Methionine enkephalin: a new cytokine--human studies.

The effects of methionine enkephalin (met-enkephalin) on human immune function are reviewed. This pentapeptide functions to upregulate, or enhance, immune function in the majority of donor samples at low doses and suppresses at high doses. The influence of this molecule is shared by the central nervous, neuroendocrine, and immune systems. Cells from each of these systems possess receptors for met-enkephalin and have the ability to process met-enkephalin from its prohormone, proenkephalin A. Studies have shown that this molecule is capable of enhancing immune function in patients with cancer or AIDS. It is proposed that this molecule be classified as a cytokine.

Anti-retroviral activity of methionine enkephalin and AZT in a murine cell culture.

Previously, this laboratory has demonstrated that azidothymidine used in combination with methionine enkephalin, an opioid pentapeptide, was more effective than AZT alone in inhibiting disease progression due to murine retrovirus infections. In order to study the mechanism(s) by which Met-ENK mediates-antiviral effects, when used in combination with AZT in Friend leukemia virus infected mice, an in vitro focus forming assay was used. AZT at 1 ng/ml inhibited FLV replication by 30-50% in the susceptible Mus dunni cell line. By contrast, the immunostimulatory neuropeptide, Met-ENK, displayed no direct inhibition of viral replication. This suggests that Met-ENK does not have any direct anti-retroviral activity. Subsequent testing of Met-ENK in the presence of AZT showed no ability of this peptide to promote inhibition of viral replication due to AZT. By contrast, in the presence of mouse spleen cells, as a source of lymphocytes, in vitro combination treatments using AZT and Met-ENK reduced FLV replication by 67%, compared to 47% using AZT alone. The inhibition due to Met-ENK was abrogated when spleen cells were pretreated with naloxone, an opioid antagonist. Therefore, we conclude that Met-ENK effects are mediated via opioid receptors on spleen cells and that the observed anti-FLV activity is dependent on the use of Met-ENK stimulated spleen cells in combination with AZT.

Methionine enkephalin combined with AZT therapy reduce murine retrovirus-induced disease.

AZT (7.5 or 15 mg/kg/dose) and the neuropeptide methionine enkephalin (Met-ENK, 1 or 3 mg/kg/dose) were used in a combined protocol for therapy of established murine retroviral infection. In both models used, Friend virus leukemia (FV) and BM5 complex (lymphadenopathy and immune deficiency), the drug combination was able to reduce mortality and splenomegaly. While increasing mean survival time of those animals that did not survive infection by FV, when compared to infected control mice or mice treated with AZT alone, Met-ENK used alone at 1 and 3 mg/kg/mouse had no effect in reducing morbidity or mortality due to either virus. This suggested that Met-ENK had no direct antiviral effect at the concentrations used. In fact, mice treated with either single drug therapy or the combination still yielded virus in their spleen, even when splenomegaly was absent. The data suggest that Met-ENK, which has been reported to be immunostimulatory, acts in combination to improve the efficacy of AZT in reducing progression of disease in murine retrovirus models for human AIDS.

Activation of the lipoxygenase pathway in the methionine enkephalin induced respiratory burst in human polymorphonuclear leukocytes.

In comparative studies of f-met-Leu-Phe (FMLP) and methionine enkephalin (ME) induced polymorphonuclear leukocyte (PMNL) stimulation the following results were obtained: (i) both FMLP and ME increased the intracellular killing (IK) capability of human PMNLs probably through NADPH oxidase activation, (ii) the ME-induced respiratory burst (RB) differed from the chemotactic peptide FMLP-triggered superoxide generation because the former was not accompanied by the activation of the glutathione system and the duration of the superoxide production was prolonged. The reaction was dependent on lipoxygenation, was potentiated by indomethacin (IM) and was inhibited by nordihidro-guairetic acid (NDGA), (iii) both 14C-arachidonic acid (14C-AA) release and leukotriene B4 (LTB4) synthesis of ME-treated PMNLs were elevated as compared to those of FMLP triggered cells. Our results suggest that lipoxygenation and even an increased LTB4 synthesis are involved in the ME-induced RB of leukocytes.

Psychoneuroimmunology, new approaches to neurobehavioral testing.

A review of the literature in the field of psychoneuroimmunology was reported by Robert Ader [1]. Basically, three research approaches have been used to demonstrate a relationship between central sites within the brain that have modulatory influences on the immune system. These include: (1) both the electrical stimulation and surgical lesioning of nuclei in the hypothalamus; (2) traditional behavioral conditioning; and (3) behavioral stress studies. The mechanisms of action for the psychoneuroimmunologic response appear to be focused in three areas: (1) the autonomic nervous system, where direct anatomical connections from the brain to the immune system have been recently demonstrated; (2) the endocrine system where the individual hormones have direct and indirect effects on the immune system; and (3) enkephalins-endorphins in the brain, pituitary, and adrenals that also have direct and indirect effects on the immune system. Examples of drug effects on the above complex systems will be centered on morphine and the enkephalins-endorphins with reference to drugs of abuse, food additives, and cosmetics.

