Jian-Ping Lai

Human lymphocytes express substance P and its receptor

We present data demonstrating the gene expression of substance P (SP) and its receptor in human peripheral blood-isolated lymphocytes. Using reverse transcribed polymerase chain reaction (RT-PCR) assay, preprotachykinin-A (substance-P) mRNA is detected in human peripheral blood-isolated lymphocytes. Among the alpha, beta, and gamma transcripts of the SP gene, only the beta and gamma transcripts are detectable in these cells. These RT-PCR amplified transcripts are recognized by Southern blot assay using a specific SP probe. Direct DNA sequence analysis of the RT-PCR products from lymphocytes also confirmed the structure of these transcripts which are identical to those found in human neuronal cells. At the protein level, human lymphocytes produced endogenous SP as determined by an enzyme immunoassay. Capsaicin, a vanillyl fatty acid amide (ingredient of hot pepper), released preformed SP from lymphocytes. In addition, using RT/nested-PCR analysis, we identified the presence of mRNA for neurokinin-1 receptor (the receptor for SP) in human peripheral blood-isolated lymphocytes, which was confirmed by Southern blot and DNA sequencing analysis. The demonstration that human lymphocytes express SP and its receptor support the notion that SP is biologically involved in regulating the functions of these cells in an autocrine fashion.

Full-length and truncated neurokinin-1 receptor expression and function during monocyte/macrophage differentiation

The substance P (SP)-preferring receptor neurokinin-1 receptor (NK-1R) has two forms: a full-length receptor consisting of 407 aa and a truncated receptor consisting of 311 aa. These two receptors differ in the length of the C terminus of NK-1R. We studied the undifferentiated and phorbol myristate acetate (PMA)-differentiated human monocyte/macrophage cell line THP-1 to investigate the expression and function of NK-1R. The expression of full-length and truncated NK-1R in this cell line was determined by using real-time PCR and immunofluorescence staining. Undifferentiated THP-1 cells expressed only truncated NK-1R. The differentiation of THP-1 cells with PMA to a macrophage-like phenotype resulted in the expression of full-length NK-1R, which was functionally accompanied by an SP (10−6 M)-induced Ca2+ increase. In contrast, the addition of SP (10−6 M) did not trigger Ca2+ response in undifferentiated THP-1 cells; however, SP did enhance the CCR5-preferring ligand RANTES (CCL5)-mediated Ca2+ increase. When a plasmid containing the full-length NK-1R was introduced into undifferentiated THP-1 cells, exposure to SP triggered Ca2+ increase, demonstrating that the full-length NK-1R is required for SP-induced Ca2+ increase. The NK-1R antagonist aprepitant (Emend, Merck) inhibited both the SP-induced Ca2+ increase in PMA-differentiated THP-1 cells and the SP priming effect on the CCL5-mediated Ca2+ increase, indicating that these effects are mediated through the full-length and truncated NK-1R, respectively. Taken together, these observations demonstrate that there are unique characteristics of NK-1R expression and NK-1R-mediated signaling between undifferentiated THP-1 cells and THP-1 cells differentiated to the macrophage phenotype.

Identification of a δ isoform of preprotachykinin mRNA in human mononuclear phagocytes and lymphocytes

We have characterized preprotachykinin (PPT-A) gene transcript splicing products and identified a fourth isoform of PPT-A mRNA transcript in human peripheral blood-isolated monocytes and PBL. Using RT-PCR, Southern blot analysis and nucleotide sequencing analysis, we have identified the four isoforms of PPT-A transcripts (alpha, beta, gamma and delta) in human peripheral blood-isolated monocytes and PBL. The delta-PPT transcript present in the immune cells lacks exons 4 and 6. The sequences of exons 3, 5 and 7 of delta-PPT transcript completely match those of beta-PPT transcript. The alpha-PPT and beta-PPT sequences in these cells are identical to those obtained by Tan and Too (GenBank accession number U37539) and Harmar et al. (Genbank accession number X54469), but differ by a single nucleotide from another entry by Chiwakata et al. (Genbank accession number M68906). In comparison to this latter sequence, there was a C-->T change at amino acid position 87 (CCT-->CTT) which may result in a Pro to Leu change. Identification of the new SP mRNA transcript in both human CNS and immune cells supports the concept of an important biological link between CNS and immune system.

