Morphological evidence that pentagastrin regulates secretion in the human parotid gland

Salivary secretion is principally regulated by autonomic nerves. However, recent evidence from in vivo animal experiments suggests that gastrointestinal peptide hormones can also influence saliva production. The aim of the present study was to define the secretagogue activity of the gastrin-analogue pentagastrin in human salivary glands. For this purpose, parotid tissues were exposed to pentagastrin in vitro. Morphological techniques were used to evaluate modifications to serous acinar cells associated with secretion. Using a variant of the osmium maceration method, high resolution scanning electron microscopy allowed assessment of the morphology of the cytoplasmic aspect of the plasmalemma to demonstrate secretory activity. To quantify responses to pentagastrin, we recorded morphometric data on microvilli, microbuds, and protrusions. Dose-dependent morphological changes were observed, whereas protein concentration increased in the incubate. The use of selective receptor antagonists showed pentagastrin to act principally via cholecystokinin-A receptors. The morphological responses observed following exposure to pentagastrin differed from those elicited following exposure to the pan-muscarinic agonist carbachol. This study provides the first demonstration of a direct secretory action of gastrointestinal peptides on salivary glands in humans.     

组胺对T细胞IL-2产生及增殖活性影响的实验研究

目的:了解组胺对 CD4 和 CD8 T细胞IL-2产生和细胞增殖活性的影响。方法:密度梯度离心及吸附法分离PBMC和PBLC,采用抗CD4 和CD8 抗体分别制备CD8 和CD4 T细胞进行培养,然后采用ELISA法和MTT比色法测上清液IL-2含量及增殖活性。结果:①组胺 CD4 （CD8 ）培养上清液中IL-2水平及MTT增殖指数与T细胞自然培养孔比较明显降低（P<0.05）。②组胺 CD4 （CD8 ） 西咪替丁培养孔上清液中IL-2水平及 MTT增殖指数明显高于未加西咪替丁孔（P<0.05）。③CD4 T细胞自然培养孔上清液中IL-2水平显著高于CD8 T细胞自然培养孔。结论:组胺可抑制T细胞IL-2产生及增殖。西咪替丁可阻断组胺对T细胞的抑制作用。CD8 T细胞也可产生IL-2,但其功能较 CD4 T细胞为低。     

Glutathione increases interleukin-2 production in human lymphocytes

It is known that glutathione (GSH) has an immunological effect on several features of the immune system. The present study investigated the effects of GSH on interleukin-2 (IL-2) production from normal human peripheral blood lymphocytes (PBL). The results showed that both exogenous GSH and 2-mercaptoethanol (2-ME) significantly increased intracellular GSH levels after PBL were incubated with both agents. IL-2 production from PBL was markedly increased at the presence of exogenous GSH (0.5–8 mmol/1) or 2-ME (12.5–50 μmol/1) which corresponded to 1.57–2.82 nmol/10⁶ cells and 1.41–1.80 nmol/10⁶ cells of intracellular concentrations of GSH, respectively. However, IL-2 production seemed to reach a steady level when exogenous GSH concentrations in cell culture were between 2 and 8 mmol/1. The findings also showed that there was a positive correlation between the IL-2 concentrations and intracellular GSH levels. This study indicated that both exogenous GSH and 2-ME were able to elevate intracellular GSH levels and the increased intracellular GSH could increase IL-2 production in vitro. It is suggested that GSH may exert its effects on the immune system via the regulation of IL-2 synthesis.     

A novel PKC regulates ERK activation and degranulation of cytotoxic T lymphocytes: Plasticity in PKC regulation of ERK

Stimulation of cytotoxic T lymphocyte (CTL) degranulation with plate-bound anti-CD3 Ab leads to two phases of ERK activation: an early PKC-independent phase followed by a later sustained PKC-dependent phase. Herein, we show that a novel PKC (nPKC) mediates the late phase of ERK activation, upstream of Ras in murine T cells. In contrast, when CTL are activated with cross-linked anti-CD3 Ab, which does not trigger CTL degranulation, there is a requirement for conventional PKC (cPKC) for ERK activation. We detect increased novel PKCtheta activation only when CTL are stimulated with plate-bound Ab and not cross-linked Ab. Interestingly, in T cells from mice lacking PKCtheta, sustained ERK activation requires the activity of cPKC, implying that PKCtheta is required for the nPKC pathway that normally mediates sustained ERK activation. CTL lines derived from PKCtheta-deficient mice degranulate and activate ERK normally, and exhibit altered expression of PKC isozymes, which may compensate for the loss of PKCtheta. Taken together, these data demonstrate that normally an nPKC participates in the sustained activation of ERK. However, if the nPKC pathway is compromised, alternate PKC pathways can compensate, suggesting that considerable plasticity exists with respect to PKC regulation of ERK activation in T cells.     

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL. Surprisingly, antibodies to beta1 or beta 3 integrins were also unable to stimulate MAP kinase activation, suggesting that although beta 1 integrins are capable of stimulating MAP kinase activation in other cells, they cannot do so in CTL. In CTL, phosphorylation of proline-rich tyrosine kinase 2 downstream of integrin stimulation did not result in recruitment of the adaptor protein Grb2. Additionally, we examined the role of MAP kinases in regulating integrin-mediated adhesion. Anti-CD3-triggered adhesion to fibronectin was largely insensitive to the MAP kinase kinase inhibitor PD98059. Triggered cell-spreading on fibronectin was inhibited by PD98059 but not by U0126. In summary, ligation of beta 3 integrin by antibodies or fibronectin or of beta1 integrin by monoclonal antibodies fails to activate ERK MAP kinases, but integrin ligation synergizes with T cell receptor stimulation upstream of MAP kinases.     

