NK cell and CTL activities can be raised via conditioning of the CNS with unrelated unconditioned stimuli.

Poly I:C, an inducer of interferon alpha and beta, and arecoline, a cholinergic muscarinic agonist, were used as unconditioned stimuli in conjunction with camphor odor to condition the augmentation of natural killer (NK) cell and cytotoxic T lymphocyte (CTL) activities. This observation suggested that two unrelated unconditioned stimuli might be used to associate the signals, and induce mediators at recall that can raise natural killer cell activity. In animals stimulated with an alloantigen to induce CTL, we observed that either poly I:C or arecoline conditioned association, and recall with the same CS raised CTL activity. It appears that conditioning with different substrates can raise either native (NK cell activity) or acquired (CTL activity) immunity. The studies suggest that communication between the CNS and NK cells or CTL appear to take place through a common pathway(s).

Immunotherapy of a murine T cell lymphoma localized to the brain.

Mouse YC8 T cell lymphoma was used as a model to determine whether an effective immunotherapy procedure could be devised for the treatment of lymphoma localized to the brain. Implantation of 5 x 10(4) YC8 cells into the left cerebral hemisphere induced rapid loss of the animal's body weight. Severe loss of weight and early deaths were observed in the untreated control group. Although resistance can be conferred to the brain by immunization of naive BALB/c mice, adoptive chemoimmunotherapy procedures were surprisingly not effective in inducing remissions in animals with lymphoma confined to the brain. Even passive transfer of effector cells from immunized, tumor resistant donor animals combined with systemic IL-2 treatment did not impart resistance to recipients with brain tumors. However, regression of the intracranial tumor and apparent cures could be accomplished, when ex vivo cultured effector cells were transferred intravenously.

Psychoneuroendocrine immunology: perception of stress can alter body temperature and natural killer cell activity.

Psychoimmunology has been credited with using the mind as a way to alter immunity. The problem with this concept is that many of the current psychoimmunology techniques in use are aimed at alleviating stress effects on the immune system rather than at direct augmentation of immunity by the brain. Studies in animals provide a model that permits us to approach the difficulties associated with gaining an understanding of the CNS-immune system connection. A particular advantage of using animals over humans is that psychological and social contributions play a less prominent role for animals than for human subjects, since the animals are all inbred and reared under identical controlled conditions. If the insightful information provided by animal studies is correct, then psychotherapy for the treatment of diseases might be made more effective if some aspect of this knowledge is included in the design of the treatment. We emphasize conditioning as a regimen and an acceptable way to train the brain to remember an output pathway to raise immunity. We propose that a specific drug or perception (mild stress, represented by rotation, total body heating or handling) could substitute and kindle the same output pathway without the need for conditioning. If this view is correct, then instead of using conditioning, it may be possible to use an antigen to activate desired immune cells, and substitute a drug or an external environmental sensory stimulus (perception) to energize the output pathway to these cells. Alternatively, monitoring alterations of body temperature in response to a drug or perception might allow us to follow how effectively the brain is performing in altering immunity. Studies with animals suggest that there are alternative ways to use the mind to raise natural or acquired immunity in man.

Afferent signals to the CNS appear not to condition the modulation of interleukin-1 receptors in the hippocampus.

Conditioned alteration of natural killer (NK) cell activity was used as an indicator of the functional bidirectional communication between the immune and central nervous systems. Poly I:C and lipopolysaccharide (LPS) from Escherichia coli and Salmonella typhimurium were used as unconditioned stimuli and odor of camphor as the conditioned stimulus. An attempt was made to demonstrate the role of central interleukin (IL-1) receptors in this communication process. Brain IL-1 receptors were down-regulated by treatment with 50 microg/mouse of LPS from S. typhimurium, but not with the same dose of LPS from E. coli or poly I:C. A significant level of conditioned augmentation of NK cell activity was observed with poly I:C. Conditioned alteration in NK cell activity was also observed with LPS from E. coli, but at much lower level than poly I:C. NK cell activity was not conditioned with LPS from S. typhimurium at the same dose as E. coli LPS, but conditioned enhancement of NK cell activity was observed with a higher dose (100 microg) of S. typhimurium LPS. These results suggest that modulation of central IL-1 receptors do not seem to play a role in the conditioned augmentation of NK cell activity.

