The immunomodulation mediated by a delta-opioid receptor for [Met(5)]-enkephalin in oyster Crassostrea gigas.

Opioid receptors (OR) are a group of G protein-coupled receptors with opioids as ligands, which play an important role in triggering the second messengers to modulate immune response in vertebrate immunocytes. In the present study, the full length cDNA of a homologue of δ-opioid receptor (DOR) for [Met(5)]-enkaphalin was cloned from oyster Crassostrea gigas (designated as CgDOR), which was 1104 bp encoding a peptide of 367 amino acids containing a conserved 7tm_1 domain. After the stimulation of [Met(5)]-enkephalin, the concentration of second messengers Ca(2+) and cAMP in the HEK293T cells decreased significantly (p <0.05) with the expression of CgDOR. However, this trend was reverted with the addition of DOR antagonist BNTX. The CgDOR transcripts were ubiquitously detected in the tested tissues including haemocytes, gonad, mantle, kidney, gill, adductor muscle and hepatopancreas, with the highest expression level in the hepatopancreas. After LPS stimulation, the expression level of CgDOR mRNA began to increase (4.05-fold, p <0.05) at 6 h, and reached the highest level (5.00-fold, p <0.05) at 12 h. Haemocyte phagocytic and antibacterial activities increased significantly after [Met(5)]-enkephalin stimulation, whereas the increase was repressed with the addition of DOR antagonist BNTX. These results collectively suggested that CgDOR for [Met(5)]-enkephalin could modulate the haemocyte phagocytic and antibacterial functions through the second messengers Ca(2+) and cAMP, which might be requisite for pathogen elimination and homeostasis maintenance in oyster.

Immunotherapy of cancer via mediation of cytotoxic T lymphocytes by methionine enkephalin (MENK).

The aim of this study was to investigate the immunological mechanisms by which synthetic methionine enkephalin (MENK) exerts therapeutic effects on tumor growth. Our findings in vivo or in vitro show that MENK treatment either in vivo or in vitro could up-regulate the percentages of CD8+T cells, induce markers of activated T cells, increased cytotoxic activity against mouse S180 tumor cells and increase secretion of IFNγ. In addition, the adoptively transferred CD8+T cells, after either in vitro or in vivo treatment with MENK, result in significantly increased survival of S180 tumor-bearing mice and significant shrinkage in tumor growth. Opioid receptors are detected on normal CD8+T cells and exposure to MENK leads to increased expression of opioid receptors. Interaction between MENK and the opioid receptors on CD8+T cells appears to be essential for the activation of CTL, since the addition of naltrexone (NTX), an opioid receptor antagonist, significantly inhibits all of the effects of MENK. The evidence obtained indicates that the MENK-induced T cell signaling is associated with a significant up-regulation of Ca2+ influx into the cytoplasm and the translocation of NFAT2 into nucleus, and these signaling effects are also inhibited by naltrexone.

Pain inhibition by blocking leukocytic and neuronal opioid peptidases in peripheral inflamed tissue.

Inflammatory pain can be controlled by endogenous opioid peptides. Here we blocked the degradation of opioids in peripheral injured tissue to locally augment this physiological system. In rats with hindpaw inflammation, inhibitors of aminopeptidase N (APN; bestatin) or neutral endopeptidase (NEP; thiorphan), and a dual inhibitor, NH(2)-CH-Ph-P(O)(OH)CH(2)-CH-CH(2)Ph(p-Ph)-CONH-CH-CH(3)-COOH (P8B), were applied to injured paws. Combined bestatin (1.25-5 mg)/thiorphan (0.2-0.8 mg) or P8B (0.0625-1 mg) alone elevated mechanical nociceptive thresholds to 307 and 227% of vehicle-treated controls, respectively. This analgesia was abolished by antibodies to methionine-enkephalin, leucine-enkephalin, and dynorphin A 1-17, by peripherally restricted and by selective µ-, δ-, and κ-opioid receptor antagonists. Flow cytometry and photospectrometry revealed expression and metabolic activity of APN and NEP on macrophages, granulocytes, and sciatic nerves from inflamed tissue. Radioimmunoassays showed that inhibition of leukocytic APN and NEP by bestatin (5-500 µM)/thiorphan (1-100 µM) combinations or by P8B (1-100 µM) prevented the degradation of enkephalins. Blockade of neuronal peptidases by bestatin (0.5-10 mM)/thiorphan (0.1-5 mM) or by P8B (0.1-10 mM) additionally hindered dynorphin A 1-17 catabolism. Thus, leukocytes and peripheral nerves are important sources of APN and NEP in inflamed tissue, and their blockade promotes peripheral opioid analgesia.

