Induction of specific transplantation immune reactions using anti-idiotypic antibodies.

B and T lymphocytes with reactivity against major histocompatibility antigens are known to express this immune potential via a display on the outer surface of antigen-specific, idiotypic receptors. Here, we show that anti-idiotypic antibodies directed against such receptors may serve as specific triggering agents of the idiotype-positive lymphocytes in the physical absence of foreign histocompatibility antigens. This was shown in vitro using normal or immune spleen T cells where anti-idiotypic antibodies would lead to the selective proliferation and development of antigen-specific cytolytic T cells as determined by short-time 51Cr release assays. Furthermore, purified anti-idiotypic antibodies in adjuvant administered in vivo to normal syngeneic animals could be shown to lead to production of high titers of specific alloantibodies. The present experiments were in most cases carried out using auto-anti-idiotypic antibodies as triggering agents. The present results thus lend further support to the concept that idiotype-anti-idiotype reactions may be normal parts of conventional immune processes with either stimulatory or inhibitory consequences, depending upon the prevailing conditions.

Idiotypic determinants on T-cell subpopulations.

Killer T cells with specificity for major histocompatibility antigens have been shown in mice and rats to display idiotypic receptors allowing the lysis of such cells at the effector phase by anti-idiotypic antibodies and complement. A comparison was made between idiotypes displayed by Lyt-1-2+3+ and Lyt-1+2-3- T blasts, generated in the same mixed leucocyte culture (MLC), across an entire H-2 locus barrier. This was done by absorption of anti-idiotypic antibodies with respective T blasts, followed by estimation of the ability of the absorbed antiserum to inhibit MLC or killer T-cell function. Further, the capacity of Lyt-purified, MLC-generated T blasts to provoke specific unresponsiveness via anti-idiotypic immunity in syngeneic recipients was analyzed. Taken together, the results demonstrate that Lyt-1+2-3- T blasts responsible for the major part of MLC proliferation have distincly different idiotypes from those on the Lyt 1-2+3+ killer T cells. That the idiotypes on the killer T-cell presursors can serve as triggering sites for induction of effector T-cell function was then suggested by experiments with Lyt-1-2+3+-purified, normal T cells as precursor cells in vitro. The fact that autoanti-idiotypic antibodies may circumvent the need for helper Lyt-1+2-3- T cells in the generation of allospecific killer T cells indicates that the former cells may normally function partly via such anti-idiotypic reactions.

Binding of purified, soluble major histocompatibility complex polypeptide chains onto isolated T-cell receptors. I. Reactivity against allo- and self-determinants.

In this study, we tried to get information about the fine antigen-binding ability of purified, soluble, idiotype-positive T-cell receptor molecules. Lewis anti-DA T-cell receptors were purified from normal Lewis serum by the use of anti-idiotypic immunosorbent and sodium dodecyl sulfate-polyacrylamide gel, and were coupled to cyanogen bromide-activated Sepharose 4B. In parallel, Lewis anti-DA, Lewis anti-BN, and DA anti-Lewis alloantibody immunosorbents were prepared. The major Ag-B chain (44,000 daltons) and the two polypeptide chains (34,000 and 27,000 daltons) of Ia were purified from Lewis, DA, and BN lymphocytes and absorbent on the above-mentioned immunosorbents. We found that the major Ag-B chain as well as the two Ia chains were bound to the alloantibody columns if they were derived from the corresponding allogeneic strain. No retaining ability for self-major histocompatibility complex (MHC) or third-party MHC chains was noted with the alloantibody immunosorbents. When using immunosorbents made up of idiotypic T-cell receptors, only two MHC polypeptides of the relevant allo-MHC type were retained, namely, the Ag-B and the heavy Ia chains. No detectable activity was observed when testing the same column for reactivity against third-party MHC polypeptide chains. However, the Lewis anti-DA T-cell receptors could be shown to display weak, but significant, reactivity toward one Lewis MHC polypeptide chain, that is, the heavy chain of Ia type.

An ethological model for the study of activation and interaction of pain, memory and defensive systems in the attacked mouse. Role of endogenous opioids.