Psychoneuroendocrine immunology: site of recognition, learning and memory in the immune system and the brain.

How the interaction between the brain and immune system takes place has not been clearly defined. Because multiple changes are occurring simultaneously in all organ systems (e.g., cardiovascular, gastrointestinal, reproductive, renal, respiratory, immune, CNS), how many single systems interacts with the brain becomes extraordinarily difficult to understand. The problem boils down to developing an approach that not only allows one to study the whole organism and define the mediators of the interacting systems, but also permit one to establish the connection and physiologic relevance of the responses that are being evaluated. Conditioning, a phenomenon made popular by the work of Pavlov (1906, 1927), may provide insight into the pathways of communication between the brain and possibly any organ system of the body. Conditioning allows one to separate the afferent from the efferent circuits. That is, signals from the immune system to the CNS (IS-->CNS) can be effectively separated from signals from the CNS to immune system (CNS-->IS). This permits one to study each pathway individually. Simple, single association trial models to condition fever, natural killer (NK) cell and cytotoxic lymphocyte (CTL) activities have been developed to evaluate the pathways. Single trial learning is not new. Pavlov has observed that "The electric buzzer set going before administration of food established a conditioned alimentary reflex after only a single combination," whereas the reverse order of presentation failed to condition the animal (Pavlov 1927 p. 27). Thus, conditioning can be used to train the brain to activate the immune system and other organ systems participating in the response. During the course of the conditioned response, presumably the CNS via the hypothalamus integrates in a cohesive orderly fashion all input and output signals and coordinates the responses made by the brain to the organ systems. The odor of camphor, the conditioned stimulus (CS) can be associated with the response produced by an unconditioned stimulus (US). The unconditioned stimuli used are poly I:C to raise fever and nonimmunospecific NK cell activity or alloantigens to raise immunospecific CTL activity. The unconditioned stimulus serves only as a means to activate the immune system and unbalance the homeostasis so that a transient but new bidirectional communication loop can be established between the immune system and the CNS (IS<-->CNS). The expression of the conditioned response (i.e., elevation of fever, NK cell, or CTL activity) induced with the CS (odor stimulus) is an outcome of neural activity (CNS-->IS). This infers that during conditioning, the signals generated by the CS and US imprints a neural pathway located within the central nervous system and leaves behind a CS/US memory of the association. The immune activity (NK cell or CTL activity) which is modulated indicate that the memory pathway was activated in the brain of the animal expressing the conditioned response. The immune cells that are modulated can be considered to be casual bystander cells. These cells however must be in the proper (ready) state of activation to receive salient signals from the brain. Along with changes in the indicator cell population, other complex physiological processes are altered by the brain via sympathetic and neuroendocrine pathways to raise the fever response. These observations suggest that the physiological changes which are being evaluated such as fever, NK cell or CTL activities or perhaps blood pressure, heart rate, fat metabolism, oxygen consumption serve only as indicators (readouts), and infer that the CNS has made a coordinated reply in response to the CS signal.

Conditioning of body temperature and natural killer cell activity with arecoline, a muscarinic cholinergic agonist.

Arecoline, a muscarinic cholinergic agonist, was found to depress body temperature and elevate the activity of preactivated natural killer (NK) cells. To demonstrate that the unconditioned responses produced by arecoline were mediated through central nervous system pathways, we used the drug as an unconditioned stimulus. By pairing camphor odor (conditioned stimulus) with arecoline (unconditioned stimulus), it was possible to simultaneously condition both a decrease in body temperature and augmentation of NK cell activity. The observations suggest that although both the modulation of body temperature and NK cell activities are integrated at the level of the hypothalamus, these pathways of regulation can be differentiated.