Immune modulation in response to stress and relaxation.

Traditional medical science has kept the mind separate from the body. Recently people realize the effect of mind on health and psychoneuroimmunology is the new evolved science that describes the interactions between psyche and soma. In this review through a typical psycho-neuro-endocrino-immune network the effects of psychological stress (acute, brief naturalistic and chronic) and relaxation on immune modulation has been shown. From this network Corticotrophin Releasing Factor (CRF), Adrenocorticotrophic Hormone (ACTH), Glucocorticoids (GC), alpha-endorphin and Met-enkephalin are found as important endocrine components and T cells, B cells, monocytes/macrophages, Natural Killer (NK) cells and their cytokines that is Tumor Necrosis Factor-alpha (TNF-alpha), Interferon Gamma (IFN-alpha) and interleukins such as IL-1, IL-2, IL-4, IL-6, IL-10, IL-12 etc. are found as important immune components. Finally, it has been shown that, acute, brief naturalistic and chronic stress have different immune modulatory activities which are harmful to one's homeostasis and relaxation can help to maintain that homeostasis.

Methionine-enkephalin secreted by human colorectal cancer cells suppresses T lymphocytes.

The role of methionine-enkephalin (MENK) as an immunomodulator in colorectal carcinomas (CRC) was examined. MENK was produced in CT26, IEC6A, Colo320, and HT29 CRC cell lines but not in IEC6 intestinal cells. MENK secretion was associated with tumorigenicity and metastasis of CRC cells in syngeneic rodent models. The MENK concentration in subcutaneous tumors of CT26 and IEC6A CRC cells exhibited an inverse correlation with the number of tumor-infiltrating T lymphocytes. MENK inhibited the growth of MOLT-4 T-lymphoblastic cells in a dose-dependent manner. Furthermore, it increased the phosphorylation level of c-Jun N-terminal kinase and induced apoptosis in MOLT-4 cells. MENK-induced apoptosis was abrogated by a c-Jun N-terminal kinase inhibitor. Immunohistochemical analysis revealed moderate to strong expression of MENK in 33 (54%) of 61 CRC. MENK expression was associated with Dukes' staging, nodal metastasis, and liver metastasis. The MENK concentration in tumor tissues was higher in Dukes' C cases than in Dukes' B cases. MENK expression was associated with tumor-infiltrating T lymphocytes, especially those belonging to the CD4(+) subset. These findings suggest that MENK secreted by CRC cells caused escape of the host from the effects of immunity.

The involvement of spinal bovine adrenal medulla 22-like peptide, the proenkephalin derivative, in modulation of nociceptive processing.

Bovine adrenal medulla 22 (BAM22), one of the cleavage products of proenkephalin A, possesses high affinity for opioid receptors and sensory neuron-specific receptor (SNSR). The present study was designed to examine the expression of BAM22 in the spinal cord and dorsal root ganglion (DRG) of naive rats as well as in a model of inflammation. BAM22-like immunoreactivity (BAM22-IR) was expressed in fibers in the spinal cord, with high density seen in lamina I in naïve rats. The expression of BAM22-IR in the superficial laminae was greatly reduced following dorsal rhizotomy. BAM22-IR was also located in 19% of DRG cells, mainly in the small- and medium-sized subpopulations. Following injection of complete Freund's adjuvant (CFA) in the hindpaw, the expression of BAM22-IR in the superficial laminae of the spinal cord and small-sized DRG neurons on the ipsilateral side was markedly increased. Double labeling showed that the Fos-positive nucleus was surrounded by BAM22-IR cytoplasm in the spinal dorsal horn neurons or closely associated with BAM22-IR fibers in the superficial laminae. Furthermore, CFA-induced mechanical allodynia in the inflamed paw was potentiated by intrathecal administration of anti-BAM22 antibody. Together, these results demonstrate for the first time that BAM22-like peptide is mainly located in the superficial laminae of the spinal cord and mostly originates from nociceptive DRG neurons. BAM22 could thus act as a ligand for presynaptic opioid receptors and SNSR. Our study also provides evidence suggesting that BAM22 plays a role in the modulation of nociceptive processing at the spinal level under normal and inflammatory conditions.

