Morphine upregulates kappa-opioid receptors of human lymphocytes.

Opioids such as morphine are potent analgesic and addictive compounds. Chronic morphine use also induces immunomodulatory and immunosuppressive effects, as especially evident in HIV-infected patients. Morphine acts on the immune cells primarily through its binding to mu-opioid receptors on the plasma membrane. However, morphine modulation of immune functions still exists in mu-opioid receptor knockout mice, suggesting that in addition to the mu opioid receptors, morphine may also act by mechanisms mediated by either delta or kappa opioid receptors. To determine whether morphine activates kappa opioid receptors (KOR), a quantitative competitive RT-PCR procedure was utilized to quantify the KOR gene expression of morphine-treated cells. A segment of KOR transcript spanning the second extracellular loop, which has the reported dynorphin specificity, and the seventh transmembrane domain of the receptor was amplified from the total RNA of morphine-treated CEM x174 lymphocytes, along with a competitor molecule. The competitor was constructed by deleting a 33-nucleotide fragment from KOR. The results of the competitive RT/PCR indicated that CEM x174 cells expressed KOR mRNA constitutively, in the order of femto-grams. Treatment of 10 microM of morphine resulted in the up-regulation of KOR gene expression 24 hr post-treatment. The observed morphine effect could be reversed by treating the cells with either naloxone (a KOR-partially selective antagonist) or nor-Binaltorphimine (a KOR-selective antagonist).

Kappa-opioid receptors on lymphocytes of a human lymphocytic cell line: morphine-induced up-regulation as evidenced by competitive RT-PCR and indirect immunofluorescence.

We have previously shown that classical brain-like kappa opioid receptors (KOR) are constitutively expressed in lymphocytic cells. including human CEM x174 T-B hybrid cells, Jurkat -T4 cells, human peripheral blood mononuclear cells (PBMC), human CD4+ cells and monkey PBMC (Biochem. Biophys. Res. Commun. 209 (1995) 1003). The present study further demonstrates that the KOR of lymphocytes are activated in the presence of extracellular morphine or U50,488H, a KOR selective agonist, and the activation causes an increase in the expression of KOR mRNA, as determined by a quantitative competitive Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) procedure. The observed agonist-induced KOR up-regulation was blocked by treating the cells with either naloxone (a KOR-partially selective antagonist) or nor-binaltorphimine (a KOR-selective antagonist). Up-regulation of lymphocytic KOR by morphine was also evidenced by flow cytometric analysis of phycoerythrin (PE) amplification of fluorescein isothiocyanate-conjugated arylacetamide labeling of the KOR. Although morphine binds primarily to mu-opioid receptors, together with the previously reported phenomenon that morphine modulation of immune functions also exists in mu-opioid receptor knockout mice, the present study confirms that opioids such as morphine may exert their effects through multiple opioid receptor types and that the effects of morphine or endogenous opioids on immune cells could not be simply adduced from the anticipated effects of a synthetic, selective opioid receptor ligand.

Morphine upregulates mu opioid receptors of human and monkey lymphocytes.

Opioid receptors of subtypes delta, kappa, and mu similar to those found in brain cells have been identified in immune cells. The current study demonstrates by competitive polymerase chain reaction the treatment of human lymphocytic cells with morphine resulting in an increased amount of gene expression of mu opioid receptors. Antibodies against the MOR-1, the neuronal mu opioid receptor, were used in Western blot analysis of mu proteins and the results revealed a single band of approximately 50 kDa, the intensity of which was increased by morphine treatment. Similar results of mu opioid receptor activation were observed when monkey lymphocytes were treated with morphine. These studies suggest that in addition to causing an immune effect through communication with the neuroendocrine system, the psychoactive drug morphine may modulate immune functions by acting directly on the mu opioid receptors expressed on lymphocytes.

Morphine induces gene expression of CCR5 in human CEMx174 lymphocytes.

