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Neuropeptides in the Immune System: Mediators of Stress and Inflammation
Abstract
Neuropeptides and cytokines expressed within the brain and the immune system can act in association with circulating cytokines,
catecholamines, and glucocorticoids to form an elegant bidirectional symmetry of brain–immune system communications. Interactions
between these compounds are critical for the maintenance of homeostasis during immune system activation and during chronic
inflammatory stress. Now that many fundamental principles governing the general anatomy of this network have been established,
a major research challenge lies in elucidation of the functional roles of cytokines in the brain and neuropeptides in immune
tissues. This chapter will focus on immunoneuropeptides, expression of many of which is altered in autoimmune diseases and
also in models of chronic stress associated with inflammation. This has led to the proposition that immunoneuropeptides may
be involved in underlying processes in the development of, and the responses to, acute and chronic inflammation. In support
of this concept, potential for modulation of inflammation in vivo and in vitro has been demonstrated for many neuropeptides
synthesized within immune cells. The purpose of this chapter is to summarize the evidence for involvement of immunoneuropeptides
in disease processes and to discuss potential antiinflammatory therapies that may arise from our increased understanding of
the roles these compounds play in immune responses.
... It should be emphasized that hemorphins, as members of the endogenous protective system of the organism, play a significant role mainly in response to pathophysiological conditions (e.g., stress, inflammation, cancer). In this case, hemorphins, similar to other pleiotropic neuropeptides [62], serve as one of the homeostatic factors that activate compensatory systems within the organism. This effect could be observed as the occurrence of interactions between CRP, opioids and hemorphins in the hypothalamus [5] and pituitary gland [4] in response to stressful conditions [3]. ...
During the last three decades, a variety of different studies on bioactive peptides that are opioid receptor ligands, have been carried out, with regard to their isolation and identification, as well as their molecular functions in living organisms. Thus, in this review, we would like to summarize the present state-of-the art concerning hemorphins, methodological aspects of their identification, and their potential role as therapeutic agents. We have collected and discussed articles describing hemorphins, from their discovery up until now, thus presenting a very wide spectrum of their characteristic and applications. One of the major assets of the present paper is a combination of analytical and pharmacological aspects of peptides described by a team who participated in the initial research on hemorphins. This review is, in part, focused on the analysis of endogenous opioid peptides in biological samples using advanced techniques, description of the identification of synthetic/endogenous hemorphins, their involvement in pharmacology, learning, pain and other function. Finally, the part regarding hemorphin analogues and their synthesis, has been added.
Defense of the vertebrate brain against infection must be accomplished without the generation of potentially harmful inflammation since the brain has a limited capacity to repair damage. Neuropeptides are multifunctional neuronal signaling molecules that regulate physiological processes and behaviors. Recently, their potential roles in modulating immune responses and inflammation have been discovered in mammals. However, the role of neuropeptides in teleost brain immune response mediation is unknown. Herein, the brain transcriptional profiles of economically important fish, Nile tilapia (Oreochromis niloticus), against Streptococcus agalactiae, a common pathogen causing serious meningitis, was first revealed. Twelve cDNA libraries of O. niloticus brain from four treatment time points (0, 6, 24, and 48 h) were constructed. A total of 517 differentially expressed genes (DEGs) were obtained via pairwise comparison of the brain transcriptome data at different time points. KEGG pathway analysis showed that most DEGs were in “neuroactive ligand-receptor interaction” (ko04080). Besides, 37 neuropeptide families with 143 neuropeptide precursor genes (producing 204 different transcripts) were identified. Also, 15 neuropeptide precursor genes (ADCYAP1A, AVP, CCK2, GAL, GNRH3, NPY1, OXT, PDYN, PMCH1, PMCH2, POMC1A, SST1, TAC1, UCN3, and HAMP1) were differentially expressed (P < 0.05) during S. agalactiae infection. Among them, five mature neuropeptides (AVP, OXT, UCN3, SP, and NKA) were prepared and used to determine and characterize the heterogeneous regulation of immune response. Specifically, neuropeptides promoted inflammatory response, immune pathways, and the cAMP–PKA pathway to Lipopolysaccharide (LPS) stimulation. However, the responses to Lipoteichoic acid (LTA) were first promoted and soon reduced with neuropeptides treatment. This study represents the first comprehensive identification of neuropeptides in tilapia, and would contribute to a better understanding of the evolution and immunological roles of neuropeptides in teleosts.
The pig is a biomedical model to study human and livestock traits. Many of these traits are controlled by neuropeptides that result from the cleavage of prohormones by prohormone convertases. Only 45 prohormones have been confirmed in the pig. Sequence homology can be ineffective to annotate prohormone genes in sequenced species like the pig due to the multifactorial nature of the prohormone processing. The goal of this study is to undertake the first complete survey of prohormone and prohormone convertases genes in the pig genome. These genes were functionally annotated based on 35 gene expression microarray experiments. The cleavage sites of prohormone sequences into potentially active neuropeptides were predicted.
We identified 95 unique prohormone genes, 2 alternative calcitonin-related sequences, 8 prohormone convertases and 1 cleavage facilitator in the pig genome 10.2 assembly and trace archives. Of these, 11 pig prohormone genes have not been reported in the UniProt, UniGene or Gene databases. These genes are intermedin, cortistatin, insulin-like 5, orexigenic neuropeptide QRFP, prokineticin 2, prolactin-releasing peptide, parathyroid hormone 2, urocortin, urocortin 2, urocortin 3, and urotensin 2-related peptide. In addition, a novel neuropeptide S was identified in the pig genome correcting the previously reported pig sequence that is identical to the rabbit sequence. Most differentially expressed prohormone genes were under-expressed in pigs experiencing immune challenge relative to the un-challenged controls, in non-pregnant relative to pregnant sows, in old relative to young embryos, and in non-neural relative to neural tissues. The cleavage prediction based on human sequences had the best performance with a correct classification rate of cleaved and non-cleaved sites of 92% suggesting that the processing of prohormones in pigs is similar to humans. The cleavage prediction models did not find conclusive evidence supporting the production of the bioactive neuropeptides urocortin 2, urocortin 3, torsin family 2 member A, tachykinin 4, islet amyloid polypeptide, and calcitonin receptor-stimulating peptide 2 in the pig.
The present genomic and functional characterization supports the use of the pig as an effective animal model to gain a deeper understanding of prohormones, prohormone convertases and neuropeptides in biomedical and agricultural research.
Integrity of the hypothalamo-pituitary-adrenal (HPA) axis is essential to survival of vertebrate species. This neuroendocrine axis functions to coordinate neural, endocrine and immune responses to diverse stressful stimuli that threaten homeostasis. The final products of activation of the HPA axis are the glucocorticoids that exert widespread effects on body functions, including cellular metabolism and immune function. Inappropriate secretion of endogenous glucocorticoids is potentially damaging and may predispose to disease. Homeostatic regulation of the HPA axis is complex and involves coordination of multiple systems of the body, in part mediated by the bi-directional communication network between the brain, endocrine and immune systems. Health and integrity of the individual relies on the appropriate integration of stress signals, including pro-inflammatory messages, generated at central and peripheral sites. Functional balance between pro- and anti-inflammatory mediators is fundamental to the appropriate control of the HPA axis and the prevention of dysregulation in its activity, a characteristic of numerous stress-related disorders including chronic inflammatory disease.
