Amber D. Steele

μ-opioid modulation of HIV-1 coreceptor expressionand HIV-1 replication

A substantial proportion of HIV-1-infected individuals are intravenous drug users (IVDUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the μ-opioid receptor. Our results show that DAMGO, a selective μ-opioid agonist, increases CXCR4 and CCR5 expression in both CD3+ lymphoblasts and CD14+ monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the μ-opioid receptor based on the ability of a μ-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of μ-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression.

Bidirectional Heterologous Desensitization of Opioid and Chemokine Receptors

Abstract: Opioids are known to suppress a number of elements of the immune response, including antimicrobial resistance, antibody production, and delayed‐type hypersensitivity. Phagocytic cells may be particularly susceptible to opioid administration, since reduced production of the cytokines IL‐1, IL‐6 and TNF‐α, monocyte‐mediated phagocytosis, and both neutrophil and monocyte chemotaxis have all been well established. Earlier studies have shown that both μ‐ and δ‐opioid agonists induce a chemotactic response in monocytes and neutrophils. In addition, μ‐ and δ‐opioid administration inhibited the chemotactic response of these cell populations to a number of chemokines through a process of heterologous desensitization. We report here that μ‐, δ‐, and κ‐opioid agonists also induce a chemotactic response in T lymphocytes. Using the human T‐cell line Jurkat, we have confirmed previous observations that pre‐incubation with met‐enkephalin (MetEnk), an endogenous opioid agonist, prevents the subsequent chemotactic response to the chemokine RANTES. On the other hand, treatment with MetEnk does not alter the response to the chemokine SDF‐1α. Moreover, we found that pretreatment with RANTES prevented a subsequent response of monocytes to the μ‐opioid agonist DAMGO. These results suggest that activation of members of the opioid and chemokine receptor families leads to downregulation of each others leukocyte migratory activities.

Selective inactivation of CCR5 and decreased infectivity of R5 HIV-1 strains mediated by opioid-induced heterologous desensitization.

The opiates are well‐established immunomodulatory factors, and recent evidence suggests that μ‐ and δ‐opioid receptor ligands alter chemokine‐driven chemotactic responses through the process of heterologous desensitization. In the present report, we sought to examine the capacity of μ‐ and δ‐opioids to modulate the function of chemokine receptors CCR5 and CXCR4, the two major human immunodeficiency virus (HIV) coreceptors. We found that the chemotactic responses to the CCR1/5 ligand CCL5/regulated on activation, normal T expressed and secreted, but not the CXCR4 ligand stromal cell‐derived factor‐1α/CXCL12 were inhibited following opioid pretreatment. Studies were performed with primary monocytes and Chinese hamster ovary cells transfected with CCR5 and the μ‐opioid receptor to determine whether cross‐desensitization of CCR5 was a result of receptor internalization. Using radiolabeled‐binding analysis, flow cytometry, and confocal microscopy, we found that the heterologous desensitization of CCR5 was not associated with a significant degree of receptor internalization. Despite this, we found that the cross‐desensitization of CCR5 by opioids was associated with a decrease in susceptibility to R5 but not X4 strains of HIV‐1. Our findings are consistent with the notion that impairment of the normal signaling activity of CCR5 inhibits HIV‐1 coreceptor function. These results have significant implications for our understanding of the effect of opioids on the regulation of leukocyte trafficking in inflammatory disease states and the process of coreceptor‐dependent HIV‐1 infection. The interference with HIV‐1 uptake by heterologous desensitization of CCR5 suggests that HIV‐1 interaction with this receptor is not passive but involves a signal transduction process.

μ-Opioid Induction of Monocyte Chemoattractant Protein-1, RANTES, and IFN-γ-Inducible Protein-10 Expression in Human Peripheral Blood Mononuclear Cells