All HIV-1 strains studied to date use CCR5, CXCR4, or both receptors to enter cells. Simian immunodeficiency virus (SIV) infection of non-human primates has served as a useful model for understanding AIDS pathogenesis in humans. Research on several genetically divergent SIV isolates has revealed that SIV uses CCR5, and not CXCR4, for entry. CEM x174, a human lymphoid cell line, has been routinely used to cultivate and maintain various SIV strains. However, questions have arisen about how CEM x174, which reportedly was unable to express detectable amounts of CCR5 transcripts, efficiently supports the growth of SIV. In searching for an answer, we resorted to a sensitive competitive reverse transcriptase-polymerase chain reaction procedure in an attempt to detect as well as quantify the amount of CCR5 expression. Here we present our findings, which indicate that CEM x174 indeed expresses CCR5 and that the amount of CCR5 is increased in cells pretreated with morphine. These results correlate well with our previous observations that morphine treatment causes CEM x174 cells to be more susceptible to SIV infection. Similar morphine effect was not observed on CEM x174 cells infected with simian retroviruses, which do not depend on CCR5 for entry. These findings suggest a plausible mechanism whereby opiate drug users render themselves more susceptible to HIV infection, thereby explaining the vast prevalence of HIV infection among endemic drug use populations.

Opioids suppress chemokine-mediated migration of monkey neutrophils and monocytes - an instant response.

Opioid users having acquired human immunodeficiency syndrome (AIDS) are at a greater risk than non-users of contracting opportunistic infections. Opioid-administered and simian immunodeficiency virus (SIV)-infected rhesus monkeys have been an excellent model for studying AIDS and drug abuse in humans. In this study, chemotaxis of monkey leukocytes was evaluated using the chemokines interleukin-8 (IL-8) and regulated upon activation, normal T cell expressed (RANTES) as the chemoattractants, and the effects of various opioid agonists and antagonists on the efficiency of chemotaxis were examined. Opioids were either incubated with monkey leukocytes or added directly to chemokines, and the number of cells migrating toward IL-8 (for neutrophils) or RANTES (for monocytes) was scored. Inhibition of chemotaxis was seen with both assay conditions, and the inhibition was mediated by opioids binding to mu or kappa receptors. Binding to delta opiod receptors was rarely, if ever, observed. Although opioids themselves may act as weak chemoattractants for monkey leukocytes, addition of opioid agonists to chemokines would reduce the chemoattractant ability of the chemokines. Opioids did not cause the same inhibitory effect on the chemotactic migration of neutrophils when the complement component C5a or the chemotactic peptide N-formyl-MET-LEU-PHE (fMLP) was used as chemoattractant. These studies suggest that the presence of opioids during SIV infection immediately alters chemokine-mediated immune functions.

Modulation of CPP32 activity and induction of apoptosis in Human CEM X 174 lymphocytes by heptachlor, a chlorinated hydrocarbon insecticide.

Heptachlor is an organochlorine insecticide used worldwide for the control of pests both agriculturally and domestically. Its lipophilic structure allows it to bioaccumulate and pass through the food chain, exposing those who come in contact with it to its tumor promoting and possible carcinogenic effects. As a mechanism of tumor promotion, we explored the possibility of heptachlor suppressing the apoptotic process in human CEM x 174 lymphocytes. In this article, we describe the effect of heptachlor on the activity of the apoptosis protease CPP32. We show that heptachlor by itself was able to stimulate CPP32 activity at relatively high concentrations. When combined with the chemotherapeutic agent doxorubicin, a known CPP32 activator, a dual effect was observed. Low concentrations of heptachlor (5 microM-10 microM) suppressed doxorubicin-induced CPP32 activity, and high concentrations of heptachlor (80 microM-120 microM) augmented it. We also showed that heptachlor alone at relatively high concentrations induced apoptosis-associated changes in CEM x 174 cells including high molecular weight (HMW) DNA cleavage and chromatin condensation. From these results, it appears that heptachlor has tumor promoting-like effects at lower concentrations, and at higher concentrations induces apoptosis as a mechanism of cytotoxicity.

Detection of SIV DNA in rhesus macaque polymorphonuclear neutrophils.