The effect of synthetic LVV-hemorphin-7 and hemorphin-7 on hypothalamo-pituitary-adrenocortical axis activity in response to endotoxin-induced stress was studied. The intraperitoneal (ip) endotoxin (lipopolysaccaride, LPS) (0.5 mg/kg) administration in combination with hemorphin (1 mg/kg) induce significant decrease in plasma corticosterone and modest decrease in plasma levels of tumor necrosis factor-alpha (TNFalpha) in compare with elevated levels of both corticosterone and TNFalpha in plasma of rats received LPS administration alone. Increased activity of calcineurin in both plasma and brain of rats received ip administration of LPS, was recovered under LPS + hemorphin treatment. In two independent proteome analysis, using 2-dimensional fluorescence difference gel electrophoresis and the isotope coded protein label technology, peptidyl-prolyl cis-trans-isomerase A (cyclophilin A) was identified as regulated by hemorphins protein in mouse brain. A therapeutic potential of hemorphins and mechanisms of their homeostatic action in response to endotoxin-induced stress are discussed.
Morphine and the endogenous opioid peptide β-endorphin exert neuromodulatory as well as immunomodulatory effects, which are
transduced by μ-opioid receptors. In this report we show that stimulation with interleukin-4 induces μ-opioid receptor transcripts
in human primary blood cells (T cells and polymorphonuclear leukocytes), immune cell lines (Raji, U-937, and HMEC-1), and
dendritic cells. In nonstimulated immune cells this gene is silent. In addition, μ receptor transcription is up-regulated
by interleukin-4 in cultures of primary rat neurons. Transient transfection experiments in Raji and SH SY5Y neuronal cells
with human and rat reporter gene constructs linked the interleukin-4 effect directly to cis-active μ receptor promoter elements
located at nucleotide −997 on the human gene and nucleotide −727 on the rat gene. The interleukin-4 response elements function
orientation independently. They bind STAT6 transcription factors as shown by electrophoretic mobility shift assays. In the
human gene, a single nucleotide polymorphism within the interleukin-4 response element reduces the trans-activating potential
of this element by 50%, which may affect the phenotype of persons carrying this variation. These findings provide a molecular
basis for understanding bidirectional interactions between the opioid system and the immune system.
Corticotropin-releasing hormone (CRH), the principal regulator of the hypothalamic-pituitary-adrenal axis, is also secreted in peripheral inflammatory sites, where it acts as a local proinflammatory agent. Arthritis-susceptible LEW/N rats have profoundly deficient hypothalamic CRH responses to inflammatory stimuli and other stressors. Arthritis-resistant F344/N rats, on the other hand, have a robust increase in hypothalamic CRH in response to the same stimuli. Contrasting with these hypothalamic CRH responses, we now show that CRH expression is markedly increased in the joints and surrounding tissues of LEW/N rats with streptococcal cell wall- and adjuvant-induced arthritis, whereas it is not increased in similarly treated F344/N rats and is only transiently increased in congenitally athymic nude LEW.rnu/rnu rats. Glucocorticoid treatment suppressed, but did not eliminate, CRH immunoreactivity in the joints of LEW/N rats. CRH mRNA was present in inflamed synovia, as well as in spinal cord, and inflamed synovia also expressed specific CRH-binding sites. We compared CRH expression in inflamed joints with another well-characterized proinflammatory neuropeptide, substance P (SP), and found that SP immunoreactivity paralleled that of CRH. In summary, although LEW/N rats have deficient hypothalamic CRH responses to inflammatory stimuli compared with F344/N rats, they express relatively high levels of CRH at the site of inflammation. Analogous to SP, CRH may be delivered to the inflammatory site by peripheral nerves and/or synthesized at the inflammatory site. These data provide further support for the concept that CRH not only triggers the pituitary-adrenal antiinflammatory cascade, but also functions as an antithetically active local mediator of acute and chronic inflammatory arthritis. These data also illustrate the complex interrelationships of the nervous, endocrine, immune, and inflammatory systems.
We have previously demonstrated low levels of immunoreactive (ir)-beta-endorphin (beta-EP) and ir-ACTH in a subpopulation of mouse spleen macrophages, which is consistent with an involvement of opioid peptides in modulation of immune responses. Gel chromatography studies suggested the presence of an approximately 3.5,000-molecular weight (mol wt) species, putatively beta-EP, an approximately 11.5,000-mol-wt species, putatively beta-lipotropin, and a higher molecular weight species (putative beta-EP precursor, pro-opiomelanocortin (POMC). In this study we have extended our original findings by demonstrating the presence of messenger RNA for POMC by the use of a complementary DNA probe and Northern blot analysis of extracts of mouse and rat spleen. In addition, using high performance liquid chromatography (HPLC), we have shown that the major endorphin species in mouse spleen macrophages is beta-EP1-31, and that there are smaller amounts of each of the acetylated forms, N-acetyl-beta-EP1-16 (alpha-endorphin), N-acetyl-beta-EP1-17 (gamma-endorphin), N-acetyl-beta-EP1-27, and N-acetyl-beta-EP1-31. We interpret these studies as showing that (a) the spleen is an organ of POMC synthesis and that (b) the predominant COOH-terminal product of macrophage POMC is the opiate-receptor active species beta-EP1-31.
Objective. To determine whether endorphin (END) and enkephalin (ENK) modulate excessive synovial cell functions in patients with rheumatoid arthritis (RA). Methods. Effects of leucine-enkephalin (leu-ENK), methionine-enkephalin (met-ENK), and beta -endorphin (END) on proinflammatory cytokine and matrix metalloproteinase (MMP) production by RA synovial cells were analyzed by immunoblotting, and their mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) using limiting dilution of complementary DNA. Expression of opioid receptors on RA synovial cells was assessed by immunohistochemical staining, radioreceptor assay, and RT-PCR. Results. Leu-ENK, met-ENK, and END inhibited tumor necrosis factor-alpha and interleukin 1 beta production at the level of mRNA expression. ENK and END inhibited MMP-9 production and its enzymatic activity by RA synovial cells. The p-subtype opioid receptor was expressed in the RA synovial lining and sublining cells. Radioreceptor assay suggested expression of high affinity receptor for END on RA synovial cells. The p-subtype opioid receptor-specific antagonist, naloxone, reversed the inhibitory effect of the opioid peptides. The opioid peptides inhibited nuclear translocation and phosphorylation of the transcription factor, cyclic AMP responsive element binding protein (CREB) in RA synovial cells. Conclusion. Leu-ENK, met-ENK, and END inhibited excessive RA synovial cell functions in vitro. The opioid hormones may have not only antinociceptive action, but also antiinflammatory effects on synovitis itself in RA.
