The lipoxygenase inhibitor, baicalein, modulates cell adhesion and migration by up-regulation of integrins and vinculin in rat heart endothelial cells.

BACKGROUND AND PURPOSE: Endothelial cell proliferation, migration and adhesion are necessary for the formation of new blood vessels. We reported previously that baicalein strongly inhibited proliferation of rat heart endothelial cells and here we assess effects on migration and adhesion of these cells. EXPERIMENTAL APPROACH: Effects of baicalein on endothelial migration and adhesion were determined by in vitro wound assays and in modified Boyden chambers. Protein expression and subcellular distribution in rat heart endothelial cells were analysed by immunoblots and immunofluorescence staining. RESULTS: Pretreatment with baicalein for 48 h resulted in a concentration-dependent inhibition of endothelial migration, with an IC(50) of approximately 20 microM. Adhesion assays revealed that baicalein stimulated endothelial cell adhesion to fibronectin and vitronectin, effects blocked by the synthetic peptide Arg-Gly-Asp (RGD). Moreover, treatment with a blocking antibody against integrin alpha5beta1 drastically attenuated baicalein-mediated endothelial adhesion to fibronectin, but not to vitronectin. Furthermore, baicalein-mediated anti-migration effect and adhesion promotion could be partially reversed by the addition of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE). Western blot analysis indicated that baicalein increased expression levels of integrin-alpha5beta1, -alphavbeta3 and vinculin proteins. Immunofluorescence staining showed that baicalein induced a marked reorganization of actin stress fibres and the recruitment of vinculin and integrins to focal adhesion plaques, with consequently increased formation of focal adhesion contacts. CONCLUSIONS AND IMPLICATIONS: Baicalein markedly inhibited the migration and enhanced the adhesion of rat heart endothelial cells, possibly by up-regulation of the integrins (alpha5beta1 and alphavbeta3) and vinculin and by promotion of actin reorganization and focal adhesion contact formation.

The involvement of glutamate in recall of the conditioned NK cell response.

The molecular mechanisms responsible for the conditioned enhancement of natural killer (NK) cell activity were investigated. The primary goal of the study was to examine the roles of glutamate and gamma-aminobutyric acid (GABA) in recall of the conditioned NK cell response. Both neurochemical blocking assay and high performance liquid chromatography (HPLC) technique were used in the study. Results from the neurochemical blocking assay demonstrated that glutamate but not GABA was required in recall of the conditioned NK cell response. NMDA but not the kainate/AMPA receptors, are believed to be involved. The levels of glutamate that were released and/or taken up also appeared to be critical in that interruption of glutamate release and/or uptake blocked the conditioned NK cell response. Results from the HPLC analysis, however, did not show any significant difference in the glutamate content between the conditioned and control brains.

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.

Involvement of catecholamines in recall of the conditioned NK cell response.

The primary goal of the study was to identify the types of catecholamines and the associated receptors which might be involved in the recall of the conditioned NK cell response. Specific catecholamine receptor antagonists were selected to block the conditioned NK cell response at the recall step. The regional contents of dopamine (DA), norepinephrine (NE), and epinephrine were determined in the brain of the conditioned animals by using the high performance liquid chromatography with electrochemical detection (HPLC/ED). Results showed that pre-disruption of the central alpha1-, alpha2-, beta1-, beta2-, D1-, or D2-receptors at the conditioned recall stage, interrupted the conditioned enhancement in NK cell activity. The NE contents at the cerebellum, and DA contents at the striatum and hippocampus, were significantly higher in the brain of the conditioned animals when compared to that of the control animals. These information indicated the possible roles of the central noradrenergic and dopaminergic systems in regulating the recall of the conditioned NK cell response.

The central effect of methionine-enkephalin on NK cell activity.

The central effect of opioid peptide on natural killer (NK) cell activity in BALB/c mice was investigated. Injection of methionine-enkephalin (Met-Enk), 0.02 microgram/mouse or 1 microgram/kg, directly into the cisterna magna (CM) of the brain, resulted in a significant enhancement of NK cell activity. This enhancement was blocked by opiate antagonists, naltrexone and quaternary naltrexone. The same dose of Met-Enk had no effect on NK cell activity when given to the mouse intraperitoneally or intravenously. Moreover, des-tyrosine-methionine-enkephalin injected into the CM at 1 microgram/kg, had no effect on NK cell activity. The results indicate that activation of an opioid-mediated pathway in the central nervous system is capable of activating the pathways that stimulate the NK cell response in the periphery.