The present work reviews neurochemical, physiological and behavioral data recorded from the attacked mouse and integrates them into a model of coping mechanisms during social conflict. More specifically, the possible relationships between systems of pain, memory and defense are presented, with special emphasis on the role of endogenous opioid peptides (EOPs). In recipients of attack, decreased beta-endorphin-like immunoreactivity and changes in opiate and benzodiazepine binding characteristics are found in structures of the brain defensive system. EOPs mediate the social conflict-induced increase of dopamine synthesis in the periaqueductal grey and frontal cortex. Social conflict analgesia in attacked mice is under the control of central opioid and nonopioid (e.g., benzodiazepine, glutamate) mechanisms, and is modified by experience (e.g., long-term analgesic reaction; tolerance). EOPs and pain-inhibitory mechanisms participate in the organization of behavioral defense, recuperative behavior and the memory of attack experience. The data are considered in relation to the perceptual-defensive-recuperative model of fear and pain, forwarded by Bolles and Fanselow.

Long-term analgesic reaction in attacked mice.

Four experiments were designed to characterize long-term analgesic (LTA) reaction in attacked mice. In Experiment 1 we showed that analgesic reaction in DBA mice, induced by the stress of being attacked (30 or 50 bites), is reinstated upon reexposure to seven bites 24 hr later. The magnitude of the LTA response depended on the level of analgesia on Day 1 and was smaller than the original response. In Experiment 2 we showed that LTA was prevented by naloxone or beta-chlornaltrexamine given before exposure (50 bites) on Day 1. Results of Experiment 3 revealed that naloxone or beta-chlornaltrexamine injected before reexposure to seven bites on Day 2 antagonized LTA measured 10 min, but not 1 min, after reexposure. In Experiment 4 we showed that morphine substituted for being attacked on Day 1 failed to produce LTA. We concluded that pain inhibitory mechanisms remain in a state of increased readiness for at least 24 hr after attack stress and that activation of opioid systems is necessary but not sufficient to produce LTA, a response that is only partly sensitive to opioid antagonists.

Preexposure to a nonaggressive opponent prevents low-intensity, social-conflict analgesia in mice.

In a first experiment, exposure of DBA/2 mice to a small number of attack bites by a C57BL/6 mouse resulted in low-intensity analgesia as assessed by the tail-flick test. The analgesia dissipated within 10 min and was insensitive to naloxone (10 mg/kg, sc) but was antagonized by the irreversible opioid antagonist beta-chlornaltrexamine (5 mg/kg, sc). In a second experiment, preexposure to a nonaggressive C57BL/6 opponent prevented low-intensity analgesia induced by a small number of attack bites 24 hr later. The preexposure effect was abolished by naloxone (10 mg/kg, sc) given before the nonaggressive confrontation. This suggests that the release of endogenous opioids during preexposure interferes with the subsequent activation of endogenous opioid-mediated pain control mechanisms.

Relationship between behavioral and nociceptive changes in attacked mice: effects of opiate antagonists.

The relationship between analgesia and behavior during and after an aggressive encounter was investigated in saline- and opiate antagonist-treated DBA mice. A low number of bites induced an analgesia that was reversed by beta-chlornaltrexamine but not by naloxone, and that correlated positively with increased displays of defensive upright and immobility upon contact with the opponent. Extended attacks induced a naloxone-sensitive analgesia that was linked to a delayed occurrence of "panic" escape behavior. In the post-conflict phase, the degree of immobility and analgesia correlated positively in attacked mice. Naltrexone prevented this analgesia and lowered immobility. Endogenous opioids released during social conflict may induce analgesia and immobility in DBA mice.

Behavioral effects of hashish in mice. III. Social interactions between two residents and an intruder male.

The acute and subchronic effects of hashish extract (20 mg delta 9-THC/kg) on the social interactions between two drug-treated residents and an untreated intruder male were investigated. In this analysis 28 different behavioral elements were recorded. A single drug application suppressed all categories of behavior, except submissive behavior and flight, in dominant and subordinate residents. Treated animals were less active than controls and immobility was very frequent. An elevated total activity, due to an increase in non-social activities, was observed in the untreated intruder males of this group. Social investigation as well as submissive behavior and flight were reduced in these animals. On introduction of an untreated male after the fourth drug treatment of the residents, the drugged males showed tolerance to the sedative and most of the other behavioral effects of the drug, and intruder males behaved quite normally. The formation of a dominant-subordinate relation within the group was influenced neither by a single nor by repeated drug treatment. The acute and subchronic effects of hashish extract on social, especially aggressive behavior of males are compared to those described in previous papers and the variation in the results of the different studies is discussed.

Behavioral effects of hashish in mice. I. Social interactions and nest-building behavior of males.