Strong evidence for the direct modulation of the immune system by opioids is well documented. μ-Opioids have been shown to alter the release of cytokines important for both host defense and the inflammatory response. Proinflammatory chemokines monocyte chemoattractant protein-1 (MCP-1), RANTES, and IFN-γ-inducible protein-10 (IP-10) play crucial roles in cell-mediated immune responses, proinflammatory reactions, and viral infections. In this report, we show that [d-Ala2,N-Me-Phe4,Gly-ol5]enkephalin (DAMGO), a μ-opioid-selective agonist, augments the expression in human PBMCs of MCP-1, RANTES, and IP-10 at both the mRNA and protein levels. Because of the proposed relationship between opioid abuse and HIV-1 infection, we also examined the impact of DAMGO on chemokine expression in HIV-infected cells. Our results show that DAMGO administration induces a significant increase in RANTES and IP-10 expression, while MCP-1 protein levels remain unaffected in PBMCs infected with the HIV-1 strain. In contrast, we show a dichotomous effect of DAMGO treatment on IP-10 protein levels expressed by T- and M-tropic HIV-infected PBMCs. The differential modulation of chemokine expression in T- and M-tropic HIV-1-infected PBMCs by opioids supports a detrimental role for opioids during HIV-1 infection. Modulation of chemokine expression may enhance trafficking of potential noninfected target cells to the site of active infection, thus directly contributing to HIV-1 replication and disease progression to AIDS.

Interactions between opioid and chemokine receptors: heterologous desensitization

The opioid and chemokine receptors are both members of the seven transmembrane G protein-coupled receptor (GPCR) superfamily. Desensitization is believed to be a major element of the regulation of the function of these receptors, and recent findings suggest that both agonist-dependent (homologous) desensitization and heterologous desensitization can control receptor activity. The cross-desensitization between opioid and chemokine receptors has significant implications for our understanding of both the regulation of leukocyte trafficking, as well as the regulation of chemokine receptor function in inflammatory disease states. We also review findings which suggest that pro-inflammatory chemokine receptor-induced heterologous desensitization of opioid receptors has important implications for the regulation of opioid receptor function in the nervous system.

DAMGO-induced expression of chemokines and chemokine receptors: the role of TGF-β1

Studies from a number of laboratories suggest that modulation of cytokine expression plays an integral role in the immunomodulatory activity of opioids. Previously, our laboratory reported that activation of the μ‐opioid receptor induced the expression of CCL2, CCL5, and CXCL10, as well as CCR5 and CXCR4. Previous work has also suggested the possibility that TGF‐β may participate in the opioid‐induced regulation of immune competence, and in the present study, we set out to determine the role of this cytokine in the control of chemokine and chemokine receptor expression. We found that D‐ala2,N‐Me‐Phe4‐Gly‐ol5enkephalin (DAMGO), a highly selective μ‐opioid agonist, induced the expression of TGF‐β1 expression at the protein and mRNA levels. In turn, the addition of TGF‐β1 was found to induce CCL5 and CXCR4 expression but not CCL2, CXCL10, or CCR5. Further analysis showed that pretreatment with neutralizing anti‐TGF‐β1 blocked the ability of DAMGO to induce CCL5 or CXCR4. Similarly, pretreatment with cycloheximide prevented CCL5 or CXCR4 mRNA expression, consistent with the observation that DAMGO induction of chemokine and chemokine receptor expression requires newly synthesized TGF‐β1 protein. These results describe a common molecular basis for the activation of chemokine and chemokine receptor expression and may permit the development of strategies to inhibit certain undesirable immunological properties of μ‐opioid agonists such as morphine and heroin.

Nucleotides Part LXX

A new labelling technique attaching fluorescein via a carbamoyl linker directly to the amino groups of the nucleobases was developed. The amino groups were first converted to the phenoxycarbonyl derivatives (10, 15, 19, 58), which reacted under mild conditions with 5-aminofluorescein to give the corresponding N-[(fluorescein-5-ylamino)carbonyl] derivatives (11–14, 16, 17, 20, 59, 60). The introduction of the 5-aminofluorescein residue into properly protected adenylyl-adenosine dimers (39, 40) and trimer (50) worked well, and final deprotection of these uniformly blocked precursors led on treatment with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), in one step to dimer 41 and trimer 51. Synthesis of an appropriately protected monomeric phosphoramidite building block (75) was more difficult, since introduction of the 2-(4-nitrophenyl)ethyl residue into the fluorescein moiety in 59 led mainly to trisubstitution to give 61 including the urea function. Formation of the adenylyl dimer 66 and trimer 67 proceeded in the usual manner by phosphoramidite chemistry; however, deprotection of 67 with DBU was incomplete since the O-alkyl group at the urea moiety was found to be very stable. Finally, the appropriate phosphoramidite building block 75 could be synthesized by the sequence 5972737475. The phosphoramidite 75 was used for the synthesis of dimer 77 and trimer 79 by solution chemistry, as well as for that of various oligonucleotides by the machine-aided approach on solid support carrying the fluorophore at different positions of the chain (84–87). The attachment of the fluorescein fluorophor via a short carbamoyl linker onto the 6-amino group of 2′-deoxyadenosine enables such molecules to function very well in fluorescence-polarization experiments.