Lipopolysaccharide and IL-1 alpha activate CNS pathways as measured by NK cell activity.

The effect of lipopolysaccharide (LPS) on murine unstimulated and prestimulated natural killer (NK) cells and its ability to serve as an unconditioned stimulus was investigated. LPS injection induced a statistically significant increase in NK cell activity when compared with saline-treated control groups. To demonstrate the existence of communication between the peripheral immune system and the central nervous system (CNS), we used a single-trial conditioning paradigm in which camphor served as the conditioned stimulus (CS) and LPS as the unconditioned stimulus (US). Once a CS/US association is made, exposure of animals to the CS alone results in the conditioned response (i.e., increase in NK cell activity). Using 50 micrograms of LPS as the US produced a low but significant increase in NK cell activity when compared to control groups. However, 10 micrograms of LPS did not show a significant increase in NK cell activity. We also observed that interleukin-1 alpha (IL-1 alpha) injected intracisternally can serve as a US to condition a central neuroendocrine pathway. Because the dose of IL-1 alpha employed was too small to raise NK cell activity in the spleen, the NK cells themselves were formally not subjected to conditioning. These observations suggest that LPS and IL-1 alpha conditions the brain and that NK cell activity can be used as an indicator system to detect neuroendocrine signals arising from the activated pathway(s).

Arecoline a muscarinic cholinergic agent conditions central pathways that modulate natural killer cell activity.

The central nervous system plays an active role in the regulation of the immune system. Modulation of immune activities appears to be in part under the control of the hypothalamic-pituitary-adrenal (HPA) axis. We investigated the effect of a muscarinic cholinergic agonist, arecoline, which stimulates the secretion of corticotropin-releasing hormone (CRF) and adrenocorticotropic hormone (ACTH) on the immune system. In this report we demonstrate that peripherally administered arecoline or ACTH can increase activity of pre-activated NK cells. Second, we show that central administration of arecoline at a dose too low to alter peripheral events is sufficient to induce a significant increase in the activity of pre-activated natural killer (NK) cells. Finally, we demonstrate by using a Pavlovian conditioning paradigm that the pairing of a novel odor (camphor) with administration of arecoline can be used to alter NK cell activity. Subsequent to the conditioning trial, exposure to the odor alone is sufficient to raise NK cell activity. From these observations, we infer that the pathway(s) that are conditioned reside in sites located within the CNS and the conditioned response is evoked in the peripheral compartment (NK cell activity).

Conditioning of the secondary cytotoxic T-lymphocyte response to YC8 tumor.

Studies from our laboratory demonstrated that conditioned resistance to the syngeneic YC8 lymphoma was established by multiple conditioned stimulus (CS)/unconditioned stimulus (US) associations. The conditioned stimulus used was exposure to the odor of camphor for 1 h and the unconditioned stimulus was an injection of DBA/2 spleen cell alloantigen that shares minor histocompatibility determinants with the YC8 lymphoma. To demonstrate a cellular basis for immune resistance to the YC8 tumor, BALB/c mice primed with DBA/2 spleen cell alloantigen were conditioned using a single trial CS/US association paradigm. Conditioned animals showed a measurable conditioned elevation of the cytotoxic T-lymphocyte (CTL) response to the YC8 tumor. Control groups in which the CS and US were not given in the proper sequence were unable to mount a conditioned response. These studies show that a secondary CTL response can be upregulated by the central nervous system (CNS).

Use of an allogeneic antigen to induce resistance and regression of an autologous tumor.