The extent of infection of monkey polymorphonuclear neutrophils (PMN) by simian immunodeficiency virus (SIV) has not yet been determined. Using the polymerase chain reaction (PCR) technique, we detected the presence of SIVmac239 DNA in rhesus macaque-derived PMN after 24 hrs of in vitro incubation of the cells with SIVmac239. Infection by SIVmac239 also down-regulated the expression of the bcl-2 apoptosis-blocking gene in the infected PMN. These SIVmac239-induced PMN intracellular alterations were correlated with an accelerated decrease in PMN viability over a period of 120 hrs compared to non-infected PMN. Evidence of chromatin condensation characteristic of programmed cell death (apoptosis) was also observed in SIVmac239-infected PMN. The results of this study provide a mechanism for the reduced chemotaxis/phagocytosis activities of PMN of SIVmac239-infected macaques and suggest that PMN is one of the target cells for SIVmac239 infection.

Effect of the chlorinated hydrocarbons heptachlor, chlordane, and toxaphene on retinoblastoma tumor suppressor in human lymphocytes.

Organochlorine use over the past 50 years has resulted in the contamination of soil, water, plant and animal species. This contamination has created a long-lasting environmental problem, as the members of the organochlorine class of pesticides are resistant to degradation and have been labeled as persistent bioaccumulators. Studies have shown certain organochlorines to be tumor promoters, liver toxicants and to induce immune cell dysfunction in rats and mice. Our laboratory has shown that the organochlorines heptachlor and chlordane affect leukocytic gene expression and differentiation. In this study, experiments with CEM x 174 cells, a hybrid of human T and B cells, were performed to investigate the effects of the tumor promoter heptachlor and its congeners chlordane and toxaphene on retinoblastoma (Rb) gene expression. The results indicated that heptachlor, chlordane or toxaphene, in the range of 10-50 microM, were able to reduce Rb protein levels in a concentration-dependent manner. In the case of heptachlor, the reduction could be seen as early as 12 h and was time-dependent. Analysis of Rb mRNA levels revealed no detectable difference over the same concentration range. These results suggest that members of the organochlorine class are able to downregulate Rb expression at the post-transcriptional level, an effect similar to that on p53 tumor suppressor previously reported by our laboratory.

Differential regulation of the major cyclin-dependent kinases, cdk2 and cdc2, during cell cycle progression in human lymphocytes exposed to heptachlor.

Heptachlor, a chlorinated hydrocarbon insecticide, has been considered an environmental contaminant with potential adverse health effects. Exposure to heptachlor may impair immune functions including the inhibition of leukocyte chemotaxis, which causes defects in host defense mechanisms. This study addresses the effects of heptachlor on the cell cycle progression of human lymphocytes. It has been found that addition of heptachlor to cultured lymphocytic cells prevents the cells from progression into the S phase of the cell cycle, with a concomitant accumulation of cells in G1 phase. An accompanying decrease (deactivation) in cyclin-dependent kinase cdk2 and dephosphorylation (activation) of cdc2 was observed. The altered cell cycle progression may trigger the cell's apoptotic potential, as indicated by the reduced amount of Bcl-2 synthesized inside heptachlor-treated cells. The interference of cell cycle progression by heptachlor was also seen with chlordane and toxaphene, two other chlorinated hydrocarbon insecticides.

Inhibition of chemokine-induced chemotaxis of monkey leukocytes by mu-opioid receptor agonists.

It is recognized that chemotaxis and phagocytosis constitute the first line of defense in the immune system, and chemokines function mainly as chemoattractants for phagocytic cells, recruiting monocytes and neutrophils from the blood to sites of infection. In this study, chemotaxis of monkey leukocytes was evaluated using human chemokines IL-8 (interleukin-8), MIP-1 beta and RANTES as the chemoattractants, and the effects of micro-opioid receptor agonists, morphine, DAMGO, methadone and endomorphine, on the efficiency of chemotaxis were examined. It was found that human chemokines served well as chemoattractants for monkey leukocytes, and similar to the human system, chemokine-induced chemotaxis of monkey leukocytes was inhibited in the presence of micro-opioid receptor agonists. The inhibition could be reversed by naloxone, a specific micro-opioid receptor antagonist. These studies further support the value of the monkey model for drug abuse studies in humans, as well as suggest that opioids such as morphine may alter immune functions through micro-opioid receptors on leukocytes.