The acute and subchronic effects of hashish extract (20 mg delta 9-THC/kg) on the behavior of male mice encountering a control partner was studied by ethological methods. A single administration of the extract resulted in general sedation, suppressing all the individual and social activities with the exception of some submissive elements. The locomotive and the overall activity of drugged males was drastically reduced and immobility occurred frequently. After four applications, tolerance to the sedative effects had developed and behavioral drug effects were recognizable. Drugged males showed an increase in nonsocial activities as well as in submissive behavior and flight, whereas social investigation was less frequent. Sexual and aggressive behavior was not significantly affected by the drug and immobility no longer occurred. In spite of behavioral changes after a single or repeated drug treatment, drugged males became dominant in about half the experiments. The nest-building behavior of males was disturbed in the same way after one or four drug applications. Drugged males generally refrained from carrying and working up the nesting material. The acute behavioral effects of hashish extract are compared to those described in previous papers and the difference between acute and subchronic drug effects is discussed.

Behavioral effects of hashish in mice. II. Nursing behavior and development of the sucklings.

Adult mice were treated from parturition to weaning of their first litter with a hashish extract containing 40% delta 9-tetrahydrocannabinol (delta 9-THC), 45% cannabidiol, 9% cannabinol, and 6% other cannabinoids. Oral administrations of 20 mg delta 9-THC/kg three times a week decreased the weight gain of pups from days 3-6 and 6-10 significantly, resulting in about 15% lower body weights on days 6 and 10 compared with control sucklings. Other parameters of development such as the general appearance of the pups were little affected, except for a slight tendency by day 13, when some additional control pups already had both eyes open. The effects of hashish in sucklings might be caused by drug intake with mother's milk, as well as by a decreased lactation of drugged dams. In addition, our pup retrieving tests at the day 3, 1.5-2 h after the second application of hashish extract, showed a decrease in the mother's locomotive and nonsocial activities and pointed to at least transient impairment of the maternal behavior. By day 10, after the fifth administration of hashish extract, a partial tolerance occurred, with normal care for the young, but still decreased nonsocial activities of the drugged dams. Thus our experiments showed distinct effects of cannabis on mice litters when the parents were drugged postnatally during the period of lactation only.

Behavioral effects of hashish in mice. IV. Social dominance, food dominance, and sexual behavior within a group of males.

Within groups of three adult male mice the acute and subchronic effects of hashish extract (20 mg delta 9-THC/kg) on social dominance, food dominance, and sexual interactions with a female were investigated. An initial drug treatment of only the dominant male weakened his social position, but dominance was regained after treatment 2 or 3. In contrast, a persistent change in dominance was found when only the male which was dominant in the feeding test was treated with the extract. Simultaneous drug treatment of all three males did not affect the social dominance relationship but resulted in a reversible change in food dominance. The original feeding order was reestablished after drug treatment 3. Upon meeting an estrous female, no male of the group was distinctly dominant in mating. After treatment 1 was given to all members of the group, all types of behavior were impaired and total activity was significantly reduced. After treatment 2, animals showed tolerance to the sedative effects, and after treatment 3, sexual behavior was even more frequent in drugged animals than in controls. The results are discussed in relation to a possible dependence of behavioral drug effects and tolerance development on the experimental situation.

Hashish extract impairs retention of defeat-induced submissive behavior in mice.

The effects of hashish extract on adaptive behavior of male mice were studied in a paradigm which allows the investigation of learning mechanisms in a social context. Mice of the C3H strain, which were not submissive in a confrontation with a nonaggressive DBA mouse on day 1, were defeated on day 2 over 3 min by aggressive, isolated DBA mice, and showed conditioned submissive behavior upon mere contact with a nonaggressive DBA mouse on day 3. A hashish extract containing 38.6-39.4% delta 9-tetrahydrocannabinol (delta 9-THC), 11.6-12.0% cannabinol and 47.7-48.5% cannabidiol was administered orally in all experiments. Hashish extract given 90 min before defeat on day 2, in dosages corresponding to 1, 5, and 10 mg delta 9-THC/kg, impaired retention of defensive upright, defensive sideways and immobility on day 3 (experiment 1). Experiment 2 showed that the drug (5, and 10 mg delta 9-THC/kg) had no antinociceptive potency in mice and did not modify defeat-induced analgesia. Experiment 3, with drug (5 mg delta 9-THC/kg) or solvent administration on day 2 and day 3, showed that the retention deficit was neither due to state-dependent learning, nor to impaired retrieval. It is suggested that hashish extract administered before learning may interfere with memory processing.

