Nancy S. Hiramoto

Identification of specific pathways of communication between the CNS and NK cell system

The specific signals and pathways utilized by the natural killer (NK) cell system and the central nervous system (CNS) that results in the conditioned response (CR) is not clearly understood. Single trial conditioning of the NK cell activity provides us with a model to probe the mechanisms of communication between two major systems (Immune and CNS) which are involved in the health and disease of the individual. The studies show that the IFN-beta molecules possess the properties attributed to the unconditioned stimulus (US). IFN-beta can penetrate the CNS and evoke the elevation of NK cell activity in the spleen. This unconditioned response (UR) can be linked to a specific conditioned stimulus (CS). Specific odors such as camphor provide a neural pathway for the CS to associate with the US. Evidence is presented that in conditioning there are two locations where memory develops. The CS/US association is made centrally and its memory is stored at a central location, but the memory for the specificity of the odor is presumably stored in the olfactory bulbs. The CS recalls the CR by triggering the olfactory neural pathway which, in turn, signals the hypothalamic-pituitary axis to release mediators that modulate the activity of NK cells in the spleen. These results imply that through conditioning one has direct input into the regulatory hypothalamus that controls the internal environment of the organism and the health and disease of the individual. Consequently, it is not inconceivable that through this approach we might be able to alter the course of a disease process.

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.

A Simple, Single, Trial-Learning Paradigm for Conditioned Increase in Natural Killer Cell Activity

Abstract 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 < 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.

Regulation of Natural Immunity (NK Activity) by Conditioning

A paradigm is described for the conditioning of the NK response. Mice were exposed to a classical conditioning procedure in which saccharin and lithium chloride (LiCl) served as the conditioned stimulus (CS) paired with an injection of either poly I:C or cytoxan, the unconditioned stimulus (US). Poly I:C was used as the US for enhancement and cytoxan served as the US for suppression. Subsequent exposure to the CS either enhanced or suppressed the NK activity in the conditioned animals. Our studies showed that only one association was needed for the learning to take place, training to consumption of water was not necessary, and the conditioned (enhanced) response could be recalled as soon as 7 days after pairing of the US and CS.

Augmentation of natural immunity and regulation of tumor growth by conditioning.

We have reported the effect of classical (Pavlovian) conditioning of natural immunity on survival of tumor-bearing mice. In the first study, we have observed that mice conditioned, transplanted with tumor, and re-exposed to conditioned stimulus (camphor odor) had an increase in median survival (day 43, as compared to days 34, 38, and 37 of various control groups). Two of these conditioned mice lived more than 120 days and showed early tumor growth, but were free of disease at day 97. We report the observations of a repeat study. Two groups of conditioned mice were used for these studies. One group was re-exposed to the conditioning stimulus following transplantation with tumor (CND) and the second group was not re-exposed to odor of camphor (CNDo). Statistically significant delay in growth of MOPC 104E in the CND group was observed when compared with the CNDo group. The survival data supports the observations of tumor IgM values. In an independent study, we investigated the possible mechanisms of MOPC 104E regulation in vitro. Plastic adherent spleen cells (macrophage cells) from mice primed in vivo with MOPC 104E tumor cells suppressed tumor IgM production by MOPC cells by 98% and also reduced colony formation by MOPC cells. The possible mechanism(s) of regulation of tumor growth in conditioned mice might be mediated by plastic adherent activated macrophages.

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).

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.

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).

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.