Objective. The sensory nervous system with the 2 neurotransmitters substance P (SP) and calcitonin gene related peptide (CGRP) is proinflammatory in experimental models of arthritis. The role of the sympathetic nervous system with norepinephrine (NE), adenosine, beta -endorphin, and methionine enkephalin (MENK) is not clearly understood. We studied the influence of these neurotransmitters; on secretion of interleukin 6 (IL-6) and IL-8 in primary cultures of synovial fibroblasts of patients with rheumatoid arthritis (RA) compared to osteoarthritis (OA). Methods. Fibroblasts were isolated using fresh synovial tissue of 5 patients with RA and 5 with OA who underwent knee joint replacement surgery. Modulation of spontaneous secretion of IL-6 and IL-8 was investigated in vitro using the neurotransmitters noted above. Results. In RA fibroblasts, CGRP increased IL-6 and IL-8 secretion at 10(-10) to 10(-8) M (p at least < 0.01), which was not observed in OA fibroblasts. SP had no effect on either cytokine in RA fibroblasts but stimulated IL-8 secretion at 10(-8) M in OA fibroblasts (p < 0.01). In RA fibroblasts, adenosine and NE inhibited secretion of both cytokines at low concentrations (10(-8) M: p < 0.01). However, in OA fibroblasts there was a NE induced increase of IL-8 and IL-6 secretion at 10(-7) and 10(-6) M (p < 0.01), but no inhibition at lower concentrations (10(-8) M: p = NS). In RA fibroblasts. beta -endorphin and MENK inhibited IL-8 secretion at 10(-9) to 10(-7) M (p < 0.01), whereas in OA fibroblasts the dose response curve was shifted to lower concentrations (10(-12) M, 10(-11) M; p < 0.01). Conclusion. In OA fibroblasts, the sympathetic neurotransmitters were stimulatory at higher concentrations. CGRP was the most potent stimulatory neurotransmitter in RA fibroblasts: whereas the sympathetic adenosine, NE, beta -endorphin. and MENK were inhibitory. This indicates a dualism of action of sympathetic and sensory neurotransmitters, with inhibitory and stimulatory effects on cytokine secretion of RA fibroblasts.
Endomorphin (EM)-1 and EM-2 are opioid tetrapeptides recently located in the central nervous system and immune tissues with high selectivity and affinity for the µ-opioid receptor. Intracerebroventricular (i.c.v.) administration of morphine stimulates the hypothalamo–pituitary–adrenal (HPA) axis. The present study investigated the effect of centrally administered EM-1 and EM-2 on HPA axis activation. Rats received a single i.c.v. injection of either EM-1 (0·1, 1·0, 10 µg), EM-2 (10 µg), morphine (10 µg), or vehicle (0·9% saline). Blood samples for plasma corticosterone determinations were taken immediately prior to i.c.v. administration and at various time points up to 4 h post-injection. Trunk blood, brains and pituitaries were collected at 4 h. Intracerebroventricular morphine increased plasma corticosterone levels within 30 min, whereas EM-1 and EM-2 were without effect. In addition, pre-treatment of i.c.v. EM-1 did not block the rise in corticosterone after morphine. Corticotrophinreleasing factor (CRF) mRNA and arginine vasopressin (AVP) mRNA in the paraventricular nucleus (PVN) and POMC mRNA in the anterior pituitary were found to be unaffected by either morphine or endomorphins. Since release of other opioids are elevated in response to acute stress, we exposed rats to a range of stressors to determine whether plasma EM-1 and EM-2 can be stimulated by HPA axis activation. Plasma corticosterone, ACTH and -endorphin were elevated following acute restraint stress, but concentrations of plasma EM-1-immunoreactivity (ir) and EM-2-ir did not change significantly. Corticosterone, ACTH and -endorphin were further elevated in adjuvant-induced arthritis (AA) rats by a single injection of lipopolysaccharide (LPS), but not by restraint stress. In conclusion, neither EM-1 or EM-2 appear to influence the regulation of the HPA axis. These data suggest that endomorphins may be acting on a different subset of the µ-opioid receptor than morphine. The failure to induce changes in plasma EM-ir in response to the chronic inflammatory stress of AA, the acute immunological stress of LPS, or the psychological stress of restraint, argues against an important role for endomorphins in mediating HPA axis activity.
Acute stress stimulates the expression and release of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus, and the pro-opiomelanocortin products P-endorphin and ACTH from the anterior pituitary. These neuropeptides are also expressed in immune tissues, and it has been proposed that they may modulate immune responses to stress through paracrine mechanisms. We subjected rats to restraint stress or central injection of interleukin (IL)-lβ to determine whether these acute stimuli can alter the expression of neuropeptides in the spleen and thymus. Restraint stress significantly increased the contents of all these neuropeptides in thymic, but not splenic, extracts. A single icv injection of IL-lβ increased contents of CRH, AVP, ACTH and P-endorphin in the spleens of both sham-operated and adrenalectomised (ADX) rats. IL-lβ increased thymic contents of CRH and ACTH in sham-operated rats but these increases were not observed in ADX rats. These results suggest that the effects of IL-ip on neuropeptide expression in the spleen are independent of glucocorticoids, whereas IL-lβ stimulation of neuropeptide expression in the thymus is dependent on circulating glucocorticoids. There were significant correlations between increases in CRH, ACTH and β-endorphin in the spleen, and between CRH and ACTH in the thymus, consistent with the suggestion that IL-lβ-induced increases in ACTH and β-endorphin may be mediated through CRH. These results provide evidence that stressors can directly influence neuropeptide expression in immune tissues. Thus stress may influence immune functions through paracrine mechanisms involving locally synthesised neuropeptides as well as through activation of the hypothalamo-pituitary-adrenal axis.
We have previously demonstrated that the chronic inflammatory stress of adjuvant-induced arthritis in rats can alter levels of the neuropeptides adrenocorticotropin (ACTH), β-endorphin, arginine vasopressin (AVP) and corticotropin-releasing hormone (CRH) in tissues of the immune system. We now present data showing that the patterns of these changes in the spleens and thymuses of Piebald-Viral-Glaxo rats are quite dissimilar throughout the course of the disease. Immunoreactive (ir)-CRH, AVP, ACTH and β-endorphin were measured by radioimmunoassays in spleen and thymic extracts taken at days 3, 7, 11 and 14 following injection of adjuvant. AVP was increased in the spleen at day 14 compared to the controls (79.4 ± 4.4 and 60.0 ± 9.0 fmol/g tissue respectively), but no change occurred in the thymus. CRH contents were increased in the spleen at day 14 (33.4 ± 3.5) compared to controls (22.1 ±2.4 fmol/g tissue), and in the thymus at day 11 (24.0 ±2.3) compared to controls (14.1 ± 2.5 fmol/g tissue). Increases in ACTH content were observed in spleens from arthritic rats at days 3 (365 ± 23), 11 (359 ± 32) and 14 (355 ± 45 fmol/g tissue) compared to controls (198 ± 37 fmol/g tissue). In the thymus, however, ACTH was elevated only at day 14. β-Endorphin levels in the spleen were elevated only at day 14 (289 ± 41) compared to controls (97 ± 22 fmol/g tissue). We conclude that the responses of ir-CRH, AVP, ACTH and β-endorphin to an inflammatory stress in the spleen and thymus are activated at different time-points and by different events which occur during the course of a chronic stress. It is also apparent, at least in the spleen, that unlike the hypothalamo-pituitary axis, immune POMC activation is not necessarily consequent to increases in immune CRH or AVP.