Morphine Enhances Hepatitis C Virus (HCV) Replicon Expression

Little information is available regarding whether substance abuse enhances hepatitis C virus (HCV) replication and promotes HCV disease progression. We investigated whether morphine alters HCV mRNA expression in HCV replicon-containing liver cells. Morphine significantly increased HCV mRNA expression, an effect which could be abolished by either of the opioid receptor antagonists, naltrexone or beta-funaltrexamine. Investigation of the mechanism responsible for this enhancement of HCV replicon expression demonstrated that morphine activated NF-kappaB promoter and that caffeic acid phenethyl ester, a specific inhibitor of the activation of NF-kappaB, blocked morphine-activated HCV RNA expression. In addition, morphine compromised the anti-HCV effect of interferon alpha (IFN-alpha). Our in vitro data indicate that morphine may play an important role as a positive regulator of HCV replication in human hepatic cells and may compromise IFN-alpha therapy.

Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP–neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.

Interleukin‐1β upregulates functional expression of neurokinin‐1 receptor (NK‐1R) via NF‐κB in astrocytes

Cytokines and neuropeptides are modulators of neuroimmunoregulation in the central nervous system (CNS). The interaction of these modulators may have important implications in CNS diseases. We investigated whether interleukin‐1β (IL‐1β) modulates the expression of neurokinin‐1 receptor (NK‐1R), the primary receptor for substance P (SP), a potent neuropeptide in the CNS. IL‐1β upregulated NK‐1R expression in human astroglioma cells (U87 MG) and primary rat astrocytes at both mRNA and protein levels. IL‐1β treatment of U87 MG cells and primary rat astrocytes led to an increase in cytosolic Ca2+ in response to SP stimulation, indicating that IL‐1β‐induced NK‐1R is functional. CP‐96,345, a specific non‐peptide NK‐1R antagonist, inhibited SP‐induced rise of [Ca2+]i in the astroglioma cells. Investigation of the mechanism responsible for IL‐1β action revealed that IL‐1β has the ability of activating nuclear factor‐κb (NF‐κB). Caffeic acid phenethyl ester (CAPE), a specific inhibitor of NF‐κB activation, not only abrogated IL‐1β‐induced NF‐κB promoter activation, but also blocked IL‐1β‐mediated induction of NK‐1R gene expression. These findings provide additional evidence that there is a biological interaction between IL‐1β and the neuropeptide SP in the CNS, which may have important implications in the inflammatory diseases in the CNS.

Detection of substance P and its receptor in human fetal microglia

Substance P, the most abundant neurokinin in the CNS, is a major modulator of the immune system. We have examined the gene expression of substance P and its receptor in human fetal brain microglia. Using reverse transcription-polymerase chain reaction and Southern blotting assay, the four isoforms of preprotachykinin-A gene transcripts (alpha, beta, gamma and delta) were detected in the microglia. The human fetal microglia produced significantly higher levels of endogenous substance P protein (640-850 pg/10(6) cells) than did human peripheral blood monocyte-derived macrophages (25-50 pg/10(6) cells), as determined by an enzyme immunoassay. Using immunohistochemical staining with an anti-substance P antibody, cell membrane substance P immunoreactivity was observed. In addition, we identified the presence of messenger RNA for neurokinin-1 receptor, a primary receptor for substance P in human fetal microglia.From these data, we propose that substance P and its receptor are biologically involved in regulating the functions of microglia, and potentially play an important role in host defense of the central nervous system.

