day. The response of 'TDL in an irradiated recipient was almost two orders of magnitude greater than the response in a nonirradiated adult host. The affinity of the antibody in the adult recipients was significantly reduced but increased with time. Nonirradiated young recipients (3 weeks old) also supported an adoptive memory response which was comparable in quantity and quality to that found in irradiated hosts. The response of 'TDL declined progressively when transferred into 2-week-old or 4-week-old radiation chimeras, or was reduced in irradiated hosts when the memory cells were mixed together with nonimmune TDL. The experiments indicate that the differential response between the adult and irradiated recipient is the result of a restrictive control in the former host rather than an enhancing factor in the latter. Lymphocytes in the nonimmune population regulate both the expansion and maturation of the adoptive memory response, the high energy-binding B cells being at a selective disadvantage. Host irradiation effectively liberates the adoptive response from this cellular control mechanism. A comparative study of transferred, [14C]leucine-labeled TDL showed that distribution and homing of lymphocytes to lymph nodes and spleen was not altered by irradiating the recipients. However, adoptively transferred cells almost completely failed to recirculate in irradiated rats, in contrast to normal recipients. But, the injection of large numbers of unlabeled TDL following irradiation forced more labeled cells into the recirculating pool, suggesting that saturation of depleted lymphoid tissue with lymphocytes is an important factor regulating lymphocyte traffic. The relevance of this "saturation effect" in regulating the adoptive memory response is discussed.