The opioid β-endorphin (BE) is synthesized in immunocytes and exerts a tonic inhibitory effect on the immune system. Human and experimental data suggest a role for BE in the T helper 1 (Th1)-Th2-Type esponse switch.
Many neuropeptides and their receptors have been found within immune tissues, and a host of functions have been assigned to them. Some information has been gained from observations of immune responses to exogenously administered neuropeptides, but many immunoneuropeptides differ in structure and probably also in function from their analogous forms found in hypothalamic and pituitary tissues. New strategies, such as oligonucleotide antisense probes and gene-targeting models, can be used to determine physiologic roles for immunoneuropeptides. Immunoneuropeptides can respond to physiologic or pharmacologic manipulation, and evidence is mounting for important roles in mediating immune responses to inflammation and stress, as well as being instrumental in controlling cell growth. The ability to modulate immune functions through autocrine and paracrine mechanisms confers cytokine status on immunoneuropeptides, which, activated in response to stress or infection, have the potential to maintain a homeostatic balance between the immune, neural, and endocrine systems during periods of stress or illness.
(C) Lippincott-Raven Publishers.
In addition to the well-established effects of corticotrophin-releasing factor (CRF) synthesized in the hypothalamic paraventricular nucleus in modulating the stress response, CRF has also been implicated in a number of other functions. In particular both pro- and anti-inflammatory effects have been proposed for CRF. In the present study we have investigated the effects of 14 day peripheral infusion of CRF on the development of adjuvant-induced arthritis (AA) in the rat. To avoid confounding effects of the CRF stimulating endogenous corticosterone, all animals in the study were adrenalectomized. We were unable to determine any effect of the treatment at either of the doses of CRF investigated. The bioactivity of the CRF was confirmed in vitro and the presence of substantial circulating levels of CRF in plasma in the high dose group at time of sacrifice suggest that the CRF was successfully infused over the 14 day experimental period. These data suggest that CRF is not important in the mediation of AA. Alternatively either intact adrenal glands may be required for the action of CRF to become apparent or locally produced endogenous CRF may have more subtle effects than chronic infusion of synthetic CRF.
Objective
To examine the regulation and mode of action of peripheral corticotropin-releasing hormone (CRH) in human inflammatory arthritis.MethodsCRH messenger RNA (mRNA) levels were measured in normal and inflamed synovial tissue and in primary synoviocytes prior to and following cytokine stimulation. Primary synoviocytes were transiently transfected with CRH promoter/reporter constructs, and promoter activity in response to cytokines was assessed. Immunohistochemical staining established CRH receptor expression, and Northern blot analysis confirmed that the nuclear transcription factors NUR77 and NURR1 contributed to synovial CRH receptor–mediated signaling. Primary synoviocytes were treated with pro- and antiinflammatory mediators, and the time course of NURR1 and NUR77 modulation was examined. Nuclear extracts were analyzed by electrophoretic mobility shift assay to determine NURR1 binding to the CRH promoter/enhancer.ResultsCRH mRNA was up-regulated in the synovial tissue in rheumatoid arthritis (RA), psoriatic arthritis (PsA), and sarcoid arthritis, but not in normal synovium. Inflammatory cytokines, such as interleukin-1β and tumor necrosis factor α, enhanced the transcriptional activity of the human CRH promoter and increased levels of CRH mRNA in primary synoviocytes. Synovial CRH functioned in a paracrine manner to induce NURR1 and NUR77. NURR1 was abundantly expressed in the inflammatory cells of both RA and PsA synovium. NURR1 and NUR77 were differentially regulated, and NURR1 was the major cytokine-regulated member of the NURR subfamily as well as the mediator of cytokine- and CRH-dependent inflammatory responses in synovium. Furthermore, glucocorticoids dramatically suppressed cytokine- and CRH-induced synovial NURR1 mRNA.Conclusion
These data demonstrate the involvement of the transcription factor NURR1 in the regulation of CRH expression and activity in human inflammatory arthritis.
Opioid peptides affect different immune functions. We present evidence that these effects could be mediated by the modulation of TH1/TH2 cytokine production. The acute and chronic treatment with the opioid receptor antagonist naloxone decreased the production of IL-4 by splenocytes of C57BL/6 and BALB/CJ mice, that present a Th1/ Th2 dominance, respectively, immunized with the protein antigen KLH. In contrast, IL-2 and IFN-γ levels were increased after naloxone treatment. These results indicate that naloxone increases Th1 and decreases Th2 cytokine production. Moreover in C57BL/6 mice, naloxone treatment was able to accelerate skin-graft rejection, a Th1-mediated phenomenon, by increasing Th1 cytokine production. The effect of naloxone could be ascribed to the removal of the regulatory effects exerted by endogenous opioid peptides, which could activate Th2 and suppress Th1 cytokines.
In this paper, we describe that met-enkephalin and/or enkephalin-containing intermediary peptides of the prohormone pro-enkephalin A are produced and secreted by human peripheral blood T cells and monocytes. The peptides are produced after stimulation with the mitogenic monoclonal antibodies anti-CD2.1/2.2 and anti-CD28. In monocytes, enkephalin synthesis was induced by stimulation with lipopolysaccharide. We demonstrate here that these immune cell-derived enkephalins play an important regulatory role in the immune response. By using an anti-sense oligonucleotide strategy we could block the production of enkephalins. Blockade of the production of met-enkephalin and enkephalin-containing intermediary peptides resulted in enhancement of the proliferative T cell response and inhibition of monocyte IL-6 secretion. In vitro reconstitution of the anti-sense treated cultures with synthetic met-enkephalin or the delta-type specific opioid receptor agonist deltorphin could reverse inhibition of monocyte IL-6 production, suggesting that endogenous enkephalins act via membrane opioid receptors. In contrast, addition of met-enkephalin or deltorphin to the anti-sense treated T cell cultures did not have any effect on T cell proliferation.
Opioids produce analgesia by interacting with local opioid receptors in peripheral inflamed tissue. This study investigated whether endogenous ligands of these receptors are present in synovia and whether such opioid peptides can inhibit pain by activation of intra-articular opioid receptors. Samples of synovium from 8 patients undergoing arthroscopic knee surgery were examined by immunohistochemistry for the presence of β-endorphin, met-encephalin, and dynorphin. All tissue samples showed synovitis. Inflammatory cells stained strongly for β-endorphin and met-encephalin but not for dynorphin. To find out whether blockade of intra-articular opioid receptors affected pain, we randomly assigned 22 patients undergoing arthroscopic knee surgery to receive naloxone (0 04 mg) intra-articularly (n = 10) or intravenously (n=12); each patient received a placebo injection into the other site. Postoperative pain was assessed by visual analogue scale, a numerical rating scale, the McGill pain questionnaire, and supplementary analgesic consumption during the next 24 h. All pain scores were higher in the intra-articular naloxone group than in the intravenous naloxone group. The differences were significant (p<0·05) during the first 4 h. Supplementary analgesic consumption was significantly higher in the intra-articular group (52·5 [14·0] vs 15·6 [8·0] mg diclofenac, p<0·05). Opioid peptides are present in inflamed synovial tissue and can inhibit pain after knee surgery through an action specific to intra-articular opioid receptors. These findings expand the gate control theory of pain and suggest new approaches such as the development of peripherally acting opioid analgesics without central side-effects.