Overexpression of the opioid growth factor receptor downregulates cell proliferation of human squamous carcinoma cells of the head and neck.

The opioid growth factor (OGF) is a constitutively expressed negative growth regulator whose action is mediated by the OGF receptor (OGFr). The OGF-OGFr axis tonically regulates the growth of human squamous cell carcinoma of the head and neck (SCCHN). To examine the repercussions of amplifying OGFr in SCCHN, constructs were prepared to overexpress OGFr in SCC-1 cells; six clonal lines were examined. OGFr binding assays of clonal cells revealed a 2.4- to 8.4-fold increase in binding capacity compared to wild-type (WT) and empty vector (EV) controls; binding affinity was comparable in all groups. OGFr protein expression, as measured by quantitative immunohistochemistry and Western blotting, was increased in clonal cell lines compared to controls. Under standard growth conditions the cell number of the OGFr clonal lines was reduced by 11 to 68% from the WT group, and doubling times were 7 to 67% longer. Addition of exogenous OGF further reduced (8 to 37%) cell growth of the clonal lines. Depletion of endogenous OGF with antibodies to this peptide increased growth 2-fold in cells amplifying OGFr relative to increases of 32 and 34% for the WT and EV groups, respectively. DNA synthesis of cells overexpressing OGFr was reduced from the WT group by 46 to 75%. These data indicate that the OGF receptor is integral to cell replication of SCCHN, and support treatment modalities that amplify OGFr in order to decrease the growth of these neoplasias.

Influence of pain treatment by epidural fentanyl and bupivacaine on homing of opioid-containing leukocytes to surgical wounds.

Endogenous opioids released from leukocytes extravasating into injured tissue can interact with peripheral opioid receptors to inhibit nociception. Animal studies have shown that the homing of opioid-producing leukocytes to the injured site is modulated by spinal blockade of noxious input. This study investigated whether epidural analgesia (EDA) influences the migration of beta-endorphin (END) and/or met-enkephalin (ENK)-containing leukocytes into the subcutaneous wound tissue of patients undergoing abdominal surgery. In part I patients received general anesthesia combined either with intra- and postoperative EDA (with bupivacaine and fentanyl) or with postoperative patient controlled intravenous analgesia (PCIA; with the opioid piritramide). In part II patients received general anesthesia combined with either epidural fentanyl or bupivacaine which was continued postoperatively. Samples of cutanous and subcutanous tissue were taken from the wound site at the beginning, at the end and at various times after surgery, and were examined by immunohistochemistry for the presence of END and ENK. We found that (i) epidural bupivacaine, fentanyl and PCIA provided similar and clinically acceptable postoperative pain relief; (ii) compared to PCIA, epidural bupivacaine or fentanyl did not change the gross inflammatory reaction within the surgical wound; (iii) opioid-containing leukocytes were almost absent in normal subcutaneous tissue but migrated to the inflamed wound tissue in ascending numbers within a few hours, reaching a peak at about 24 h after surgery; (iv) compared to PCIA, EDA resulted in significantly decreased homing of END-containing leukocytes to the injured site at 24 h after surgery; and (v) the magnitude of this decrease was similar regardless of the epidural medication. These findings suggest that nociceptive but not sympathetic neurons are primarily involved in the attraction of opioid-containing leukocytes during early stages of inflammation.

In vitro modulation of cytokine expression by enkephalin-derived peptides.

OBJECTIVE: We have previously reported that low doses of [Met(5)]-enkephalin (YGGFM, met-enkephalin) and two of its derivatives (YGG and YG) enhanced and accelerated delayed-type hypersensitivity responses while much higher doses of these compounds suppressed these reactions. Since the underlying mechanisms by which this and other immunomodulatory effects occur have not been established, this report explores the in vitro modulation of Th1 and Th2 cytokine expression by these peptides. METHODS: Murine splenocytes were stimulated with suboptimal concentrations of concanavalin A (ConA) in serum-free medium in the absence or presence of met-enkephalin, YGG, YG, [des-Tyr(1)]-met-enkephalin (GGFM), [D-Ala(2)], [D-Met(5)]-enkephalin or tyrosine (Y). Cell-conditioned supernatants were assayed for interferon-gamma (IFN-gamma), interleukin (IL)-2 and IL-4. Relative IFN-gamma and IL-2 mRNA levels were assessed by reverse transcription-polymerase chain reaction. The enhancing and suppressive effects of met-enkephalin and YG on IFN-gamma production were also tested in the presence of naloxone (Nx). RESULTS: Met-enkephalin, YGG and YG modulated the in vitro production of IFN-gamma in a biphasic manner: stimulation at low doses and inhibition at high doses. At higher concentrations, met-enkephalin and YG also suppressed the production of IL-2 (type 1) and IL-4, a type 2 cytokine. Nx reversed the enhancing effect of met-enkephalin on IFN-gamma production without affecting its suppressive action or any of the immunomodulating effects of YG. The degradation-resistant analog [D-Ala(2)], [D-Met(5)]-enkephalin enhanced IFN-gamma production but did not suppress it. CONCLUSIONS: YG, the minimal molecular requirement for enhancement and suppression of immune responses by these metabolites, appears to mediate exclusively an across-the-board suppression via low-affinity, nonclassical, nonopioid receptors.

