Takeshi Fujii

Expression of non-neuronal acetylcholine in lymphocytes and its contribution to the regulation of immune function.

Lymphocytes express most components of the cholinergic system including acetylcholine (ACh), muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively), choline acetyltransferase (ChAT), high affinity choline transporter and acetylcholinesterase. ACh and mAChR agonists elicit intracellular Ca2+ signaling, up-regulation of c-fos expression and nitric oxide synthesis within T and B cells probably via M3 and M5 mAChRs. Stimulation of nAChRs with ACh or nicotine causes a rapid and transient Ca2+ signaling in T and B cells, probably via alpha7 nAChR subunit-mediated pathways. Phytohemagglutinin- or antigen-induced T cell activation via cell surface molecules (e.g., T cell receptor/CD3 complexes) enhances lymphocytic cholinergic transmission by up-regulating ChAT and M5 mAChR expression. It is thus likely that a local lymphocytic cholinergic system is involved in regulating immune function. This idea is supported by the findings that lymphocytic cholinergic activity is altered in animal models exhibiting immunological abnormalities. In addition, it appears likely that during interactions mediated by cell surface molecules T cells communicate via ACh with thymic epithelial cells and vascular endothelial cells, which also express ChAT and nAChRs or mAChRs. This interaction leads to T cell selection and maturation in the thymus and local vascular smooth muscle relaxation. Collectively, these data provide a compelling picture in which lymphocytes constitute a cholinergic system that is independent of cholinergic nerves, and which is involved in the regulation of immune function and local circulation.

The lymphocytic cholinergic system and its biological function

Lymphocytes are now known to possess the essential components for a non-neuronal cholinergic system. These include acetylcholine (ACh); choline acetyltransferase (ChAT), its synthesizing enzyme; and both muscarinic and nicotinic ACh receptors (mAChRs and nAChRs, respectively). Stimulating lymphocytes with phytohemagglutinin, a T-cell activator; Staphylococcus aureus Cowan I, a B-cell activator; or cell surface molecules enhances the synthesis and release of ACh and up-regulates expression of ChAT and M(5) mAChR mRNAs. Activation of mAChRs and nAChRs on lymphocytes elicits increases in the intracellular Ca(2+) concentration and stimulates c-fos gene expression and nitric oxide synthesis. On the other hand, long-term exposure to nicotine down-regulates expression of nAChR mRNA. Abnormalities in the lymphocytic cholinergic system have been detected in spontaneously hypertensive rats and MRL-lpr mice, two animal models of immune disorders. Taken together, these data present a compelling picture in which immune function is, at least in part, under the control of an independent non-neuronal lymphocytic cholinergic system.

Pirfenidone attenuates expression of HSP47 in murine bleomycin-induced pulmonary fibrosis

Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen. The present study was undertaken to investigate whether treatment with the antifibrotic drug pirfenidone attenuates the bleomycin (BL)‐induced overexpression of HSP47 in the lungs. Male ICR mice were intravenously injected with BL or saline (SA). Pirfenidone or control drug (CD) was administered 14 days after commencement of BL or SA, and continued throughout the course of the experiment. The mice were randomly divided into three experimental groups: 1) SA‐treated with CD (SA group); 2) BL‐treated with CD (BL group); and 3) BL‐treated with pirfenidone (pirfenidone group). Lungs of the pirfenidone group showed a marked reduction of fibrotic lesions compared with the corresponding BL group. Immunohistochemical studies showed that BL treatment significantly increased the number of macrophages, myofibroblasts, HSP47‐positive type II pneumocytes and HSP47‐positive interstitial spindle-shaped cells. Treatment with pirfenidone significantly reduced the number of these cells compared with the corresponding BL group. Furthermore, treatment with pirfenidone significantly suppressed the BL‐induced increase of the positive ratio of HSP47 and α‐smooth muscle actin to interstitial spindle-shaped cells. The present study results showed that pirfenidone inhibited heat shock protein 47‐positive cells and myofibroblasts, the principal cells responsible for the accumulation and deposition of extracellular matrix seen in pulmonary fibrosis.

The Non-neuronal Cholinergic System

Acetylcholine (ACh) is a well characterized neurotransmitter occurring throughout the animal kingdom. In addition, both muscarinic and nicotinic ACh receptors have been identified on lymphocytes of various origin, and their stimulation by muscarinic or nicotinic agonists elicits a variety of functional and biochemical effects. It was thus initially postulated that the parasympathetic nervous system may play a role in modulating immune system function. However, ACh in the blood has now been localized to lymphocytes; indeed expression of choline acetyltransferase (ChAT), an ACh synthesizing enzyme, has been shown in human blood mononuclear leukocytes, human leukemic T-cell lines and rat lymphocytes. Stimulation of T-lymphocytes with phytohemagglutinin activates the lymphoid cholinergic system, as evidenced by increased synthesis and release of ACh and increased expression of mRNAs encoding ChAT and ACh receptors. The observation that M3 muscarinic receptor stimulation by ACh and other agonists increases the intracellular free Ca2+ concentration and upregulates c-fos gene expression strongly argues that ACh, synthesized and released from T-lymphocytes, acts as an autocrine and/or paracrine factor regulating immune function. These findings present a compelling picture in which immune function is, at least in part, under the control of an independent lymphoid cholinergic system.