Two highly selective μ-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for μ-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the μ1-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with κ-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the δ2-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the δ1-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met5]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of μ1-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met5]enkephalin which subsequently act on κ-opioid receptors and δ2-opioid receptors to produce antinociception.
This study was undertaken to examine the presence of functional nociceptin/orphanin FQ (N/OFQ) receptors in the immune system. Receptor mRNA signals were detected by RT-PCR in porcine thymus, lymph nodes, spleen and freshly-isolated splenocytes; the distribution of prepro-nociceptin/-orphanin FQ (PP-N/-OFQ) mRNA was similar, with the exception of lymph nodes. However, specific [3H]nociceptin binding sites were not detected in rat or porcine lymphoid tissues, and 0.1–100 nM nociceptin had no effect on forskolin-stimulated cyclic AMP concentrations in porcine splenocytes. Thus, it appears that nociceptin/orphanin FQ receptor mRNA, but not a functional receptor protein is expressed in the immune system.
During phylogeny and ontogeny, the thymus appears as a crucial meeting point between the neuroendocrine and immune systems; through cryptocrine intercellular communication, thymic neuroendocrine-related precursors can influence the early steps of the immune response, while T-cell precursors are educated to recognize the principal neuroendocrine families. Here we summarize the observations that support the dual role of the thymic repertoire of neuroendocrine-related polypeptide precursors in T-cell differentiation.
This study shows the expression at the cell surface of human thymic epithelial cells (TEC) of a neurotensin (NT)-like immunoreactivity. NT radio-immunoassay (RIA) revealed that cultured human TEC contain ±5 ng immunoreactive (ir) NT/106 cells, of which 5% is associated with plasma cell membranes. HPLC analysis of NT-ir present in human TEC showed a major peak of NT-ir corresponding to NT1–13. NT-ir was not detected in the supernatant of human TEC cultures. Using an affinity column prepared with a anti-MHC class I monoclonal antibody, NT-ir-related peptides were retained on the column and eluted together with MHC class I-related proteins. According to the elution time on HPLC of these peptides, they correspond to intact NT1–13, as well as to smaller fragments of NT1–13.
The POMC-derived peptides β-endorphin and ACTH are capable of modulating an immune response in physiological concentrations. These neuropeptides can either enhance or inhibit the proliferative response of human peripheral blood lymphocytes after stimulation with the mitogen concanavalin A. The modulatory action of the peptides is not only dependent on the concentration but appears to be donor dependent. The response pattern observed is not determined by a selective affinity for certain amino acid sites on the molecules with “enhancing” or “inhibiting” activities, since fragments of β-endorphin and ACTH also produce a differential donor-dependent response pattern.
Recent evidence indicates that glucocorticoids and catecholamines, the end-products of the stress system, and histamine, a product of activated mast cells, might selectively suppress cellular immunity, and favour humoral immune responses. This is mediated by a differential effect of stress hormones and histamine, on T helper 1 (Th1)/Th2 patterns and type 1/type 2-cytokine production. Thus, systemically, stress might induce a Th2 shift, while, locally, under certain conditions, it might induce pro-inflammatory activities through neural activation of the peripheral corticotropin-releasing factor-mast cell-histamine axis. Through the above mechanisms, stress may influence the onset and/or course of infectious, autoimmune/inflammatory, allergic and neoplastic diseases.
We have examined the distribution of the opioid receptor-like-1 (ORL-1) transcript in the human CNS as well as human immune cells by RT-PCR and RNAse protection. The hORL-1 mRNA was distributed throughout the brain and particularly abundant in cortical areas, striatum, thalamus and hypothalamus. In the immune system, gene transcription was observed in normal circulating lymphocytes and monocytes as well as in T, B and monocytic cell lines. A splice variant, lacking 15 nucleotides at the junction between exon 1 and exon 2, showed a distribution similar to the already known ORL-1 transcript. Altogether these results show comparable expression levels of the hORL-1 gene in both nervous and immune systems, suggesting that the ORL-1-encoded receptor may participate to neuronal and non-neuronal physiological functions in humans.
It is well known that corticotropin releasing factor （CRF） modulates immune response during infl ammation. We investigated the eff ect of CRF family peptides on host resistance to Listeria monocytogenes infection in mice. The numbers of L. monoctyogenes in the organs of Ucn2-treated mice were dramatically increased compared with CRF- or Ucn-treated mice. CRF receptor type 2 is involved in the suppressive eff ect of Ucn2 on L. monocytogenes infection. Interferon （IFN）-γ and tumor necrosis factor （TNF）-α production were decreased and interleukin （IL） -10 production was signifi cantly increased in the spleens of Ucn2-treated mice compared with those in Ucn2-untreated control mice. The eff ect of Ucn2 was canceled by depleting endogenous IL-10 using anti-IL-10 monoclonal antibody and in IL-10 deficient mice. The expression and activation of signal transducers and activators of transcription 3 （STAT3） were up-regulated and the expression and activation of STAT1 were down-regulated in the spleens from Ucn2-treated mice compared with vehicle-treated mice. Moreover, suppression of TNF-α production and augmentation of IL-10 production and expression and activation of STAT3 by Ucn2 treatment were observed in heatkilled L. monocytogenes-stimulated macrophages. These results suggested that Urn2 suppresses host resistance to L. monocytogenes infection via up-regulation of IL-10 production.
Cells of the immune system produce biologically active adrenocorticotropic hormone (ACTH). Many laboratories, however, have been unable to replicate experiments which demonstrate ACTH in immune cells. Sensitive immunohistochemical staining and digital scanning, confocal microscopy were used to study regulation of ACTH-like immunoreactivity (ACTH-IR) in human mononuclear cells. Cytoplasmic ACTH-IR was induced by corticotrophin releasing factor (CRF)/arginine vasopressin (AVP), and also by protein kinase C (PKC) activation and by the interferon (IFN-alpha beta inducer, Na-polyinosinic-polycytidylic acid (polyIC). Induction of cytoplasmic ACTH-IR was maximal within 6 hr of stimulation with CRF/AVP or phorbol myristate acetate (PMA). Recombinant human interleukin-1 beta (rhIL-1 beta) was also stimulatory, but rhIL-1 alpha had minimal effect. Regulation of ACTH-IR production in immune cells parallels the regulation of ACTH in the anterior pituitary, and ACTH-like material may affect immune responses.
Exogenous kappa-opioid agonists have been shown to produce peripheral antinociceptive effects in inflamed tissue. This study sought to determine whether endogenous kappa-receptor ligands are present at the site of inflammation. In Freund's adjuvant-induced hindpaw inflammation in the rat, we show, by immunohistochemistry, that dynorphin is detectable within inflammatory cells and in the cutaneous nerves in a similar distribution as calcitonin gene-related peptide, a specific marker for sensory neurons. These findings extend our previous observations in that not only beta-endorphin and Met-enkephalin (mu- and delta-receptor ligands), but also a preferential kappa-ligand is present within inflamed subcutaneous tissue.
