Kathy McAllen

Nicotine administration enhances NPY expression in the rat hypothalamus

Epidemiological studies have shown an inverse relationship between cigarette smoking and body weight. In rodents, a negative correlation between nicotine and body weight has been reported, but this observation was largely derived from studies where relatively high doses of nicotine ( approximately 12 mg/kg/day) were used. In the current study, we showed that a negative relationship also holds for low doses of nicotine that are comparable to that consumed by average human smokers (<6 mg/kg/day). We also demonstrated that 14 days of nicotine administration (4 mg/kg/day) reduced average daily food intake by 19.5% (P<0.01) in the free-feeding nicotine-treated group compared to saline controls. No significant differences in body weight were detected between the nicotine-treated and pair-fed groups. To determine whether the effects of nicotine on food intake and body weight were related to neuropeptide Y (NPY) expression, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay were utilized to measure NPY mRNA and peptide levels in various regions of the hypothalamus. Significantly higher levels of NPY mRNA (ca. 20-50%) and peptide (ca. 24-69%) were only detected in the nicotine-treated groups. In addition, significantly higher NPY contents were also obtained in two hypothalamic areas of pair-fed control animals. In summary, our data suggest that the pharmacological effects of nicotine on food intake and body weight may be mediated by changes in hypothalamic NPY levels, a neuropeptide that is pivotal to the hypothalamic regulation of food intake.

Immunofluorescence Detection of δ Opioid Receptors (DOR) on Human Peripheral Blood CD4+ T Cells and DOR-Dependent Suppression of HIV-1 Expression

The δ opioid receptors (DORs) modulate T cell proliferation, IL-2 production, chemotaxis, and intracellular signaling. Moreover, in DOR-transfected Jurkat cells, δ opioids have been shown to suppress HIV-1 p24 Ag expression. These observations led us to characterize the expression of DORs by human peripheral blood T cells and to determine whether a specific DOR agonist, benzamide,4-{[2,5-dimethyl-4-(2-propenyl)-1-piperazinyl](3-methoxyphenyl)methyl]-N,−,{2S[1(S*),2α,5β]}-(9Cl) (SNC-80), can suppress p24 Ag expression by HIV-1-infected CD4+ T cells obtained from normal donors. By immunofluorescence flow cytometry, PHA stimulated the expression of DOR from 1.94 ± 0.70 (mean ± SEM) to 20.70 ± 1.88% of the PBMC population by 48 h (p < 0.0001). DOR expression was ∼40% of both the PHA-stimulated CD4+ and CD8+ T cell subsets, and virtually all DORs were found on these subsets. To determine whether activated DORs suppress HIV-1 expression, PBMC were prestimulated with PHA, and then CD4+ T cells were purified, pretreated with SNC-80, and infected with HIV-1. In a concentration-dependent manner, SNC-80 inhibited production of p24 Ag. SNC-80 10−10 M maximally suppressed (∼50%) both lymphocytotropic (HIV-1 MN) and monocytotropic (SF162) strains; higher concentrations were less effective. Naltrindole, a selective DOR antagonist, abolished the inhibitory effects of SNC-80. Kinetic studies indicated that 24-h pre- or postincubation with SNC-80, relative to infection with HIV-1, eliminated its suppressive effects. Thus, stimulating the DORs expressed by activated CD4+ T cells significantly suppressed the expression of HIV-1. These findings suggest that opioid immunomodulation directed at host T cells may be adjunctive to standard antiviral approaches to HIV-1 infection.

Regulation of delta opioid receptor expression by anti-CD3-epsilon, PMA, and ionomycin in murine splenocytes and T cells.

Previous studies have shown that low levels of delta opioid receptor (DOR) mRNA were detectable by reverse transcription polymerase chain reaction (RT‐PCR) in unstimulated splenocytes from BALB/c female mice. This study demonstrates that DOR transcripts can be detected in freshly obtained splenocytes from CD1 female mice as well. The results of studies using quantitative competitive RT‐PCR showed that DOR transcripts in splenic T cells increased from < 1 copy/cell to 22 and 42 copies/cell, respectively, after stimulation with anti‐CD3‐ε for 24 and 48 h compared to the level in freshly obtained T cells. In the presence of actinomycin D, anti‐CD3‐ε did not affect the rate of degradation of DOR mRNA, suggesting that its stability is not altered by anti‐CD3‐ε. After incubation with phorbol myristate acetate (PMA) and ionomycin, the expression of DOR mRNA in splenocytes and T cells was significantly reduced compared with unstimulated cells in culture. In addition, the inhibitory effect of PMA prevented anti‐CD3‐ε‐stimulated DOR expression. These data suggest that signaling through the T cell receptor complex by anti‐CD3‐ε regulates DOR expression differently than PMA and ionomycin. J. Leukoc. Biol. 65: 707–714; 1999.