Suppression of chemokine-induced chemotaxis of monkey neutrophils and monocytes by chlorinated hydrocarbon insecticides.

Chemokines, characterized as pro-inflammatory chemicals made by the immune system, consist of a family of low molecular weight proteins with potent in vitro chemotactic activity causing leukocyte accumulation in vivo. This study determines the effects of organochlorine pesticide exposure on the chemotactic functions of monkey neutrophils and monocytes, using a 48-well chemotaxis chamber. Chemokines IL-8 (interleukin-8) and RANTES were used as the chemoattractants to induce chemotaxis among these monkey leukocytes. Monkey neutrophils or monocytes were first treated with heptachlor, chlordane or toxaphene for 1 hour at 37 degrees C, and the number of cells migrating toward 200 ng/ml IL-8 (for neutrophils) or 100 ng/ml RANTES (for monocytes) were scored. Inhibition of chemotaxis was seen with all samples after treatment with heptachlor, chlordane and toxaphene at concentrations from as low as 10(-14) M to 10(-5) M. Among the three compounds studied, toxaphene was the least effective in preventing monocytes from migrating toward RANTES. The ability of these pesticides to inhibit chemotaxis did not correlate directly with their potential apoptotic effects on the monkey leukocytes. These studies suggest that exposure to organochlorine pesticides may alter leukocyte-related immune functions.

Heptachlor and the mitogen-activated protein kinase module in human lymphocytes.

The organochlorine pesticide heptachlor constitutes a potential health hazard because of its persistence in nature, its reported contamination in food and milk, and its possible carcinogenic effects. As a tumor promoter, heptachlor induces human myeloblastic leukemia cells to differentiate, and also down-regulates the tumor suppressor gene p53 in human immune cells. In this study, the heptachlor signaling pathway in human lymphocytes was studied. Addition of heptachlor to human CEM x174 lymphocytic cells reduced the cellular levels of MAP kinase (MAPK, mitogen-activated protein kinase) cascade proteins, including ERK1 (a 44-kDa MAPK), ERK2 (a 42-kDa MAPK), a 85-kDa and a 54-kDa MAP kinase, MEK1 (a 45-kDa ERK kinase) and MEKK (a 78-kDa MEK kinase). However, heptachlor treatment caused a marked increase in the expression of the activated (Thr- and Tyr-dually phosphorylated) ERK1 and ERK2 in the cells. These studies indicate that mitogen-activated protein kinases are important intermediates in the signal transduction pathway of immune cells upon heptachlor exposure, and the observation of stimulation of activated MAP kinases without a simultaneous accumulation of basal enzymes may suggest the involvement of a negative feedback control mechanism in the pathway.

The effect of heptachlor, a chlorinated hydrocarbon insecticide, on p53 tumor suppressor in human lymphocytes.

Previous studies have shown that heptachlor, a chlorinated hydrocarbon insecticide, is a liver tumor promoter in rats and mice and induces tumor promoting-like alterations in human myeloblastic leukemia cells. The nature of tumor promotion is multifaceted and has recently been shown to include suppression of programmed cell death (apoptosis) as a mechanism by which a tumor promoter can prolong cell viability. The ability of tumor promoters to suppress apoptosis prompted us to address the question of whether heptachlor is capable of effecting the expression of genes involved in lymphocyte apoptosis, in particular, the p53 tumor suppressor gene. Experiments with a CEM x 174 cell line, a hybrid of human T and B cells, revealed that heptachlor downregulated p53 gene expression at the post-transcriptional level without changing levels of mRNA in the cells. The heptachlor-induced reduction in the basal levels of expression of this gene was both in a concentration and time-dependent manner.

Induction and activation of mitogen-activated protein kinases of human lymphocytes as one of the signaling pathways of the immunomodulatory effects of morphine sulfate.