In addition to their characterizing secretory products, both magnocellular and parvocellular neurosecretory neurons are now known to express other neuroactive substances. Parvocellular neurons that make corticotropin-releasing factor (CRF) for example are capable of synthesizing at least seven neuropeptides. Some of these, like arginine vasopressin (AVP), interact with CRF at the level of the anterior pituitary to promote corticotropin secretion, and, like CRF, are regulated negatively by glucocorticoids and positively by at least some stressors. others are inert in these two contexts but are responsive to various challenges. Magnocellular neurosecretory oxytocin- and AVP-containing neurons are capable of producing similarly broad and distinctive complements of neuroactive principles. These are typically expressed at levels far lower than those of the nonapeptides, suggesting local modulatory effects on oxytocin and/or AVP secretion at the level of the posterior lobe. Differential regulation of coexisting molecules within magnocellular neurons by systemic challenges and steroid hormones has also been described. Secretory products of magnocellular neurons may gain access to the anterior pituitary via exocytotic release at the level of the median eminence or through vascular links between the posterior and anterior lobes, suggesting another form of 'co-localization' by which the two neurosecretory cell types may interact in the control of stress and perhaps other pituitary-mediated responses.
Our previous studies indicate that endogenous opioids (primarily beta-endorphin) released during stressful stimuli can interact with peripheral opioid receptors to inhibit nociception in inflamed tissue of rats. This study sought to localize opioid precursor mRNAs and opioid peptides deriving therefrom in inflamed tissue, identify opioid containing cells and demonstrate their functional significance in the inhibition of nociception. In rats with Freund's adjuvant-induced unilateral hindpaw inflammation we show that: (i) pro-opiomelanocortin and proenkephalin-mRNAs (but not prodynorphin mRNA) are abundant in cells of inflamed, but absent in non-inflamed tissue; (ii) numerous cells infiltrating the inflamed subcutaneous tissue are stained intensely with beta-endorphin and [Met]enkephalin (but only few scattered cells with dynorphin) antibodies; (iii) beta-endorphin is present in T- and B-lymphocytes, monocytes and macrophages; and (iv) whole-body irradiation suppresses stress-induced antinociception in the inflamed paw. Taken together, these data suggest that endogenous opioid peptides are synthesized and processed within various types of immune cells at the site of inflammation. Immunosuppression abolishes the intrinsic antinociception in inflammatory tissue confirming the functional significance of these cells.
Mouse thymocytes incubated in vitro with increasing concentrations of interleukin-1 (IL-1) in the presence of phytohemagglutinin (PHA) exhibited a dose-dependent increase in cell proliferation, as measured by [3H]thymidine incorporation. Under these conditions, there was a parallel dose-dependent increase in specific [3H]morphine binding, with a maximum increase of approximately 5-fold over basal levels. The binding sites differ from classical opioid receptors in that they are not stereo-selective. Interleukin-2 was ineffective in promoting either cell proliferation or enhanced opioid binding, but the effects of IL-1 could be mimicked by phorbol myristate acetate (PMA), suggesting the involvement of tyrosine phosphorylation. These results indicate that morphine-binding sites on immune cells can be regulated by cytokine activation.
Lymphocytes harbor a pro-opiomelanocortin (POMC) mRNA. In this report, a novel procedure was used to study the exonic arrangement of this transcript in lymphocytes. Poly(A)+ mRNA, purified from both corticotropin-releasing factor (CRF)-treated and nontreated lymphocytes, was selectively reverse-transcribed using an antisense oligonucleotide primer complementary to the 3' junction of the translated/nontranslated region of exon 3 of POMC. Alkaline agarose gel analysis of first-strand cDNA synthesis showed an upregulation of POMC transcripts in CRF-treated cells. This first-strand cDNA was amplified in a polymerase chain reaction (PCR) using the complementary antisense primer and selective sense primers homologous to the 5' ends of exons 1, 2, and 3, as well as a region immediately 5' to the ACTH/beta-lipotropin coding region of exon 3 of pituitary POMC. Primers directed at exons 1 and 2 did not amplify a POMC product in nontreated control or CRF-treated cells. However, with both treated and nontreated cells, the internal exon 3 primer amplified the expected size exon 3 DNA fragment (approximately 549 bp). Interestingly, a primer directed at the 5' end of exon 3 apparently did not amplify a POMC product in nontreated cells but did amplify a full-size POMC exon 3 from CRF-treated cells (approximately 615 bp). However, upon reamplification of the original PCR products from nontreated cells, full-length exon 3 product was also observed. Southern gel analysis using a pituitary POMC cDNA probe showed that all of the above PCR products were POMC-related. The results of this study show that lymphocytes basally transcribe at least two POMC transcripts that are upregulated by CRF. These two transcripts lack exons 1 and 2 but contain either part or all of exon 3. The smaller exon 3 transcript was the most abundant transcript under all conditions examined.
Corticotropin-releasing hormone (CRH) functions as a regulator of the hypothalamic-pituitary-adrenal axis and coordinator
of the stress response. CRH receptors exist in peripheral sites of the immune system, and CRH promotes several immune functions
in vitro. The effect of systemic immunoneutralization of CRH was tested in an experimental model of chemically induced aseptic
inflammation in rats. Intraperitoneal administration of rabbit antiserum to CRH caused suppression of both inflammatory exudate
volume and cell concentration by approximately 50 to 60 percent. CRH was detected in the inflamed area but not in the systemic
circulation. Immunoreactive CRH is therefore produced in peripheral inflammatory sites where, in contrast to its systemic
indirect immunosuppressive effects, it acts as an autocrine or paracrine inflammatory cytokine.
Naloxone-resistant binding sites for beta-endorphin have previously been observed on transformed peripheral blood mononuclear cells and on the EL4-thymoma cell line. These sites may be related to the naloxone-insensitive immunomodulatory effects of beta-endorphin. The present study was performed 1) to determine whether these sites are present on normal splenocytes and 2) to characterize them. Ficoll-hypaque-purified murine splenocytes were used in a RRA with [125I]beta-endorphin. Neither fresh intact cells obtained from viral antibody-free mice nor membrane preparations showed evidence of binding. However, splenocytes cultured in 5% fetal bovine serum for 24-96 h showed sites on intact cells or membranes (after 3 h in culture no sites were present). Intact cultured splenocytes demonstrated a saturable binding isotherm, and Scatchard analysis showed a single site (Kd = 4.1 X 10(-9) M). Competition studies showed that N-acetyl-beta-endorphin (N-Ac-beta-endorphin)-(1-31) was equipotent to beta-endorphin-(1-31). beta-Endorphin-(6-31) and beta-endorphin-(28-31) were approximately 10- and 1000-fold less potent, respectively, whereas beta-endorphin-(1-27) and naloxone were completely ineffective. Covalent cross-linking of [125I]beta-endorphin to splenocytes and resolution by gel electrophoresis showed bands at 66K and 57K which were displaced equipotently by increasing amounts of beta-endorphin and N-Ac-beta-endorphin. beta-Endorphin-(18-31) or (28-31) were less potent, and naloxone or other opioid ligands selective for receptor subtypes were ineffective. Thus, high affinity, naloxone-insensitive binding sites for beta-endorphin, which show competition characteristics distinctively different from brain opiate receptors, are inducible on normal mouse splenocytes. N-Ac-beta-endorphin, presumed to be an inactivation product of beta-endorphin because it fails to bind brain opiate receptors, may be functional at this naloxone-insensitive binding site.