Signaling through Delta Opioid Receptors on Murine Splenic T Cells and Stably Transfected Jurkat Cells

Abstract: β‐Endorphin (β‐EP) and delta opoid receptor (DOR) agonists affect immune functions such as lymphocyte chemotaxis, proliferation, and cytokine production. Recent studies indicate that both neuronal DOR and novel G‐protein‐coupled receptors with high affinity for β‐EP and DOR agonists are expressed by mononuclear cells. In addition, proenkephalin A mRNA and enkephalin‐related peptides are expressed by lymphocytes. These investigations were conducted to identify signal transduction pathways that mediate the effects of β‐EP and DOR agonists on T cells. Calcium mobilization was studied because it is central to T‐cell activation initiated by antigen presentation to the T‐cell receptor (TCR). Using the calcium‐sensitive dye Fluo‐3 and flow cytofluorometry to determine the concentration of free intracellular calcium, physiological concentrations of β‐EP were shown to enhance concanvalin. A (con A)‐stimulated calcium mobilization by murine splenic T cells (p < 0.01). The DOR antagonist, naltrindole, inhibited this, whereas CTAP, a selective mu OR antagonist, was ineffective. In addition, N‐Ac‐β‐EP and the μ OR agonist DAMGO, failed to mimic the effects of β‐EP. Although it was less potent than β‐EP, DADLE, a DOR agonist, also enhanced Con‐A‐induced calcium mobilization (p < 0.01). A DOR‐transfected human T‐cell line (DOR‐Jul.1) was developed to study signal transduction. Both DADLE and the selective DOR agonist, deltorphin, rapidly increased intracellular free calcium concentrations; ED50s were 10−9 M. Pertussis toxin prevented the response, and EGTA significantly reduced it. In addition, DADLE inhibited forskolin‐stimulated cAMP production (ED50: 10−11 M). These findings with normal splenic T cells and DOR‐transfected T‐cell line indicate that β‐EP and DOR agonists affect calcium mobilization. This is likely to modulate downstream pathways that regulate T‐cell activation and function.

Phosphorylation of activating transcription factor in murine splenocytes through delta opioid receptors.

Delta opioid receptors (DORs) modulate TCR signaling through the mitogen-activated protein kinases (MAPKs), ERKs 1 and 2. These studies determined whether a DOR agonist alone ([D-Ala(2)-D-Leu(5)]enkephalin; DADLE) affects phosphorylation of the activating transcription factor (ATF-2) and its interaction with the MAPK, c-Jun NH(2)-terminal kinase (JNK). DOR expression was induced on murine splenocytes by anti-CD3 and then quiescent cells were treated with DADLE. DADLE, itself, dose-dependently induced maximal phosphorylation of ATF-2 within 5-10min; naltrindole, a specific antagonist, abolished this. Anti-ATF-2 immunoprecipitates from control and DADLE-treated splenocytes showed a dominant 59kDa phosphorylated band and a 71kDa band. DADLE stimulated phosphorylation of both bands, although the 71kDa band was selectively immunoprecipitated by anti-JNK. Thus, DADLE stimulated phosphorylation of 71kDa ATF-2 and its association with JNK, suggesting that JNK is activated through DORs. Along with previous observations, these studies suggest that lymphocyte DORs can affect the activation of MAPKs by TCR-independent stimulation (e.g., JNK) or indirectly by modulating TCR-dependent stimulation (e.g., ERK).

Stromal cell-derived factor 1-α (SDF)-induced human T cell chemotaxis becomes phosphoinositide 3-kinase (PI3K)-independent: role of PKC-θ

Stromal cell‐derived factor 1α (SDF‐1α) is the exclusive ligand for the chemokine receptor CXCR4. This receptor plays a pivotal role in immune responses, the pathogenesis of infection such as HIV, and cellular trafficking. However, the signaling mechanisms regulating SDF‐driven T cell migration are not well defined. In this study, we determined the role of PI3K and protein kinase C‐ θ (PKC‐θ) in SDF‐induced human T cell migration in fresh versus cultured T cells. Purified human T cells (fresh vs. 48 h in media, unstimulated or activated by anti‐CD3+anti‐CD28) were used. Western blots showed that SDF induced phospho‐(p)‐Akt [threonine (Thr)308 and serine 473], a proxy for PI3K activity, in fresh cells and p‐PKC‐θ in 48 h unstimulated cells. LY294002 (PI3K inhibitor) reduced SDF‐induced chemotaxis in fresh cells by 51%, whereas it minimally affected chemotaxis in 48 h unstimulated or activated cells. However, a specific PKC‐θ inhibitor, pseudosubstrate for PKC‐θ, reduced chemotaxis in 48 h unstimulated and stimulated T cells by 72% and 87%, respectively. Thus, chemotaxis becomes independent of PI3K signaling in human T cells cultured for 48 h. Under these conditions, PKC‐θ is phosphorylated (Thr538) by SDF, and chemotaxis becomes largely PKC‐θ‐dependent.

Expression of Delta Opioid Receptors and Transcripts by Splenic T Cells

Abstract: Delta opioid receptors (DORs) and preproenkephalin‐A‐derived opiate peptides are expressed by mononuclear cells in various lymphoid organs. DOR ligands modulate a variety of immune functions, such as T‐cell proliferation, calcium mobilization, and cytokine production. recently, quiescent T‐cells were found to express low levels of DOR transcripts, which increased due to the following: cell culture of unstimulated murine splenocytes (depending on cell density); cross‐linking the T‐cell receptor (TCR) with anti‐CD3‐ε and a single in vivo exposure to staphylococcal enterotoxin B (SEB). Enhanced expression of DOR mRNA was mediated transcriptionally. Moreover, PMA + ionomycin, which mimic the proliferative signal of anti‐CD3, inhibited the expression of DOR mRNA. Using semiquantitative immunofluorescence to detect DORs, SEB was found to increase the fraction of T cells that expressed DOR and to enhance the relative level of DOR expression per T cell. Previous studies have shown that DOR agonists inhibited the anti‐CD3‐stimulated production of interleukin‐2 and T‐cell proliferation. Therefore, the enhanced expression of DORs by activated T cells may be capable of downregulating the T‐cell activation program.