Morphine sulfate causes immunomodulatory and immunosuppressive effects in human. In this study, the signaling pathway involved in these morphine effects was studied. Addition of morphine sulfate to human CEMx174 lymphocytic cells resulted in increased expression of mitogen-activated protein kinase cascade proteins. Morphine enhanced the cellular levels of ERK1 (44 kDa), ERK2 (42 kDa), a 54-kDa ERK, MEK1 (45 kDa), and MEKK (78 kDa). A time-dependent increase in the activated (Thr and Tyr dually phosphorylated) state of ERK1 and ERK2 was also observed. Naloxone, a morphine antagonist, reversed the observed morphine effects, implicating a micro opioid receptor-mediated process. These findings suggest that mitogen-activated protein kinases are important intermediates in signal transduction pathways initiated by morphine receptors in immune cells.

SIV infection of macaques: a model for studying AIDS and drug abuse.

Rhesus monkeys (Macaca mulatta) and the simian immunodeficiency virus (SIV) were used as an animal model system to evaluate longitudinally the effects of opioid dependence on the development of AIDS. Results have shown that in addition to weakening the host's T cell and PMN functions, chronic opioid treatment of SIVmac239-infected animals caused (a) an increased virus replication rate, (b) an increased rate of viral mutation, (c) a nascent humoral (antibody) response against mutated autologous strains of virus, (d) an alteration of CD8 + cell-mediated immunity toward the mutant virus, (e) an increased tolerance of AZT among the infectious virus and (f) a shorter life span for the infected animals. These results suggest that chronic administration of opioids alters general fundamental aspects of the AIDS viremia.

Mu opioid receptor gene expression in immune cells.

We have previously demonstrated that administering morphine sulfate to rhesus monkeys alters the cell-mediated as well as humoral immune responses of these primates. Furthermore, morphine treatment greatly reduces the chemotactic and phagocytotic activities of primate polymorphonuclear (PMN) cells. The present study describes the identification and isolation of mRNA encoding the mu opioid receptor gene sequence from human and monkey immune cells. Through the use of primer sequences designed from the human brain mu opioid receptor cDNA sequence, specific opioid receptor segments in mRNA transcripts were amplified, cloned, and sequenced. The mu opioid receptor gene was therefore found expressed in the following cell types: CEM x174 (a hybrid of human T and B cells), Raji (human B cells), human CD4+ cells, human monocytes/macrophages, human PMN, monkey peripheral blood mononuclear cells (PBMC), and monkey PMN. These studies present the first evidence to demonstrate that cells of human and monkey immune systems constitutively express mu opioid receptor mRNA.

Expression of kappa opioid receptors in human and monkey lymphocytes.

mRNA encoding the kappa opioid receptor gene sequence was identified and isolated from various human lymphocytic cells: CEM x174 (a hybrid of T and B origin) cells, Jurkat-T4 cells, human peripheral blood mononuclear cells (PBMC) and purified CD4+ cells. Analyzing the cDNA sequences of RNA transcripts spanning the putative second extracellular loop, which has reported dynorphin specificity, and the seventh transmembrane domain revealed a 100% and 95% homology in amino acid sequence to corresponding kappa opioid receptor sequences in human placenta and rat brain, respectively. Expression of a similar kappa opioid receptor sequence could be detected in normal monkey PBMC but not in monkey PBMC in which the CD4+/CD8+ cell ratio (or CD4+ cell number) was significantly reduced due to prolonged SIV infection. These findings suggest that human and monkey lymphocytes constitutively express kappa opioid receptor mRNA.

Inhibition of simian immunodeficiency virus (SIV) replication by CD8+ cells of SIV-infected rhesus macaques: implications for immunopathogenesis.

The ability of the CD8+ cells from simian immunodeficiency virus (SIV)-infected rhesus macaques to inhibit SIV replication was investigated. Inhibition was produced by a heat-stable soluble factor of molecular size greater than 10kDa. CD8+ supernatants from some macaques were found not only to suppress SIV growth but also to be cytolytic toward both infected and uninfected CD4+ cells. Such indiscriminate CD8+ cell-mediated cell killing may therefore account for DC4+ cell depletion in certain SIV-infected macaques.