Studies were initiated to determine if it was possible to use a tumor-bearing animal's own spleen cells to impart resistance to its neoplasm. YC8 T-cell lymphoma-bearing BALB/c mice (TBAs) were immunized with allogeneic DBA/2 spleen cells, which share cross-reacting antigens with the YC8 tumor. Animals immunized with the alloantigen were splenectomized and their spleen cells co-cultured with additional alloantigens for 2 days in media containing 2% polyethylene glycol (PEG) before being returned to the cyclophosphamide (cytoxan) (Ctx) pretreated autologous host. Treatment with Ctx was used to reduce suppressor factors in the TBA. It was found that when cultured spleen cells were returned to the autologous TBA, much greater resistance was imparted to the host and, in many instances, regression of the YC8 tumor was observed.

Role of arcuate nucleus of the hypothalamus in the acquisition of association memory between the CS and US.

A single trial association protocol was used to demonstrate a conditioned increase in natural killer (NK) cell activity. The signals used were odor of camphor as the conditioned stimulus (CS) and polyinosinic-polycytidylic acid (poly I:C) as the unconditioned stimulus (US). This model has been used to dissect the underlying mechanisms of interaction between the central nervous system (CNS) and the immune system (IS) and vice versa. Here, we demonstrate the potential role played by the arcuate nucleus of the hypothalamus in the acquisition of association memory between the CS and the US. Chemical destruction of the arcuate nucleus with monosodium glutamate (MSG) was used for this purpose. Mice with arcuate nucleus lesion prior to the association protocol did not demonstrate a conditioned increase in NK cell activity. However, the lesion has no effect if produced prior to exposure to the CS at recall. These studies demonstrate the significant role played by the hypothalamus (arcuate nucleus) in a conditioned response.

A multiple modality approach combining the effect of conditioning with adoptive chemoimmunotherapy.

It has been demonstrated that significant protection against YC8 lymphoma can be induced in mice preimmunized with normal DBA/2 spleen cells. The DBA/2 spleen cells used as an alloantigen share minor histocompatibility determinants with the YC8 tumor. Our observations showed that once tumor was present in vivo, the use of a potent tumor specific vaccine that can confer 100% protection to preimmunized animals, can help in increasing survival but can no longer produce high incidence of regression and cure. We have used this model to show that adoptive chemoimmunotherapy (ACIT) can be used to regress tumors in mice with large body burden of tumor and that combination of conditioning with ACIT appears to enhance the effectiveness of treatment. The nature of the immunity conferred by conditioned resistance might be due to cytotoxic T-lymphocytes.

Conditioning of the allogeneic cytotoxic lymphocyte response.

Allogeneic cytotoxic T-lymphocyte (CTL) response can be obtained following immunization of BALB/c mice with C57BL/6 spleen cells. We investigated the possibility of behaviorally conditioning this response by associating the C57BL/6 spleen cell immunization [unconditioned stimulus (US)] with camphor odor [conditioned stimulus (CS)]. We reported the possible mechanisms involved in the conditioning of natural killer cell activity. Similar approaches were used to investigate the mechanisms that participate in the conditioned CTL activity. The first mechanism of investigation utilized opioid receptor blockers naltrexone and quaternary naltrexone. Naltrexone, which blocks both the central and peripheral opioid receptors, blocked the recall of the conditioned response, whereas quaternary naltrexone, which does not penetrate the blood-brain barrier, was unable to block the conditioned response, demonstrating that centrally located opioid receptors play a role in the recall of the conditioned response. The studies are of interest because they indicate that resistance or susceptibility to various diseases such as cancer, autoimmunity, and infectious diseases might be influenced by the regulatory network of the CNS.

A simple, single, trial-learning paradigm for conditioned increase in natural killer cell activity.

A change in natural killer (NK) cell activity can be conditioned with one trial learning when conditioned stimulus (CS) precedes the unconditioned stimulus (US). To avoid the problems associated with two reexposures in our earlier studies, we have developed a reliable and simple conditioning protocol utilizing the one trial learning and one reexposure to the odor CS. The conditioned change in NK cell activity was significantly different (P less than 0.05) from the control groups of mice. The paradigm is short and simple in that the conditioned change could be demonstrated within 3 days. We have also compared the effects of temporal association of CS and US on conditioned increase in NK cell activity. Forward conditioning (CS preceded the US) demonstrated a conditioned change, but the backward conditioning protocol did not. The paradigm provides a reliable approach to the study of mechanisms of the phenomenon of odor-NK conditioning.