Nicotine-induced Ca2+ signaling and down-regulation of nicotinic acetylcholine receptor subunit expression in the CEM human leukemic T-cell line

We previously showed that T- and B-lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChR and nAChR, respectively), and that stimulation of M(3) mAChRs on lymphocytes increases the intracellular free Ca(2+) concentration ([Ca(2+)](i)) and up-regulates c-fos gene expression. Little is known about the effects of nicotinic stimulation on lymphocyte function, however. We therefore investigated the acute effect of nicotine on [Ca(2+)](i) in CEM cells, a model of T-lymphocytes, using confocal laser scanning microscopy with fluo-3, a Ca(2+)-sensitive fluorescent indicator. In addition, we examined the long-term effect of nicotine on the expression of selected nAChR subunits using semiquantitative reverse transcription-polymerase chain reaction analysis. In the presence of extracellular Ca(2+), nicotine (30 microM) evoked rapid, transient increases in [Ca(2+)](i). This effect was concentration-dependently inhibited by the alpha7 nAChR subunit antagonists, alpha-bungarotoxin (0.01-10 microM) and methyllycaconitine (0.01-10 mM), suggesting that the alpha7 nAChR subunit mediates Ca(2+) signaling in T-lymphocytes. Nicotine (0.01-10 microM) also concentration-dependently down-regulated expression of mRNAs for all the nAChR subunits tested: expression of the alpha6 and alpha7 subunits was down-regulated within 1 week, while expression of the alpha3 and alpha5 subunits declined gradually throughout the 8-week experimental period. These findings indicate that nicotine--and therefore likely smoking--affects immune function by suppressing expression of the neuronal nAChR subtype involved in Ca(2+) signaling in lymphocytes.

The endogenous, immunologically active peptide apelin inhibits lymphocytic cholinergic activity during immunological responses

We investigated the effects of apelin, an immunologically active peptide ligand for orphan receptor APJ, on acetylcholine (ACh) synthesis in MOLT-3 human leukemic T cells. We initially confirmed expression of APJ mRNA in several human T- and B-cell lines by reverse transcription-polymerase chain reaction (RT-PCR). We also found that in phytohemagglutinin (PHA)-stimulated MOLT-3 cells, an active apelin fragment, apelin-13, down-regulates expression of choline acetyltransferase (ChAT) mRNA and significantly reduces ChAT activity and cellular ACh content and release. It thus appears that apelin inhibits lymphocytic cholinergic activity via APJ during immunological responses.

Significance of IL‐1β and IL‐1 receptor antagonist (IL‐1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12.9 γδ+9.5 months (mean γδ+s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL‐1β and IL‐8 were significantly higher in DPB compared with healthy volunteers (P < 0.05, P < 0.05, respectively). IL‐1 Ra in patients is considered to have a weak inhibitory activity for IL‐1β, with approximately five‐fold concentration of IL‐1β compared with that in healthy volunteers (approx. nine‐fold concentration of IL‐1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL‐1Ra in BALF. The level of IL‐1β correlated significantly with the concentration of neutrophils in BALF (r= 0.72, P < 0.01), as well as with the level of IL‐1Ra (r= 0.688, P < 0.05) and IL‐8 (r= 0.653, P < 0.05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL‐1Ra or IL‐8 in BALF (r= 0.526, P= 0.053 or r= 0.776, P < 0.01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r= 0.524, P < 0.05). Our results suggest that the relative amounts of IL‐1β and IL‐1Ra or IL‐8 may contribute, at least in part, to the neutrophil‐mediated chronic airway inflammation in patients with chronic airway disease, and long‐term erythromycin therapy may down‐regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil‐mediated inflammatory response.

Detection of the high-affinity choline transporter in the MOLT-3 human leukemic T-cell line

We previously showed that lymphocytes possess the necessary components to constitute an independent, non-neuronal cholinergic system; these include acetylcholine (ACh) itself, choline acetyltransferase (the ACh-synthesizing enzyme), and both muscarinic and nicotinic ACh receptors (AChRs). In addition, we showed that stimulation of AChRs with their respective agonists elicits a variety of biochemical and functional effects, suggesting that lymphocytic cholinergic system is involved in the regulation of immune function. In nerve terminals, choline taken up via the high-affinity choline transporter (CHT1) is exclusively utilized for ACh synthesis. In the present study, therefore, we investigated the expression of CHT1 in T-lymphocytes. Reverse transcription-polymerase chain reaction analysis revealed that MOLT-3 cells, a human leukemic T-cell line used as a T-lymphocyte model, expressed CHT1 mRNA, but that the CEM and Jurkat T-cell lines did not. Consistent with that finding, specific binding of [3H]hemicholinium-3 (HC-3), an inhibitor of CHT1, and HC-3-sensitive [3H]choline uptake were also detected in MOLT-3 cells. These results suggest that CHT1 plays a role in mediating choline uptake in T-lymphocytes and provides further evidence for the presence of an independent lymphocytic cholinergic system.