Modulation of delayed-type hypersensitivity responses in hairless guinea pigs by peptides derived from enkephalin.

Although opioid peptides such as methionine (met)-enkephalin have been previously shown to enhance or suppress immune responses, few studies in animal models have addressed the immunomodulatory activity of their metabolic derivatives. Hairless (IAF/HA-HO) guinea pigs immunized with Freund's complete adjuvant containing Mycobacterium tuberculosis and repeatedly skin tested with purified protein derivative of tuberculin (PPD) display high levels of stable delayed-type hypersensitivity (DTH) to PPD. Met-enkephalin (YGGFM) and two of its metabolites (YGG, YG) enhanced and accelerated PPD-elicited DTH inflammatory reactions when injected together with elicitor in these animals. At 24 h, 5 x 10(-3) pmol met-enkephalin significantly enhanced DTH responses by 30% over PPD alone, while 5 x 10(-5) pmol of YGG and 5 x 10(-9) pmol of YG significantly enhanced these responses by 62 and 32%, respectively. At much higher doses (5 x 10(3) pmol), met-enkephalin and its metabolites significantly suppressed DTH reactions by 25-32%. Tyrosine and glycine had no effect on PPD-elicited DTH. All DTH reactions (control, enhanced, suppressed) displayed typical perivascular mononuclear cell infiltrates. We conclude that the immunoactivity of met-enkephalin resides in its first two amino acids and suggest that cleavage of enkephalin molecules to YG occurs in serum and/or on the cell surface.

Effects of endogenous and synthetic opioid peptides on neutrophil function in vitro.

BACKGROUND: Opioid peptides released from immunocytes during inflammation and stress in critically ill patients are associated with an altered immune response. Moreover, concentrations of opioid peptides are increased in peripheral blood and at the sites of inflammatory reactions. METHODS: Using flow cytometric assay of whole human blood, we investigated direct effects of endogenous and synthetic opioid peptides on surface expression of complement receptors CD35 and CD11b/CD18 and Fcã receptor III CD16, and superoxide anion generation of neutrophils. RESULTS: The endogenous opioid peptides beta-endorphin(1-31) and met-enkephalin, representing the N-terminal fragment of beta-endorphin(1-31), and the synthetic delta opioid receptor agonists D-Ala(2)-D-Leu(5)-enkephalin and D-Pen(2)-enkephalin produced concentration-dependent stimulation of neutrophil activity. Incubation with met-enkephalin 10(-7) M or beta-endorphin(1-31) 10(-7) M led to an increase in receptor expression of up to 10% (met-enkephalin) and 15% (beta-endorphin(1-31)). After incubation with D-Ala(2)-D-Leu(5)-enkephalin or D-Pen(2/5)-enkephalin, receptor expression was increased by up to 30%. This correlated with concentration-dependent stimulation of the production of reactive oxygen intermediates, as shown by an increase of up to 40% in oxidative burst activity. All effects were abolished after preincubation with naloxone or with the selective delta opioid antagonist naltrindole, whereas the selective micro receptor antagonist d-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH(2) showed only partial inhibitory effects. CONCLUSIONS: Our data suggest a delta opioid receptor-mediated stimulatory effect on neutrophil function. beta-Endorphin(27-31), the C-terminal fragment of beta-endorphin(1-31), did not alter neutrophil function, indicating that beta-endorphin(1-31) mediates its effect on neutrophils via the N-terminal fragment. This study may contribute to a better understanding of neuroimmune interaction.