Transcriptional regulation of the major HIV‐1 coreceptor, CXCR4, by the κ opioid receptor

Previous studies have demonstrated that KOR activation results in decreased susceptibility to infection by HIV‐1 in human PBMCs. In the present studies, we have found this effect is, in part, a result of down‐regulation of the major HIV‐1 coreceptor, CXCR4. Using a combination of biochemical approaches, our results show that CXCR4 protein and mRNA levels were reduced significantly following KOR activation. We evaluated the nature of the signaling pathway(s), which were induced by KOR activation, using transcription factor‐binding array analysis and comparing extracts from control and KOR‐activated cells. We determined that the IRFs and STATs were induced following KOR activation, and these events were important for the inhibition of CXCR4 expression. Using chemical inhibitors and siRNA constructs, we determined that JAK2, STAT3, and IRF2 were critical members of this signal transduction pathway. Immediately following KOR activation, JAK2 was phosphorylated, and this was required for the phosphorylation/activation of STAT3. Moreover, IRF2 mRNA and protein expression were also up‐regulated, and further studies using ChIP analysis showed that IRF2 was induced to bind in vivo to the CXCR4 promoter. This is the first report detailing the initiation of a KOR‐induced JAK2/STAT3 and IRF2 signaling cascade, and these pathways result in substantial down‐regulation of CXCR4 expression. The capacity of KOR to down‐regulate CXCR4 expression may provide a strategy for the development of novel therapeutics for the inhibition of HIV replication.

Novel Peptides Based on HIV-1 gp120 Sequence with Homology to Chemokines Inhibit HIV Infection in Cell Culture

The sequential interaction of the envelope glycoprotein of the human immunodeficiency virus type 1 (HIV-1) with CD4 and certain chemokine coreceptors initiates host cell entry of the virus. The appropriate chemokines have been shown to inhibit viral replication by blocking interaction of the gp120 envelope protein with the coreceptors. We considered the possibility that this interaction involves a motif of the gp120 that may be structurally homologous to the chemokines. In the amino acid sequences of most chemokines there is a Trp residue located at the beginning of the C-terminal α-helix, which is separated by six residues from the fourth Cys residue. The gp120 of all HIV-1 isolates have a similar motif, which includes the C-terminal part of a variable loop 3 (V3) and N-terminal part of a conserved region 3 (C3). Two synthetic peptides, derived from the relevant gp120 sequence inhibited HIV-1 replication in macrophages and T lymphocytes in sequence-dependent manner. The peptides also prevented binding of anti-CXCR4 antibodies to CXCR4, and inhibited the intracellular Ca(2+) influx in response to CXCL12/SDF-1α. Thus these peptides can be used to dissect gp120 interactions with chemokine receptors and could serve as leads for the design of new inhibitors of HIV-1.

Regulation of Chemokine and Chemokine Receptor Expression and Function by Opioids

Opioids exert a broad range of effects on immune responses either directly by altering immune cell function or indirectly by altering the expression of immune regulatory proteins such as cytokines, chemokines, and their respective receptors (reviewed in McCarthy et al.(1)). The expression of -, -, and -opioid receptors (MOR, KOR, and DOR, respectively) by immune cells has been definitely established on the basis of a number of parameters, including the isolation of receptor mRNA, cell binding analysis, and by flow cytometry.(2–5) The molecular basis for the immunomodulatory activities of the opioids has remained incompletely defined up to this time. However, it appears that a major mechanism of opioid-induced immunoregulation is through the control of cytokine and cytokine receptor expression and/or function. More specifically, recent reports suggest that the chemotactic cytokines (chemokines) are a significant target of the opioid-induced effects on the function of the cells of the immune system.