In vivo enhancement of NK cell activity with met-enkephalin and glycyl-glutamine: their possible role in the conditioned response.

These studies investigated the effect of met-enkephalin, glycyl-glutamine, and naltrexone on NK cell activity in vivo and in vitro. It was found that both met-enkephalin (which shares the amino-terminal end of beta-endorphin) and glycyl-glutamine (which reflects the carboxyl-terminal end of beta-endorphin) can enhance the NK cell activity of mice prestimulated with a low dose (1 microgram/mouse) of poly I:C. Naltrexone had no effect. In vivo prestimulation of the mice with 1 microgram poly I:C was necessary as mice which were not pretreated with poly I:C did not show enhanced NK cell activity when treated with either met-enkephalin or glycyl-glutamine. In vitro studies however indicate that the drugs when cultured together with the NK cells from mice preactivated with poly I:C did not have a direct stimulatory effect on the NK cells. These studies imply that while beta-endorphin released from the pituitary could be involved in enhancement of activated NK cells in vivo other indirect peripheral pathways might be involved. The results suggest beta-endorphin probably reacts with other accessory type cells which in turn release the mediators which are required for the stimulation of NK cells in vivo.

Role of immune cells in the Pavlovian conditioning of specific resistance to cancer.

It has been demonstrated that significant protection against YC8 lymphoma can be induced in mice preimmunized with normal DBA/2 spleen cells. The DBA/2 spleen cells used as alloantigens share minor histocompatibility determinants with the YC8 tumor. We have used this model to investigate the nature of the immunity conferred by treatment with the alloantigen and infer that the conditioned resistance observed was maintained by the same effector mechanism. The results demonstrated that repeated immunization of tumor bearing mice with the alloantigen had some beneficial effect as shown by the slower rate of growth of the tumor, and an increase in median survival time over controls. The observations showed however that once tumor was present in vivo, the use of potent tumor specific vaccine can help in increasing survival but can no longer produce high incidence of regressions and cures. Conditioning can potentiate the effects of this treatment by increasing survival and cure.

Effect of thymic hormone treatment on several immune functions of nude mice.

Studies of the effect of short-term, intense treatment with thymic hormone on mitogen response, cytotoxicity to EL-4 lymphoma and natural killer cell (NK) activity was investigated Balb/c nude mice (about 12-16-week-old) were treated 5 times per week for 3 weeks with: Facteur Thymic Serique (FTS) and Thymopentin (TP5, Thymopoietin 32-36) at 1 microgram and 10 ng; TM4 1 ng (an enzyme resistant variant of FTS); Thymosin Fraction V (TF5), 10 and 1 microgram; and 0.1 ml saline, and killed 2 days after the last treatment. The animals were monitored for changes in weight, hematocrit, peripheral blood lymphocyte (PBL) and spleen mitogen response. Additional groups of nude mice were immunized with 1 x 10(7) 5000 R irradiated EL-4 cells 10 days before sacrifice and tested for the presence of cytotoxic T-lymphocytes (CTL). The results show that weight and hematocrit were similar among the groups. Treatment with FTS significantly elevated the number of PBL. Spleen stimulation in mice treated with 1 microgram TP5 was depressed to mitogen concanavalin A (ConA) and lipopolysaccharide (LPS) stimulation. The phytohemagglutinin (PHA) response was not different among the treatment groups. The PBL mitogen response to ConA and LPS was generally increased over saline control in the hormone treated groups but was not statistically significant. The PHA response was only slightly elevated. No CTL was generated in nude mice in any of the groups. However, there was a statistically significant general depression of NK activity in all of the hormone treated animals compared with saline. The results indicate that the basic differentiation defect of the T-cells of nude mice cannot be restored to full functional activity by short-term treatment.

