Linda F. Chuang

Morphine Induces Gene Expression of CCR5 in Human CEM x174 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.

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 μ 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.

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.

Morphine suppresses lymphocyte apoptosis by blocking p53-mediated death signaling

Opiates such as morphine or heroin may promote cell apoptosis and cause dysfunction of immune cells. In simian immunodeficiency virus (SIV)-infected lymphocytic cells, however, morphine may protect the cells from apoptotic lysis and allow the virus to continue to replicate. To further explore this apparently antithetical effect of opiates, we evaluated in the present study the effects of morphine on human lymphocytic CEM x174 cells induced to undergo apoptosis in the presence of actinomycin D. It was found that induction of apoptosis (characterized by DNA laddering) by actinomycin D was accompanied by a stimulation of the expression of active (phosphorylated) form of p53. Pretreatment of the cells with 10nM morphine caused a transient, naloxone-reversible suppression of the appearance of activated p53 and the generation of DNA laddering. Parallel evaluation of the growth of CEM x174 indicated that morphine treatment delays the inception of cell death triggered by actinomycin D. Inasmuch as Bcl-2 suppresses while Bax accelerates apoptosis, treatment of cells with morphine reduced the expression of Bax and enhanced the expression of Bcl-2. Taken together, morphine, through binding at the opioid receptor, may protect lymphocytic cells from apoptotic lysis if cell death is initiated by apoptosis-inducing agents such as human immunodeficiency virus (HIV), SIV or actinomycin D.

Methadone induces CCR5 and promotes AIDS virus infection

Methadone, a regimen for the treatment of opioid dependency, was found to induce the expression of CCR5, a co‐receptor for human immunodeficiency virus (HIV)/simian form of HIV (SIV) entry, on human CEM x174 lymphocytes. Both CCR5 mRNA and protein were elevated in methadone‐treated cells. A concomitant increase of mu opioid receptors was also observed. Upon methadone exposure, SIVmac239‐infected CEM x174 cells released greater amounts of virus particles as revealed by both the number of syncytia formation and reverse transcriptase activities. Similar methadone effect was not observed on CEM x174 cells infected with other simian retroviruses that do not depend on CCR5 for cellular entry. These studies raise concerns considering methadone as an innocuous morphine substitute.

Cremophor EL, a widely used parenteral vehicle, is a potent inhibitor of protein kinase C

Cremophor EL, a castor oil derivative, has been considered a non-toxic solubilizer for lipophilic drugs and vitamins. Protein kinase C, a phospholipid/Ca++-dependent protein kinase, is known to phosphorylate, in response to extracellular stimuli, a variety of proteins for cellular functions. The present study shows that Cremophor EL selectively inhibits the activity of protein kinase C in vitro. The potency of this selective inhibition is greater than that of other protein kinase C-specific inhibitor thus far reported. Cremophor EL acts primarily on the enzyme activator diacylglycerol (or the phorbol ester) and prevents the latter from both interacting with the phospholipid and binding to protein kinase C. This is the first report of a significant biological activity induced by this widely used substituted castor oil solubilizer.

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.

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.

Protein kinase C phosphorylates leukemia RNA polymerase II.

Purified RNA polymerase II from chicken leukemia cells was found to be an effective substrate for protein kinase C but not cAMP-dependent protein kinase. Protein kinase C catalyzed the incorporation of 1-2 mol of phosphate per mol of polymerase II and the reaction was totally calcium and lipid dependent. Electrophoresis studies revealed a time-dependent increase of phosphate incorporation into RNA polymerase II subunits of 220 KDa, 180 KDa and 150 KDa, with a preferential phosphorylation of the 180 KDa polypeptide. The phosphorylated enzyme has a preference for using single-stranded DNA as the template for transcription, including transcription of the single-stranded myb oncogene sequence. Phosphoamino acid analysis indicated that both serine and threonine residues were phosphorylated at equal amounts. Phosphorylation by protein kinase C increased the affinity of substrate-polymerase binding and the initial rate of RNA synthesis, suggesting a mechanism by which gene expression can be activated by protein kinase C.

Detecting bluetongue virus RNA in cell culture by dot hybridization with a cloned genetic probe.

A 70% copy of genome segment 7 of bluetongue virus (BTV)-17 has been cloned into the plasmid pBR-322. This cloned BTV segment when used as a radioactive probe will hybridize to BTV double-stranded RNA extracted from cell cultures and dotted onto nitrocellulose paper. This dot hybridization technique is therefore suitable for detecting and identifying BTV in cell culture. The specificity of cloned probes is discussed in relation to detecting gene sequences specific for either the bluetongue serogroup or different serotypes of BTV.