Neuropeptides: conductors of the immune orchestra.

There is increasing evidence for a bidirectional communications system between the immune system and the brain. Many of the substances involved in this communication appear to be neuropeptides. These findings have given biochemical validity to the clinical and epidemiological studies that have suggested that psychosocial factors can modulate the response to infections and neoplasms.

Enhancement of host resistance to viral and tumor challenge by treatment with methionine-enkephalin.

Host resistance to disease is dependent upon a number of factors. Recent evidence indicates that natural killer (NK) cells play an important role in resistance to both neoplastic and virally induced disease. Treatment of C57Bl/6 mice with methionine-enkephalin (1, 3, 10, or 30 mg/kg body weight) results in significant increases in NK activity of splenic lymphocytes 20 hours after injection of the enkephalin. Enkephalin treatment also enhances host resistance. The short-term survival of A/J female mice after HSV-2 infection was significantly increased by daily subcutaneous injections (3 mg/kg body weight) of methionine-enkephalin. Similarly, daily doses of 50 micrograms of methionine-enkephalin for 7 to 14 days inhibit the local subcutaneous tumor growth of B15 melanoma in C57Bl/6 mice.

Possible correction of defective polymorphonuclear cell functions in type-2 diabetes mellitus by met-enkephalin.

In vitro pretreatment with Met-Enk of human PMNLs obtained from patients suffering from type-2 diabetes mellitus resulted in the normalization of the ROS generating system. It is assumed that Met-Enk has a modulating effect on the arachidonic acid metabolism, with a consecutive increase of the LTB4 release.

Neuroimmunomodulation with enkephalins: in vitro enhancement of natural killer cell activity in peripheral blood lymphocytes from cancer patients.

To initiate investigations into the effects of enkephalins on immune function in cancer patients, the effect of methionine-enkephalin and leucine-enkephalin on natural killer (NK) cell activity in isolated peripheral blood lymphocytes from cancer patients was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in NK cell activity. At effector:target cell ratios of 100:1, 33:1 and 11:1 leucine-enkephalin significantly (p less than 0.05) enhanced NK activity at dilutions of 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml. Similar results were obtained with methionine-enkephalin with the exception that the 10(-6) dilution gave insignificant changes at both the 33:1 and 11:1 cell ratios. The results indicate a difference in dose response to both enkephalins between lymphocytes from cancer patients and normal volunteers.

Effect of methionine-enkephalin plus ZnCl2 on active T cell rosettes.

Methionine-enkephalin (Met-Enk) and ZnCl2 in combination enhances active T cell rosette formation of human peripheral blood lymphocytes to a greater degree than either of the agents used separately. Enhancement of rosette formation by Met-Enk plus ZnCl2 was not inhibited by the opiate receptor antagonist naloxone but was completely blocked by the zinc chelator 1,10-phenanthroline. The results suggest a relationship between zinc and Met-Enk and that zinc may be a modulator of enkephalin binding and function in the immune system as well as the nervous system.

Enkephalins: immunomodulators.

Our original studies of the enkephalins were centered on behavioral stress and brain dopaminergic interactions. More recently we discovered the enkephalins to be immunomodulators as evidenced by their enhancement effects on lymphocyte blastogenesis in mice, increases in the sizes of the thymus or spleen in rodents, and prolongation of survival of BDF1 mice inoculated with attentuated L1210 cells. Finally, in studies of human blood samples from both normal volunteers and cancer patients, the enkephalins were demonstrated to stimulate active T cell rosettes and natural killer cell activities (in vitro). These studies support our hypothesis that, in stress, the enkephalins modulate the effects of steroid hormones on the immune system.

Neuroimmunomodulation with enkephalins: effects on thymus and spleen weights in mice.

The subcutaneous injection of either methionine-enkephalin or leucine-enkephalin in BDF1 mice resulted in a significant increase in thymus weight and a significant decrease in spleen weight. This study further supports earlier findings that the enkephalins are neuroimmunomodulators.

Neuroimmunomodulation with enkephalins: enhancement of human natural killer (NK) cell activity in vitro.

To further define the effects of enkephalins on immune function, the effect of methionine-enkephalin and leucine-enkephalin on natural killer cell (NK) activity in isolated human peripheral blood lymphocytes was investigated. Incubation of lymphocytes with either enkephalin resulted in significant increases in natural killer cell activity. At effector:target cell ratios of 11:1 methionine-enkephalin significantly (P less than 0.05) enhanced NK activity at dilutions of 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml, while leucine-enkephalin significantly (P less than 0.05) enhanced NK activity at dilutions of 10(-4), 10(-6), 10(-8), 10(-10), and 10(-14) mg/ml. Cells from individuals with low NK activity showed greater percentage increases in NK activity following enkephalin than did cells from individuals with high NK activity.