Met-enkephalin immunoreactive neurons recruited by acute stress are innervated by axon terminals immunopositive for tyrosine hydroxylase and dopamine-alpha-hydroxylase in the anterolateral division of bed nuclei of the stria terminalis in the rat.

The bed nuclei of the stria terminalis (BST) are highly heterogeneous forebrain structures, which play a central role in the regulation/modulation of stress responses. Studies using the inducible immediate early gene c-fos as a marker of activated neurons have demonstrated significant stress-induced neuronal activation in this limbic region. The BST also exhibit a dense network of dopamine and noradrenaline immunoreactive (ir) axon terminals. These catecholaminergic projections from various brainstem sources to the BST play an important role in a neurochemically mediated coordination of stress responses. In the anterolateral division of bed nuclei of the stria terminalis, the distribution of several Met-enkephalin immunopositive perikarya overlaps with that of catecholaminergic axon terminals. Both monoaminergic and enkephalinergic structures have been postulated to play a role in the regulation/modulation of the central regulatory pathways of endocrine, behavioural and physiological responses during stress. Therefore the aims of this study were: (i). to study the possible involvement of dopaminergic fibre terminals in stress-induced activation of BST perikarya; (ii). to investigate whether Met-enkephalin-immunoreactive neurons are recruited by acute volumen/osmotic challenge; and (iii). to demonstrate synaptic interactions between Met-enkephalin-ir neurons and fibre terminals immunopositive for dopamine or noradrenaline in the anterolateral division of the BST. From the results of this study we can conclude that depletion of dopamine in fibre terminals completely abolished stress-induced activation of perikarya in the anterolateral division of BST. Furthermore, the innervation of stress-induced Met-enkephalin-ir perikarya by dopaminergic fibre terminals in the oval nucleus of BST was demonstrated, whereas noradrenergic axons contacted enkephalinergic structures in the fusiform and subcomissural nuclei of BST. These interactions can be central in the modulatory control of the major stress regulatory pathway, the limbic hypothalamo-pituitary-adrenal axis.

Methionine-enkephalin-and Dynorphin A-release from immune cells and control of inflammatory pain.

We have previously shown that beta-endorphin (END) is contained and released from memory-type T-cells within inflamed tissue and that it is capable to control pain (J Clin Invest 100(1) (1997) 142). Methionine-enkephalin (MET) and Dynorphin-A (DYN) are endogenous opioids with preference for delta- and kappa-opioid receptors, respectively. Both MET and DYN are produced and contained within immune cells. The goal of this study was to determine the release characteristics of MET and DYN in a rat model of localized hindpaw inflammation and to examine the antinociceptive role of MET and DYN in a Freund's adjuvant induced model of inflammatory pain. We found that corticotropin-releasing factor (CRF) can stimulate the release of both MET and DYN from lymphocytes. This release is dose-dependent and reversible by the selective CRF antagonist alpha-helical-CRF. Furthermore, CRF (1.5 ng) produces analgesia when injected into the inflamed paw, which is reversible by direct co-administration of antibodies to MET. Lymphocyte content of MET was 7.0+/-1.4 ng/million cells, whilst DYN content was ~30-fold lower. Both END and DYN, but not MET, were released by IL-1. Consistently, IL-1 produced peripheral analgesic effects which were not reversed by antibodies to MET. These results indicate that both MET and DYN play a role in peripheral analgesia but have different characteristics of release. These studies further support a role of the immune system in the control of inflammatory pain. This may be particularly important in patients suffering from compromised immune systems as with cancer and AIDS.

Chronic prenatal exposure to carbon monoxide results in a reduction in tyrosine hydroxylase-immunoreactivity and an increase in choline acetyltransferase-immunoreactivity in the fetal medulla: implications for Sudden Infant Death Syndrome.