Selective Serotonin Reuptake Inhibitor and Substance P Antagonist Enhancement of Natural Killer Cell Innate Immunity in Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome

BACKGROUND Natural killer (NK) cells play an important role in innate immunity and are involved in the host defense against human immunodeficiency virus (HIV) infection. This study examines the potential role of three underlying regulatory systems that have been under investigation in central nervous system research as well as immune and viral research: serotonin, neurokinin, and glucocorticoid systems. METHODS Fifty-one HIV-seropositive subjects were recruited to achieve a representative sample of depressed and nondepressed women. The effects of a selective serotonin reuptake inhibitor (SSRI), a substance P (SP) antagonist, and a glucocorticoid antagonist on NK cell function were assessed in a series of ex vivo experiments of peripheral blood mononuclear cells from each HIV-seropositive subject. RESULTS Natural killer cell cytolytic activity was significantly increased by the SSRI citalopram and by the substance P antagonist CP-96345 relative to control conditions; the glucocorticoid antagonist, RU486, showed no effect on NK cytotoxicity. Our results suggest that the effects of the three agents did not differ as a function of depression. CONCLUSIONS Our findings provide evidence that NK cell function in HIV infection may be enhanced by serotonin reuptake inhibition and by substance P antagonism. It remains to be determined if HIV-related impairment in not only NK cytolytic activity but also NK noncytolytic activity can be improved by an SSRI or an SP antagonist. Clinical studies are warranted to address these questions and the potential roles of serotonergic agents and SP antagonists in improving NK cell immunity, delaying HIV disease progression, and extending survival with HIV infection.

Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines

The chronicity of hepatitis C virus (HCV) infection raises the question of how HCV is able to persist in hepatic cells. We show that human primary hepatocytes and human hepatic cell lines (Huh7 and HepG2) spontaneously produce interferon (IFN)‐α that is inhibited in the HCV replicon cells (Huh.8 and FCA‐1). Silencing IFN‐α gene expression by IFN‐α small interfering RNA (siRNA) in the HCV replicon cells resulted in increased HCV replicon expression. The activation of IFN‐α expression by interferon regulatory factor (IRF‐7) led to the inhibition of HCV replicon expression, whereas the anti–IFN‐α receptor antibody could partially block IRF‐7–mediated HCV replicon inhibition. In addition, the blockade of IFN‐α receptor by anti–IFN‐α receptor antibody on the replicon cells increased HCV replicon expression. Among the HCV nonstructural (NS) proteins tested, NS5A is the most potent inhibitor of IFN‐α expression by the hepatic cells. Investigation of the mechanism of HCV action on IFN‐α showed that IRF‐7–induced IFN‐α promoter activation was inhibited in the HCV replicon cells. Furthermore, IRF‐7 expression was restricted in the HCV replicon cells. In conclusion, we provide direct evidence that HCV undermines the intracellular innate immunity of the target cells, which may account for HCV persistence in hepatic cells. (HEPATOLOGY 2005;42:00–00.) (HEPATOLOGY 2005;42:819–827.)

HIV enhances substance P expression in human immune cells.

Substance P (SP), a potent modulator of neuroimmunoregulation, is expressed in human immune cells. We observed elevated plasma SP levels in HIV‐infected men compared with uninfected subjects. In the present study, we investigated the possible cellular source of the increased SP level caused by HIV infection. Using real‐time reverse transcriptase‐polymerase chain reaction, we demonstrated that monocyte‐derived macrophages (MDM) and lymphocytes from both placental cord blood and adult peripheral blood expressed SP mRNA, which was significantly increased by HIV infection. HIV‐induced SP expression was positively related to virus replication in the infected MDM. Purified recombinant HIV envelope glycoprotein 120 (gp120) derived from both the macrophage‐tropic strain (MN) and the T lymphocyte‐tropic strain (IIIB), when added to MDM cultures, enhanced SP mRNA expression. The gp120‐induced SP expression was abrogated by pretreating the cells with soluble CD 4. Furthermore, the activation of HIV in the latently infected promonocytic cell line (U1) and T‐cell line (ACH‐2) up‐regulated SP mRNA expression. These data support the hypothesis that interaction of HIV and SP may have significant in vivo relevance to the immunopathogenesis of HIV infection and AIDS.