Carolyn F. Rogers

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.

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.

Effect of Dexamethasone on Conditioned Enhancement of Natural Killer Cell Activity

It is believed that the expression of the conditioned natural killer cell activity is regulated through the hypothalamus-pituitary axis. Since the conditioned expression of both the plasma level of adrenocorticotropic hormone and natural killer (NK) cell activity show a sequential rise after exposure to the conditioned stimulus, it was of interest to determine if treatment with dexamethasone could inhibit this response. These studies suggest that treatment with dexamethasone was able to block the expression of the conditioned NK cell activity but was unable to interfere with the pairing of the conditioned stimulus with the unconditioned stimulus. We conclude that the hypothalamic pituitary axis is an integral part of the pathway for stimulation of NK cell activity in the spleen.

Indomethacin and sodium carbonate effects on conditioned fever and NK cell activity

The augmentation of natural killer (NK) cell activity and elevation of body temperature (fever) can both be conditioned using camphor odor as the conditioned stimulus (CS) and poly I:C as the unconditioned stimulus (US). While both responses can be conditioned in parallel fashion as shown previously, our results indicate the conditioned learning of these responses may not follow along a common path. We found that injection of a 1% solution of sodium carbonate was able to consistently block the CS/US learning of the NK cell response but did not block conditioning of the fever response. In contrary fashion, mice treated with indomethacin (which inhibits prostaglandin-induced fever) dissolved in the sodium carbonate solution did not learn in consistent fashion the fever response. However, indomethacin-treated animals were able to recall the NK cell response. These results support the view that although the same mediator, IFN-beta, is responsible for the conditioned learning of the NK cell and fever responses both the learning and recall of the responses are initiated along separate pathways.

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.

The Direction of the Conditioned Natural Killer Cell Response can be Re-Directed with Indomethacin and/or Handling

We have used the pairing of camphor odor conditioned stimulus (CS) and injection of poly I:C unconditioned stimulus (US) in a short 3 day single trial conditioning paradigm. Conditioning was done by exposing mice to the CS/US combination on day 0 and reexposing the conditioned animals to the CS on day 2. This results in a conditioned augmentation of the natural killer (NK) cell response. Indomethacin treatment and/or handling stress induced by simply measuring rectal temperature was found to dramatically alter the direction of the conditioned NK cell response. Conditioning of indomethacin treated mice produced a conditioned suppression of the NK cell response mimicking a conditioned tolerance response. If handling stress was superimposed on day 2 the conditioned suppression response was replaced by a conditioned augmentation of the NK cell response. Even with one trial conditioning, drugs and handling stress can serve as additional cues to alter the direction of the conditioned response. The studies also show that the conditioning of the fever response is independent of conditioning of the NK cell response.