Maternal cigarette smoking during pregnancy is associated with a significantly increased risk of Sudden Infant Death Syndrome (SIDS). This study investigated the effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, on the neuroglial and neurochemical development of the medulla in the fetal guinea pig. Pregnant guinea pigs were exposed to 200 p.p.m CO for 10 h per day from day 23-25 of gestation (term = 68 days) until day 61-63, at which time fetuses were removed and brains collected for analysis. Using immunohistochemistry and quantitative image analysis, examination of the medulla of CO-exposed fetuses revealed a significant decrease in tyrosine hydroxylase-immunoreactivity (TH-IR) in the nucleus tractus solitarius, dorsal motor nucleus of the vagus (DMV), area postrema, intermediate reticular nucleus, and the ventrolateral medulla (VLM), and a significant increase in choline acetyltransferase-immunoreactivity (ChAT-IR) in the DMV and hypoglossal nucleus compared with controls. There was no difference between groups in immunoreactivity for the m2 muscarinic acetylcholine receptor, substance P- or met-enkephalin in any of the medullary nuclei examined, nor was there evidence of reactive astrogliosis. The results show that prenatal exposure to CO affects cholinergic and catecholaminergic pathways in the medulla of the guinea pig fetus, particularly in cardiorespiratory centers, regions thought to be compromised in SIDS.

Immunocytochemical characterization of quisqualic acid- and N-methyl-D-aspartate-induced excitotoxicity in the retina of chicks.

A single, large dose of N-methyl-D-aspartate (NMDA) or quisqualic acid (QA) injected into the chick eye has been shown previously to destroy many retinal amacrine cells and to induce excessive ocular growth accompanied by myopia. The purpose of this study was to identify distinct populations of retinal cells, particularly those believed to be involved in regulating ocular growth, that are sensitive to NMDA or QA. Two pmol of NMDA or 0.2 micromol of QA were injected unilaterally into eyes of 7-day-old chicks, and retinas were prepared for observation 1, 3, or 7 days later. Retinal neurons were identified by using immunocytochemistry, and cells containing fragmented DNA were identified by 3'-nick-end labelling in frozen sections. NMDA and QA destroyed many amacrine cells, including those immunoreactive for vasoactive intestinal polypeptide, Met-enkephalin, and choline acetyltransferase, but they had little effect upon tyrosine hydroxylase-immunoreactive cells. Other cells affected by both QA and NMDA included those immunoreactive for glutamic acid decarboxylase, gamma-aminobutyric acid, parvalbumin, serotonin, and aminohydroxy methylisoxazole propionic acid (AMPA) receptor subunits GluR1 and GluR2/3. Cells largely unaffected by QA or NMDA included bipolar cells immunoreactive for protein kinase C (alpha and beta isoforms) and amacrine cells immunoreactive for glucagon. DNA fragmentation was detected maximally in many amacrine cells and in some bipolar cells 1 day after exposure to QA or NMDA. We propose that excitotoxicity caused by QA and NMDA induces apoptosis in specific populations of amacrine cells and that these actions are responsible for the ocular growth-specific effects of QA and NMDA reported elsewhere.

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.

Pretreatment of human peripheral blood lymphocytes with interleukin-2 or dexamethasone does not alter their response to Met-Enkephalin in a NK-cytotoxic assay.

The effect of Met-Enkephalin (MENK; 10(-12) - 10(-8) M) on NK-activity of peripheral blood lymphocytes (PBL) after in vitro treatment (18 h, 37 degrees C) was examined in 30 young, healthy male donors. In the group as a whole (n = 30), no significant effect of MENK was detected. At the individual level, 18 of 30 donors (60%) responded to MENK either by mild enhancement (up to 8%, 8 responders), or by mild attenuation (up to 12%, 10 responders) of the basal NK-activity. The effect of MENK was donor-related regarding the dose-response, E/T ratio, and direction of MENK action. The influence of pretreatment of PBL (1 h) with either graded doses of interleukin-2 (IL-2; 3, 25, 50 U/ml) or dexamethasone (Dex; 2.5 x 10(-9), 2.5 x 10(-8), 2.5 x 10(-7) M), on the effect of MENK was also tested. The idea was that pretreatment of PBL would result in predictable, and/or stronger response to MENK. In the group as a whole again no significant effect of MENK was detected on the NK-activity of PBL prestimulated by IL-2 (n = 16), or inhibited by Dex (n = 12). Further, pretreatment of PBL with IL-2/Dex did not significantly alter the intensity of modulation by MENK, which was generally mild. The data obtained have shown that pretreatment of PBL with IL-2 or Dex, regardless of their concentrations, did not significantly alter the frequency of responders to MENK being 50%, 62.5% and 64.3% with 3, 25 or 50 U/ml IL-2, respectively, and 50% with all concentration of Dex used, as compared to that observed with resting PBL (60%). However, at the individual level physiological concentrations of MENK (10(-12) - 10(-9) M) induced enhancement or/and attenuation of the NK-activity pretreated with IL-2/Dex, respectively. The effect of MENK at the individual level was donor-related regarding the dose-response, E/T ratio, and direction of MENK action. Thus, pretreatment of PBL with graded concentrations of IL-2/Dex did not alter the effect of MENK on NK-activity, regarding the frequency and intensity, as well as the direction of modulation: it remained bidirectional, of low intensity, and independent of the grade of PBL preactivation/inhibition, therefore unpredictable.