Vasopressin impairs or enhances retention of learned submissive behavior in mice depending on the time of application.

The effects of vasopressin on learning and memory were investigated in a paradigm using adaptive capabilities of interacting male mice. Test animals of the DBA/2 strain which were not submissive in a confrontation with a non-aggressive subordinate C57BL/6 mouse on day 1 (baseline), were defeated on day 2 (learning) by an aggressive dominant C57 mouse, and showed learned submissive behavior upon mere contact with a non-aggressive C57 mouse on day 3 (retest). Pretrial injections of lysine-vasopressin (0.01, 0.1 or 1.0 I.U., s.c.) 20 min before defeat on day 2 resulted in less submissive behavior on day 3 compared to controls, with 0.1 I.U. (equal to 370 ng) being the most effective dose. Post-trial injections of vasopressin (0.1 I.U.) immediately after defeat on day 2 significantly improved retention on day 3. Preretention injections of vasopressin (0.1 I.U.) 20 min before testing on day 3 significantly increased learned submissive behavior. The amnesic effect observed after pretrial injections of vasopressin was neither due to state dependency nor to an acquisition deficit, nor to antinociception. It is concluded that processing of the stressful experience of defeat is differently influenced by vasopressin given before or after training, resulting in an impaired or facilitated retention, respectively. Among the hypothetically discussed underlying mechanisms, one suggestion is that exogenous vasopressin interacts with an assumed discriminative stimulus function of endogenously released vasopressin. Another possibility might be that exogenous vasopressin interferes with the defeat-activated opioid peptide system.

Social conflict-induced changes in nociception and beta-endorphin-like immunoreactivity in pituitary and discrete brain areas of C57BL/6 and DBA/2 mice.

The present study characterizes the time course of social conflict analgesia and its reversibility by opioid antagonist drugs in the C57BL/6 and DBA/2 inbred strains of mice and examines the relationship between alterations in brain and pituitary levels of beta-endorphin-like immunoreactivity (beta-ELIR) and the antinociception elicited by social stress. Data revealed statistically significant strain differences in regard to beta-ELIR in control animals. The pituitary content of beta-ELIR was higher in DBA/2, while the values in the periaqueductal grey (PAG) and in the amygdala were higher in C57BL/6 mice. No interstrain differences were found in the hypothalamus. Exposure to 50 attack bites resulted in a 6-fold higher analgesia in DBA/2 mice and in a strain-independent fall of beta-ELIR in pituitary (approximately 27%) and PAG (23%). PAG but not pituitary beta-ELIR levels in C57BL/6 mice correlated positively with the increase in tail-flick latency after attack. Mere confrontation with a non-aggressive opponent failed to induce analgesia and was associated in C57BL/6 mice with a significant reduction in the beta-ELIR content of both the pituitary and the PAG. The data are discussed in terms of genotype-dependent sensitivity of the beta-endorphin system to stress and its relation to analgesia.

Recovery from opioid receptor alkylation: social conflict analgesia and brain [3H]etorphine binding in beta-chlornaltrexamine-treated mice.

The effects of beta-chlornaltrexamine (CNA, 5 mg/kg s.c.) on social conflict analgesia and brain opioid binding were investigated in mice at different times after the administration of the alkylating antagonist. The specific binding of [3H]etorphine to high-affinity binding sites and the stress-induced analgesia of attacked mice (50 bites) were prevented for 6 h after CNA administration. Stress-mediated inhibition of pain fully recovered within 3 days after CNA treatment. Brain opioid binding was still reduced to 45% at this time and reached control values 9 days after treatment.

Emergence and development of stress-induced analgesia and concomitant behavioral changes in mice exposed to social conflict.

Mice of the inbred strain DBA/2, when exposed to a social conflict, developed a low intensity, naloxone-insensitive analgesia after 15 bites, and a more pronounced naloxone-sensitive analgesia after 45 bites. The effective inhibition of the antinociceptive response following low and high number of bites by the alkylating opiate antagonist beta-chlornaltrexamine suggests participation of opioid mechanisms at both stress levels. Emergence of an increased tail-flick latency was indicated by the occurrence of defensive upright postures upon contact with the opponent, while animals displaying full analgesic response during the period of bite 31-45 increased their escape reactions without being in contact with the aggressor. Suppression of social conflict analgesia in mice by pretreatment with opiate antagonists facilitated the occurrence of these escape reactions. The display of panic escape responses is discussed in the context of increased fear and helplessness that developed under conditions of sustained attacks.