Vasopressin receptors were demonstrated on human peripheral blood mononuclear cells (PBMC) by using the radioiodinated analog of d(CH2)5[Tyr(Me2)Thr4Tyr-NH2(9)]OVT (OTA). Binding of this ligand was time-dependent, specific, and saturable. Scatchard analysis of [125I]-OTA binding at equilibrium revealed a dissociation constant of 0.47 +/- 0.17 nM. A considerable sex difference in binding capacity was observed. PBMC from female donors expressed an approximately sevenfold higher receptor density than PBMC from male donors, while no change of Kd was apparent. Throughout the menstrual cycle the maximal binding capacity was relatively constant. Competition studies with vasopressin and oxytocin analogs showed that this putative receptor site on PBMC is comparable in receptor specificity to the human V1 receptor on myometrial tissue and blood platelets, but slightly different from the rat neurohypophyseal hormone receptor classes. Our findings provide further evidence of a remarkable species and sex difference of vasopressin and oxytocin receptors, regarding their ligand selective binding properties. The presence of the putative arginine-vasopressin receptors on PBMC may provide a molecular basis for several arginine-vasopressin induced effects on the chemistry and function of circulating mononuclear cells.
The immune system and the neuroendocrine system have been shown to be functionally interactive. The neuroendocrine system can modulate the immune response and immune mediators can influence the neuroendocrine system. The present paper focuses on the capacity of lymphocytes to produce and secrete neuroendocrine substances. Lymphocytes can secrete the neuropeptide beta-endorphin in response to activation with mitogen or antigen. Moreover, mediators that are involved in the adaptation to stress have also been shown to induce the release of immunoreactive-beta-endorphin by lymphocytes. It is shown here that stimulation of human peripheral blood mononuclear cells with the beta-adrenergic agonist isoprenaline induces beta-endorphin secretion. The effect of isoprenaline can be mimicked by elevation of the intracellular concentration of cAMP with forskolin or (Bu)2cAMP. Inhibition of cAMP-dependent protein kinase PKA by the antagonist N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide abrogates isoprenaline-induced secretion of immunoreactive-beta-endorphin by peripheral blood mononuclear cells. The present data give evidence that, beta-adrenergic activation activation of lymphocytes stimulates the secretion of ir-beta-endorphin via a protein kinase A-dependent mechanism. Both beta-adrenergic agonists as well as beta-endorphin have been shown to modulate the immune response. The data presented here are indicative for a role of beta-endorphin in the modulation of the immune response after beta-adrenergic activation.
Based upon an immunomodulatory role for Corticotropin-Releasing Factor (CRF), a low molecular weight neurohormone, we investigated the effect of CRF on the production of interleukin-1 (IL-1) and interleukin-2 (IL-2) activities of mononuclear cells isolated from the peripheral blood of healthy subjects. The production of both IL-1 and IL-2 was stimulated by a nanomolar concentration of CRF by itself. In addition, CRF augmented the production of IL-1 as induced by lipopolysaccharide and the production of IL-2 as induced by phytohemagglutinin. These results suggest that CRF modulates the function of the cells of the immune system presumably by acting as a blood-borne mediator of the neuroendocrine-immune pathways.
Unstimulated normal leukocytes were studied for their expression of corticotrophin-releasing factor (CRF)-like mRNA and peptide. In situ hybridization demonstrated CRF mRNA in T and B lymphocytes and in neutrophils. Northern blot analysis also detected the presence of a CRF mRNA species of approximately 1.7 Kb from lymphocytes and a peptide that reacted with anti-CRF antiserum was also detected by radioimmunoassay. This lymphocyte-derived peptide eluted earlier than standard CRF on high performance liquid chromatography. These findings suggest that regulatory mechanisms within the hypothalamus and the immune system may utilize closely related peptides.
Experiments were conducted which compared the in vivo effects of beta-endorphin (BEP), gamma-endorphin (gamma EP), methionine-enkephalin (Met-ENK), and acetylated BEP(1-27) on the in vitro proliferative response of rat spleen cells to concanavalin A (ConA). In addition, the influence of BEP administration on the primary and secondary antibody-forming cell (AFC) response to the soluble antigen keyhole-limpet hemocyanin (KLH) was examined. Intravenous administration of BEP enhanced the spleen cell proliferative response to ConA assessed 3 hr after a single bolus infusion. Conversely, infusion with AcBEP(1-27) suppressed the proliferative response, whereas no effects of intravenous gamma EP or Met-ENK treatment were observed. The enhancing effect of BEP administration was not detectable 24 hr after a single infusion, but could be maintained over a 44 hr period by multiple infusions. The primary AFC response to KLH was suppressed by a dose of 1 nmole BEP only. On the other hand, the secondary IgG AFC response to KLH was enhanced by 10 pmoles BEP, while the IgM and IgA AFC responses remained unaltered by BEP treatment. The anamnestic in vitro proliferative response of spleen cells cultured with KLH was not altered if BEP was administered at the time of secondary KLH immunization. These results extend previous observations of BEP-induced modulation of in vitro immune function by demonstrating that opioid and nonopioid forms of BEP administered in vivo alter the capacity of spleen cells to proliferate and develop antibody responses to antigen.
Corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) stimulate the secretion of beta-endorphin by human PBMC. It is shown here that peripheral blood B cells are responsible for the production of beta-endorphin after culture with CRF and AVP. The presence of CD14+ monocytes is, however, a prerequisite for the enhancing activity of CRF and AVP. The data presented here show that rIL-1 beta can replace CRF and AVP, whereas a mAb directed against IL-1 abrogates the response to CRF and AVP. These results indicate that IL-1 mediates the effect of CRF and AVP on beta-endorphin production by human PBMC.
In response to stressors involving tissue injury, pituitary corticotroph secretion of immunoreactive beta-endorphin (iB-END) could be either due to release of hypothalamic factors such as corticotropin-releasing factor (CRF) or to release of a tissue factor from the periphery. In the present experiments, we investigated whether inflamed tissue releases a factor which evokes pituitary secretion of iB-END. In an initial experiment, rats with an inflamed hindpaw due to carrageenan injection had significantly greater levels of circulating iB-END as compared to rats with saline-injected paws. Removal of afferent input, by hindlimb denervation, failed to block the carrageenan-induced increase in iB-END levels. Subcutaneous perfusates were then collected from inflamed and control hindlimbs and applied to rat anterior pituitary cell cultures. Pituitary release of iB-END due to administration of perfusate from inflamed paws was significantly greater than iB-END release due to perfusate from saline-injected paws or to basal release. The releasing activity in the perfusates was blocked in calcium-free medium and was not due to a direct action of carrageenan, bradykinin, substance P or calcitonin gene-related peptide. The results indicate that inflamed tissue releases a CRF-like factor which stimulates iB-END release both in the denervated rat and cultured pituitary cells.