Immunomodulatory activity of met-enkephalin and its two potent analogs.

The effects of Met-enkephalin (Met-Enk), a delta receptor binding opioid peptide, and its more stable synthetic analogs, Tyr-D-Ala-Gly-MePhe-Met-NHC3H7-iso (1), Tyr-D-Ala-Gly-MePhe-Gly-NHC3H7-iso (2) and Tyr-D-Ala-Gly-MePhe-Gly-NHCH2C6H5 (3), on human T-cell transformation and natural killer (NK) cell cytotoxicity have been evaluated. Analogs 1 and 2 have been found to be as potent as Met-Enk in stimulating T-cell transformation and augmenting NK cell cytotoxicity. Analog 3 had no effect on T-cell transformation and NK cell cytotoxicity. Proliferative response was measured by 3H-thymidine uptake after 5 days of incubation. The kinetics of the T-cell transformation response (peak 5th day) is similar to those for in vitro T-cell responses to specific antigens rather than via polyclonal activation.

Interleukin 1 beta and corticotropin-releasing factor inhibit pain by releasing opioids from immune cells in inflamed tissue.

Local analgesic effects of exogenous opioid agonists are particularly prominent in painful inflammatory conditions and are mediated by opioid receptors on peripheral sensory nerves. The endogenous ligands of these receptors, opioid peptides, have been demonstrated in resident immune cells within inflamed tissue of animals and humans. Here we examine in vivo and in vitro whether interleukin 1 beta (IL-1) or corticotropin-releasing factor (CRF) is capable of releasing these endogenous opioids and inhibiting pain. When injected into inflamed rat paws (but not intravenously), IL-1 and CRF produce antinociception, which is reversible by IL-1 receptor antagonist and alpha-helical CRF, respectively, and by the immunosuppressant cyclosporine A. In vivo administration of antibodies against opioid peptides indicates that the effects of IL-1 and CRF are mediated by beta-endorphin and, in addition, by dynorphin A and [Met]enkephalin, respectively. Correspondingly, IL-1 effects are inhibited by mu-, delta-, and kappa-opioid antagonists, whereas CRF effects are attenuated by all except a kappa-antagonist. Finally, IL-1 and CRF produce acute release of immunoreactive beta-endorphin in cell suspensions freshly prepared from inflamed lymph nodes. This effect is reversible by IL-1 receptor antagonist and alpha-helical CRF, respectively. These findings suggest that IL-1 and CRF activate their receptors on immune cells to release opioids that subsequently occupy multiple opioid receptors on sensory nerves and result in antinociception. beta-Endorphin, mu- and delta-opioid receptors play a major role, but IL-1 and CRF appear to differentially release additional opioid peptides.

Modulation of lipopolysaccharide-induced production of cytokines by methionine-enkephalin.

In the present study, we have examined the effect of opioid peptide methionine enkephalin (MENK) on production of factors with interleukin-1 (IL-1) and tumor necrosis factor (TNF) activity by mouse peritoneal macrophages and assessed whether modification in the production of those cytokines could be related to alteration of phagocytosis by MENK. None of the MENK concentrations examined altered IL-1 or TNF activity alone. However, peritoneal macrophages co-stimulated with 1 microgram of lipopolysaccharide (LPS) and 10(-10) M MENK potentiated IL-1 activity, compared to LPS alone, but abrogated TNF activity induced by LPS. While MENK alone slightly decreased phagocytosis of sheep red blood cells (SRBC) by mouse peritoneal macrophages, cells simultaneously incubated with 1 microgram of LPS and 10(-10) M MENK had increased phagocytosis compared to LPS alone. Moreover, phagocytosis of SRBC by cells incubated overnight with the supernatant of the respective cell culture was significantly augmented. These results provide additional evidence for the immunoregulatory role of neuropeptides and suggest that the modulatory action of MENK could be mediated, at least in part, through the up-regulation of cytokines, most probably IL-1 and TNF.