Activation of mu-opioid receptors are required for the conditioned enhancement of NK cell activity.

The type of opioid receptors involved in the conditioned enhancement of natural killer (NK) cell activity is identified in the present study. In our previous observations, we have demonstrated that the conditioned enhancement of NK cell activity was dependent on beta-endorphin and methionine-enkephalin, but not dynorphin. Based on the interaction of opioids with their homologous receptors, we concluded that mu- and delta-opioid receptors might be involved. To further classify the type(s) of opioid receptors involved in eliciting the conditioned NK cell activity, three opioid receptor antagonists, cyprodime hydrobromide, ICI-174864, and nor-binaltorphimine dihydrochloride, were used to block the conditioned NK cell activity in BALB/c mice. Blocking was conducted by intracisternal injection of the drugs. The results showed that the activation of mu-opioid receptors was required in the conditioned enhancement of NK cell activity, but not the delta- or kappa-type of receptors.

Expression of the conditioned NK cell activity is beta-endorphin dependent.

We are interested in identifying the pathways which are responsible for triggering the conditioned enhancement of natural killer (NK) cell activity. Earlier studies have suggested that central opioid(s) are involved in eliciting the expression of the conditioned NK cell activity. The purpose of this study was to identify the central opioid peptides that allow the central nervous system (CNS) to communicate with the immune system. Mediators that activate the efferent pathway of communication between the CNS and immune system was examined by injection of the mediator via the cisterna magna (CM). Conditioning was used as a tool to show that the bi-directional communication between the CNS and the immune system does take place. We found that beta-endorphin but not dynorphin could stimulate NK cell activity, when beta-endorphin or dynorphin was injected into the CM. In addition, when anti-beta-endorphin or anti-dynorphin antibody was injected into the conditioned animals via CM the conditioned response was blocked by anti-beta-endorphin but not by anti-dynorphin antibody. These observations suggest that beta-endorphin appears to be one of the signals that is induced in the brain at the CS recall step of the conditioned response to trigger the elevation of NK cell activity.

Acquisition of enhanced natural killer cell activity under anesthesia.

An increase in natural killer (NK) cell activity can be conditioned with a one trial learning paradigm to demonstrate the interaction between the central nervous system (CNS) and the immune system. In order to demonstrate learning possibilities during 'non-conscious' state, mice were anesthetized with a ketamin/rompun mixture and underwent one trial learning with odor cue as the conditioned stimulus (CS) preceding the unconditioned stimulus (US). The results indicated that mice that were exposed to camphor odor cue under the influence of anesthesia can associate the signal with the poly I:C unconditioned stimulus and were able to recall the conditioned response upon reexposure to the CS. Secondly, the conditioned association made in a conscious state can be recalled by exposure to the same olfactory odor cue in a 'non-conscious' state. The increase in the conditioned change in NK cell activity of both situations was significantly higher than the control group. The results demonstrate that learning can take place and the learned response can be recalled under the reduced awareness caused by anesthesia. The findings we report are unusual and novel in that they demonstrate that the CNS can learn new associations under conditions where the host is apparently unaware of the signals being linked. Anesthesia combined with the long interstimulus interval indicates that certain neuronal pathways in the CNS are receptive to second signals (elicited by the US) even when the second signal is separated by one day. This means the conditioned learning of a physiological response can take place unconsciously at a separate level and under situations where the host is totally unaware of the events which the brain is processing and linking as incoming information.

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.

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.

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.

Antiplatelet effects of some aporphine and phenanthrene alkaloids in rabbits and man.

Two aporphines (boldine and laurolitsine) and five phenanthrene alkaloids (litebamine, secoboldine, N-cyanosecoboldine, N-methylsecoglaucine and N-methylsecopredicentrine) were evaluated in-vitro for their ability to inhibit platelet aggregation. All seven alkaloids inhibited aggregation of rabbit platelets and inhibited the release of ATP induced by arachidonic acid and collagen in rabbit platelets. Those aggregations induced by platelet-activating factor (PAF), thrombin, U46619 and ADP were inhibited by the three N-substituted secoboldine derivatives only. Thromboxane B2 formation caused by arachidonic acid was also suppressed by these compounds. They did not affect the generation of [3H]inositol monophosphate caused by collagen, PAF and thrombin in the presence of indomethacin. Platelet cyclic AMP level was unaffected by litebamine, but was increased by N-methylsecoglaucine. Litebamine suppressed the secondary aggregation, but not the primary aggregation, induced by ADP and adrenaline in platelet-rich plasma from man, whereas N-methylsecoglaucine inhibited both primary and secondary aggregation. It is concluded that the antiplatelet effect of these seven aporphine and phenanthrene alkaloids is mainly a result of inhibition of thromboxane A2 formation; N-methylsecoglaucine has additional antiplatelet activity as a result of increasing the levels of platelet cyclic AMP.