Using Northern blotting with a human genomic DNA probe for the pro-opiomelanocortin (POMC) gene, we have shown specific mRNA in normal human peripheral mononuclear cells (PBMC); the presence of specific mRNA was also observed in a T lymphocyte cell line derived from a patient with lymphoma. We then demonstrated that PBMC translate the message into protein. Thus, using a radioimmunoassay with an antibody for ACTH, a median of 29 pg of ACTH-like immunoreactivity (ACTH-LIR) was found in 10(7) PBMC. ACTH-LIR was also detected in seven different cell lines derived from patients with lymphoid and myeloid malignancies, two of them JM and U937 showing the highest values 135 and 108 pg/10(7) cells, respectively. The chromatographic characterization of this ACTH-LIR showed, at least, three molecular forms of immunoreactive ACTH with molecular weights of the order of 31,000 POMC, 22,000 ACTH, and 4,500 ACTH, in addition to high-molecular-weight material (greater than 43,000). We conclude that PBMC produce ACTH-LIR which may act as a paracrine immunomodulator in a similar way to lymphokines and/or may signal the adrenal gland to secrete glucocorticoids.
In an effort to investigate the presence of adrenocorticotropic hormone (ACTH) receptors on rat lymphocytes, cells were separated by a panning procedure into T and B cell populations. By using the radiolabeled ACTH agonist, (125I-Tyr23) phenylalanine2-norleucine4-ACTH1-24, substantial numbers of ACTH binding sites were detected on T and B lymphocytes, but not on thymocytes. Scatchard analysis revealed two types of binding sites on each cell population, one with Kd1 = 0.088 +/- 0.025 nM and one with Kd2 = 4.2 +/- 0.6 nM; however, the absolute number of binding sites per cell was different. B lymphocytes expressed approximately three times the number of Kd1 binding sites per cell when compared with T lymphocytes. However, ACTH receptor expression by these cell populations was not static as suggested by the ability to induce receptor expression via mitogens. B or T cells and thymocytes stimulated with the mitogens LPS or Con A, respectively, substantially increased their number of Kd1 binding sites per cell (approximately three-fold). Even more dramatic increases in Kd1 receptor expression (approximately 100-fold) were observed when comparing "normal" and stimulated thymocytes. To demonstrate that these ACTH binding sites were in fact functional, cAMP levels were measured in lymphocytes 10 min after exposure to varying concentrations of ACTH. Dose-dependent increases in cAMP levels were observed, with significant stimulation occurring with as little as 0.1 nM ACTH added. Taken together, these studies demonstrate the presence of functional ACTH receptors on normal, rat T and B lymphocytes.
The supplementation of corticotropin-releasing factor (CRF) into the cultures of human blood lymphocytes caused increased proliferation both in the absence and presence of T cell mitogens such as concanavalin A and phytohemagglutinin. The stimulation of concanavalin A response was much higher with CRF ligand as compared to Tyr-CRF, CRF-antagonist and sauvagine, and this response was blocked by CRF-antagonist. The lymphocyte proliferative response to stimulation by pokeweed mitogen or monoclonal antibody to CD3 antigen (OKT3) and the activity of natural killer (NK) cells was not affected by CRF. However, this neuroendocrine hormone, in addition to its ability to stimulate lymphocyte proliferation, enhanced expression of interleukin-2 receptors (IL-2R) on T cells (activated T cells) as revealed by a 2-fold increase in the proportion of IL-2R+T cells after the culture of lymphocytes for 3-5 days in the presence of CRF. Based on these findings, we suggest that CRF plays an important role in the modulation of the neuroendocrine-immune circuity.
THE inaccessibility of many tissues makes routine investigation for receptor defects impossible. ACTH insensitivity syndrome, first described by Shepard et al.1 in 1959 and now reported in more than 63 patients,1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 is an example of this problem. Defects in adrenal ACTH receptors have been postulated because of high circulating ACTH levels and the ability of the adrenal-cortex cells to achieve steroidogenesis through an elevation of cyclic AMP.1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 In the majority of cases, the clinical presentation includes hyperpigmentation, hypoglycemia, and extremely low glucocorticoid levels with normal mineralocorticoid concentrations.2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Differences between cases suggest that the syndrome may have multiple causes. Adrenal cells . . .
Synthetic human beta endorphin shows a biphasic effect on the production of specific anti-herpes viral antibodies in vitro. At higher concentrations antibody production is reduced, at lower concentrations it is enhanced. In a proportion of donors whose cells do not produce specific antibody when cultured with antigen alone, the lower concentrations of beta endorphin allow antigen-driven specific antibody production to occur. Specific anti-herpes viral antibodies are also made by natural killer (NK) cell-depleted populations from the same non-producer donors. beta endorphin also exerts biphasic effects on NK, although the direction of the modulation is a mirror image of the effects on antibody production. This suggests that the immunomodulatory actions of beta endorphin on specific immune responses are mediated in part by effects on NK suppressor-inducer cells.
Corticotropin releasing factor (CRF) is a key hormone in the integrated response to stress, acting both as the major regulator of pituitary adrenocorticotropic hormone (ACTH) release and as a neuropeptide in the brain. The actions of CRF are mediated by specific plasma membrane receptors in the anterior pituitary gland and in discrete brain areas including the cerebral cortex and several regions related to the limbic system. In addition to the pituitary and central actions of CRF, systemic administration of the peptide in the rat, dog, monkey and man causes hypotension and tachycardia because of a decrease in peripheral vascular resistance. These observations, in conjunction with the finding of immunoreactive and bioactive CRF in peripheral tissues, suggest that the peptide is locally released in tissues to act as a neurotransmitter or paracrine hormone. As CRF is present in the adrenal medulla and the peptide is known to modulate the central activity of the autonomic nervous system, we investigated the possibility that CRF is involved in the regulation of the peripheral autonomic nervous system. Such an action of CRF is supported by our demonstration of specific CRF receptors in the monkey adrenal medulla and sympathetic ganglia. In the adrenal medulla, these receptors are coupled to adenylate cyclase and can stimulate the secretion of catecholamines and Met-enkephalin.
Human peripheral leukocytes infected by virus or treated with endotoxin will, like unstimulated mouse spleen macrophages, synthesize immunoreactive corticotrophin (ir-ACTH) and endorphins. The ir-ACTH produced appears to be identical with authentic ACTH, while enough of the material has been produced in hypophysectomized mice infected with virus to demonstrate a steroidogenic response. Because the production of ACTH by in vivo pituitary cells and by leukocytes is suppressed by dexamethasone both in vitro and in vitro, suggesting that the production of ACTH and endorphins by leukocytes is indeed controlled, we have investigated the effects of corticotropin releasing-factor (CRF), which is known to regulate the pituitary production of both ACTH and beta-endorphin. We now report that the production of ACTH and endorphins by leukocytes is indeed induced by synthetic CRF and, in turn, suppressed by dexamethasone, suggesting that, as in pituitary cells, the proopiomelanocortin (POMC) gene may be expressed and similarly controlled in leukocytes.