Conditioning the elevation of body temperature, a host defensive reflex response.

We hypothesize that a number of host defense responses such as natural killer (NK) cell activity, cytotoxic lymphocyte (CTL) activity, antibody production, and elevated body temperature (TR) might be conditionable. We have designated such specifically learned response to be a defensive reflex response. Here we describe a simple single trial association paradigm for conditioning the TR response in BALB/c mice. Animals are conditioned on day 0 by exposing them to the odor of camphor for 1 hr, followed by injection of the pyrogen poly I:C 20 microgram ip. Control groups are injected with either poly I:C or saline and not exposed to the camphor odor. Reexposure of all groups to the conditioned stimulus (CS) on day 2 or 3 cause elevation of body temperature in the conditioned group mice but not in the nonconditioned or saline control groups. Since we have conditioned the natural killer cell response with the same paradigm, these results suggest that multiple defensive responses might be conditionable simultaneously and they might have important survival value for the species.

Survey of thymic hormone effects on physical and immunological parameters in C57BL/6NNia mice of different ages.

Immunosenescence occurs with aging, which is seen in decline in response to mitogens PHA, ConA, decline in cell-mediated immunity, increase in anemia, and increase in autoimmune antibodies to erythrocytes and DNA. These studies compared FTS, TP5, TM4, and TF5 in C57BL/6NNia mice. Mice aged 4, 26, 52, 78 and 104 wk were treated with various hormones 5x/wk for 3 wk and monitored for hormonal effects on weight; hematocrit; peripheral blood, spleen, and thymic cell numbers; spleen and peripheral blood cell mitogen responses to PHA, ConA, LPS; IgM hemolysin autoantibody; and cell-mediated cytotoxicity to P815 allogenic cells. Hormone treatments altered mitogen responses, enhanced IgM hemolysin autoantibody production, and modulated cell-mediated immune responses. The effects were not consistent for every hormone. There was a tendency for enhancement in younger mice and suppression in older animals. Treatment with FTS showed the greatest changes in either enhancing or suppressing the different parameters measured. The hormonal effects appeared to be age specific in that certain activities were altered for certain age groups but not in others. Hormone treatment did not restore any immune parameters in old mice to the level of young animals. In general, the different hormones did not consistently produce the same effects in C57BL/6NNia mice of different age groups. Even though all animals received from National Institutes on Aging (NIA) animal models program were held under strictly controlled conditions, intrinsic variations between cohorts of different ages are difficult to control. Cohorts of aging animals tested at different times might be intrinsically different. This inherent variability in the cohorts could affect the range of activity, specificity and reproducibility of hormone effects in vivo. Most importantly, it should be emphasized that cross-sectional data identifies age differences rather than age changes. There is no assurance that age changes in any individual or in all subpopulations follow this pattern. In our studies only healthy animals were used. Old, sick, or tumor-bearing animals were culled out prior to being sent to us. Therefore, the 78- and 104-wk-old mice represent selected healthy cohorts. The age changes that take place can be answered only from repeated measurements made in the same individual over time.

Conditioning: a new approach to immunotherapy.

It has been demonstrated by Parmiani et al. (Int. J. Cancer, 29: 323-332, 1982) that a significant protective effect can be obtained against the transplanted syngeneic YC8 lymphoma by prior immunization of BALB/c mice with normal allogeneic DBA/2 spleen cells. Using this well established tumor model, we investigated a novel approach, conditioning of specific immunotherapeutic activity. For this purpose, we used the odor of camphor as the conditioning stimulus and allogeneic DBA/2 spleen cells as unconditioning stimulus. We associated the conditioning and unconditioning stimuli two, three, and four times. Following this the conditioned animals were reexposed to the odor of camphor only. In each case, we observed a delay in tumor growth and in some instances the conditioned group performed better than the immunotherapy control group. These results indicate that a limited number of treatments with the antigen is better than the continuous treatment in maintaining the immunity and the homeostasis of the system.