Isolation-induced locomotor hyperactivity and hypoalgesia in rats are prevented by handling and reversed by resocialization.

Differences in locomotor activity in the open field were found between individually and group-housed rats (isol greater than soc). Daily handling, initiated at postnatal day 1, was without effect in group-housed rats but prevented the isolation-induced hyperactivity. For tail-flick latency, strikingly similar differences (isol greater than soc; prevention by handling) have been observed. The isolation-induced aberrations in both locomotor reactivity in a novel environment and in pain sensitivity could be reversed by subsequent resocialization. This indicates that the altered sensitivities to external stimuli are caused by the environmental manipulation.

Effects of repeated as compared to single aggressive confrontation on nociception and defense behavior in C57BL/6 and DBA/2 mice.

Behavioral reactions (submissive postures, escape, immobility, activity, locomotion) in C57BL/6 and DBA/2 test mice were recorded during single (50 bites) or three repeated (3 X 50 bites, separated by 24 hr) aggressive confrontations, as well as during a nonaggressive confrontation 24 hr after the last aggressive confrontation with opponents of the opposite strain. Nociception (hot plate response latency) was measured 1 min after aggressive or nonaggressive confrontations. During repeated aggressive confrontation, DBA mice reacted with a stable pattern of escape and analgesia, whereas C57 mice failed to develop an analgesic response and changed their behavioral defense strategy during repeated aggressive confrontations (decrease of escape, increase of defensive upright). The conditioned display of submission and of escape behavior during nonaggressive confrontation did not change as a function of earlier repeated aggressive confrontations in DBA mice, while C57 mice showed a significant increase of defensive upright postures and immobility. Conditioned analgesia was not observed after nonaggressive confrontations. The results point toward a dissociation between attack-elicited behavior and antinociception and suggest that encounter-induced analgesia may influence the processing of aversive experience.

Beta-endorphin-like immunoreactivity levels in the hypothalamus, the periaqueductal grey and the pituitary of the DBA mouse: determination by ELISA and relationship to nociception.

The present paper describes the development and application of an enzyme-linked immunosorbent assay (ELISA) for the assessment of beta-endorphin-like immunoreactivity (beta-ELIR) level in the hypothalamus, the periaqueductal grey (PAG) and the pituitary of DBA/2 mice that were subjected to mild social stress (aggressive confrontation). After confrontation these subjects showed elevated tail-flick latencies (TFL) when compared to controls, a finding that indicates stress-induced analgesia (SIA). A positive correlation was found between individual TFLs and beta-ELIR levels in the PAG but not in the hypothalamus and the pituitary. These results suggest that individual baseline PAG beta-ELIR levels may be taken as a predictor of high degrees of stress-induced analgesia.

Inhibition of morphine-induced analgesia and locomotor activity in strains of mice: a comparison of long-acting opiate antagonists.

The long-acting opiate antagonistic potency of naloxazone (NXZ), beta-chlornaltrexamine (beta-CNA) and beta-funaltrexamine (beta-FNA) was compared using three inbred strains of mice, in which morphine induces either analgesia (DBA/2), locomotion (C57BL/6), or both responses (C3H/He). The antagonists were applied SC 24-120 hr before morphine (10 or 20 mg/kg, IP), followed by the tests after 30 min. The minimal dose which completely antagonized morphine-induced analgesia in DBA and locomotion in C57 mice during 24 hr were: for NXZ 50 and 100 mg/kg, for beta-CNA 0.8 and 6.2 mg/kg, for beta-FNA 1.6 and 12.5 mg/kg, respectively. beta-FNA and beta-CNA more potently blocked morphine-induced analgesia in DBA mice than the activity response in the C57 strain. In contrast, beta-FNA prevented morphine-induced locomotion at a lower dose (6.2 mg/kg) than analgesia (greater than 50 mg/kg) in C3H mice, while beta-CNA was equipotent (1.6 mg/kg). In general, beta-CNA turned out to be the most reactive compound, antagonizing morphine effects in low doses up to 120 hr. beta-FNA selectively antagonized either morphine-induced analgesia or locomotion, depending on the strain used. This suggests that a given morphine response might be caused by a genetically determined multiplicity of opiate receptor types and their mutual interactions.