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.

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.

Efferent signal(s) responsible for the conditioned augmentation of natural killer cell activity.

In in vivo studies, a conditioned increase in NK cell activity can be obtained by pairing odor of camphor (conditioned stimulus, CS) with poly I:C (unconditioned stimulus, US) in a single-association paradigm. We identified interferon (IFN) as the signal that reaches the central nervous system (CNS) to make an association with the camphor CS. We have also established that the CS/US association is an IFN-dependent step, and the expression stage is an opioid-dependent pathway which can be blocked with naltrexone and dexamethasone. Here we have focused on the signals responsible for the expression of conditioned augmentation of natural killer (NK) cell activity. The possible efferent signal molecules that were considered were IFN, beta-endorphin (beta-END), and adrenocorticotropic hormone (ACTH). Plasma levels of beta-END and ACTH of conditioned and control mice were quantitated by radioimmunoassay, and the changes in IFN message in the spleen cells were determined by Northern hybridization analysis. Results indicate that the ACTH levels and IFN-alpha gene expression were higher in the conditioned animals than in the controls. These studies support the view that ACTH released from the pituitary gland is involved in the up-regulation of IFN-alpha, which in turn stimulates the NK cells in the spleen.

Involvement of cytokine gene expression in the age-dependent decline of NK cell response.

The molecular mechanism responsible for the age-dependent decline of natural killer (NK) cell response was examined. The responses of NK cells to poly(I:C), Con A, and LPS were compared between the young and the aged animals. We observed that both basal and induced NK cell activity declined with age. To further determine the basis for this change, an effort was made to identify the cytokine genes that might be involved. In addition, the capacity of acetyl-L-carnitine (ALC) to restore NK cell activity was evaluated. In this study, 51Cr release assay and reverse transcriptase-polymerase chain reaction assay were used to measure the NK cell activity and the changes in cytokine gene expression, respectively. Our results show that the decline in NK cell response to poly(I:C) and LPS in aged animals was correlated with decreased expression of the IFN-gamma gene. The decline in the NK cell response to Con A in aged animals was, however, independent of the expression of IL-2 or IFN-gamma genes. Although ALC was reported to restore functions of T cells and macrophages in aged animals, we found that long-term treatment (4 months) with ALC had no effect on either the basal or the induced age-dependent loss of NK cell activity. Our observations suggest that cytokine gene therapy could be a potential approach to improving or even preventing the decline of certain immune functions in the elderly.

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.

The use of conditioning to probe for CNS pathways that regulate fever and NK cell activity.

Immune and central nervous system (CNS) interactions are complicated because afferent signals from the immune system to the CNS in response to antigens or infections may elicit an immediate efferent response to the immune system. This communication loop is required for the homeostatic regulation of the immune system. Conditioning can be used as a tool to take the communication loop apart. In conditioned animals, the conditioned stimulus can be employed later to trigger the site of the association memory located within CNS, and set off the efferent pathway. Conditioning therefore allows one to isolate and identify the potential circuits in the brain that becomes conditioned. We have conditioned a pathway in the brain which can be used to modulate core body temperature (Tc) and natural killer (NK) cell activity. The Tc and NK cell activity are used as readouts to detect the expression of the conditioned response which is taking place in the brain. Since various cytokines (IFN, IL-1 etc) that are produced by antigenic stimulation invariably raise fever, it appears that the immune system could signal the CNS with nonspecific cytokines that activate the hypothalamic-pituitary pathway to modulate core body temperature. These observations infer that the thermoregulatory pathway in the brain becomes conditioned and points to a common pathway of communication in which interferon-beta, prostaglandin E2, CRH and ACTH appear to play a role in modulating both Tc and NK cell activity.

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.

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.