Phenotypic characterization of T cells in bronchoalveolar lavage fluid (BALF) and peripheral blood of patients with diffuse panbronchiolitis; the importance of cytotoxic T cells

We investigated the contribution of T cells in diffuse panbronchiolitis (DPB) by identifying T cell subsets in BALF of 36 patients with DPB, before and after long‐term treatment with macrolide antibiotics, and 16 healthy control subjects. The percentages of lymphocytes and CD3+γδ+ cells in BALF of DPB patients and control subjects were similar, but the absolute number of these cells was higher in DPB patients. Treatment resulted in a significant reduction in the absolute number of these cells. A further two‐colour analysis of T cell subsets in BALF showed a significantly higher ratio and number of CD8+HLA‐DR+ cells in DPB patients. Treatment resulted in a significant reduction of activated T cells. Most BALF CD8+ cells were CD8+CD11b− cytotoxic T cells. The number of these cells in BALF of DPB patients (26.69u2003±u20035.86u2003×u2003103/ml) was higher than the control (2.02u2003±u20030.38u2003×u2003103/ml; Pu2003<u20030.001), and a significant reduction was observed after treatment (7.69u2003±u20032.59u2003×u2003103/ml; Pu2003<u20030.01). The number of CD4+ cells was also higher in DPB patients than in controls, and most were CD4+CD29+ memory T cells. However, treatment did not influence the number of these cells. The number of lymphocytes, CD3+γδ+, CD8+CD11b−, CD8+HLA‐DR+, and CD4+CD29+ cells was higher in patients with bacterial infection than in those without bacterial infection, and interestingly, macrolide therapy reduced the number of lymphocytes, CD3+γδ+, CD8+CD11b− and CD8+HLA‐DR+ cells, irrespective of bacterial infection. In peripheral blood, the percentage of CD8+HLA‐DR+ cells was also higher in DPB patients than in healthy subjects, and significantly decreased after treatment. The percentage of CD8+CD11b− cells in peripheral blood was similar in DPB patients and normal subjects, and treatment significantly reduced the percentage of these cells. Finally, the expression of the adhesion molecules CD11a/CD18 (α/β‐chains of LFA‐1) on lung CD3+ cells and CD49d (α‐chain of VLA) on lung CD4+ cells was enhanced compared with that on peripheral blood in DPB patients. Our results suggest that elevation of memory T cells and activation of CD8+ cells, mainly cytotoxic T cells, in the airway lumen of DPB patients may contribute to chronic bronchial inflammation, possibly through up‐regulation of adhesion molecules. Our findings also indicate that macrolide antibiotics may have a direct or indirect suppressive effect on cytotoxic T cells, and as such, reduce inflammation and improve clinical condition.

Nitric oxide (NO) synthase mRNA expression and NO production via muscarinic acetylcholine receptor-mediated pathways in the CEM, human leukemic T-cell line

Nitric oxide (NO) is synthesized from L-arginine by neuronal, endothelial and inducible isoforms of NO synthase (nNOS, eNOS and iNOS, respectively) and is involved in the regulation of a variety of physiological functions, including immune activity. In vascular endothelial cells, stimulation of M(3) subtype of muscarinic acetylcholine receptors (mAChRs) triggers NO synthesis by eNOS. Human lymphocytes express several mAChR subtypes and their stimulation increases the intracellular free Ca(2+) concentration and up-regulates c-fos gene expression. While the above findings suggest involvement of the lymphocytic cholinergic system in the regulation of immune function, little is known on NOS expression and NO synthesis in T-lymphocytes. In the present study, using reverse transcription-polymerase chain reaction, we found that CEM cells express mRNAs encoding iNOS and nNOS, but not for eNOS. In addition, using quantitative fluorescence microscopy and a novel NO-sensitive fluorescent indicator, DAF-2, we found that oxotremorine-M (Oxo-M) (100 microM), a non-selective mAChR agonist, enhances NO production in the cells. This effect of Oxo-M was antagonized by pirenzepine (10 microM), an antagonist acting preferentially at M(1) mAChR and by atropine (10 microM). Also 4-DAMP (10 microM), an antagonist acting preferentially at M(3) mAChR, reduced significantly the effect of Oxo-M, while AFDX-116 (10 microM), an antagonist acting preferentially at M(2) mAChR, was ineffective. These findings suggest that T-lymphocytes express functional mAChRs linked to NO synthesis by nNOS and/or iNOS.