Genetic difference in the proliferative response to T mitogens between Hi/PHA and Lo/PHA lymphocytes is independent of accessory cell function

The role of the macrophage as accessory cell in the proliferative response of lymphocytes to phytohemagglutinin (PHA) was studied in two lines of mice genetically selected for high and low responsiveness to T mitogens. Adherent cell depletion of lymph node cells abrogated the low (Lo)/PHA response, but only partially inhibited the high (Hi)/PHA response. Addition of peritoneal cells provided either by Hi/PHA or by Lo/PHA mice equally restored Hi/PHA responsiveness but had only a slight reconstituting effect on the inhibited Lo/PHA response. Equivalent enhancement or suppression of proliferation of untreated lymph node cells was obtained by the addition of increasing percentages of each of the two peritoneal cell populations. However, the maximum level of the Lo/PHA response never reached that of Hi/PHA cells. These data indicate that the bidirectional selective breeding has not modified the potentialities of the macrophages as accessory cells but has resulted in an impaired response of Lo/PHA lymphocytes to the signals delivered either by accessory cells or by T mitogens.     

Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin

The present study investigated bystander suppression, specific suppression and anergy as mechanisms for oral tolerance. Oral tolerance was induced in mice by a single gastric intubation of 20 mg ovalbumin (OVA) and was evaluated in vitro by the absence of T lymphocyte proliferative responses to OVA after priming by OVA-complete Freund's adjuvant (CFA). T lymphocyte unresponsiveness was antigen specific, systemic and was not affected by the vehicle used for immunization. T lymphocytes derived from tolerant popliteal lymph nodes (PLN) responded to an acetone precipitate (AP) of mycobacteria present in CFA; this response was not suppressed by co-culture with OVA, thereby arguing against a mechanism of bystander suppression in our system. Responses of PLN T lymphocytes derived from OVA-CFA primed, non-tolerant mice, or those of an OVA-specific T lymphocyte line, were not suppressed by PLN or spleen cells derived from OVA tolerant mice. These results excluded the possibility that oral tolerance was induced and maintained by a mechanism of specific suppression. At the cellular level, we found that OVA-tolerant T lymphocytes did not produce interleukin-2 (IL-2) nor express IL-2 receptor in response to OVA stimulation in vitro; both observations are indicative of a state of anergy. Incubation of OVA-tolerant PLN T lymphocytes together with murine recombinant IL-2 for 5 days, released anergic T lymphocytes and a concomitant OVA-specific proliferative response of CD4⁺ T cells was detected. Taken together, our experimental system excludes the involvement of bystander or specific suppression in the induction of oral tolerance to OVA, and provides direct evidence to show that oral tolerance results from specific T lymphocyte anergy.     

P27kip1 regulates the cell cycle arrest and survival of activated T lymphocytes in response to interleukin-2 withdrawal

The majority of activated T lymphocytes undergo cell death at the end of a primary immune response, while a minority survive as memory cells. The mechanisms that control the decision between these two fates are unknown. In the present study we examined the response of activated T cells to interleukin-2 (IL-2) withdrawal. Within hours, the percentage of T lymphocytes in cell cycle showed a steady decrease, while the percentage arrested in G1 increased proportionally. Deprivation of IL-2 resulted in upregulation of the cell cycle inhibitor p27kip1. Comparison with resting T-cell populations revealed that the highest expression of p27kip1 occurs in activated T cells undergoing cell cycle arrest following IL-2 withdrawal. T cells deficient in p27kip1 expression showed an impaired ability to undergo cell cycle arrest in response to IL-2 deprivation. Moreover, T cells deficient in p27kip1 showed significantly more apoptosis after IL-2 withdrawal. Collectively, this study demonstrates that p27kip1 regulates both the cell cycle arrest and the apoptosis of antigen-specific T lymphocytes.     

Enhancement of human T lymphocyte function by prothymosin alpha: increased production of interleukin-2 and expression of interleukin-2 receptors in normal human peripheral blood T lymphocytes

The in vitro incubation of phytohemagglutinin (PHA)- or alloantigen-stimulated peripheral blood T cells with prothymosin alpha (ProT alpha) resulted in a marked and reproducible increase in the production of interleukin-2 (IL-2). Incubation of T cells with ProT alpha, in the absence of PHA or alloantigen, failed to induce any production of IL-2. ProT alpha by itself did not exert any IL-2 activity. Finally, ProT alpha was shown to increase the expression of IL-2 receptors on phytohemagglutinin- or alloantigen-activated T cells. These data provide the basis for understanding the in vitro immunoenhancing effects of ProT alpha in cellular immune systems.     

Interleukin-4 regulates the interleukin-2 receptors on human peripheral blood B lymphocytes.

The high-affinity interleukin-2 (IL-2) receptor (IL-2R) consists of the non-covalent association of at least two subunits, p55 and p70-75, capable of binding IL-2 with low and intermediate affinity, respectively. We studied the effects of cytokines on the IL-2R expressed on human peripheral blood B lymphocytes using monoclonal antibodies specific for IL-2R p55 and IL-2R p70-75, by means of two-colour flow cytometric analysis. In freshly isolated peripheral blood B lymphocytes, the p55 subunit was expressed only in a small population (7.0% of CD20+ cells), whereas the p70-75 subunit was expressed in a large population (89.0% of CD20+ cells). Of the cytokines studied, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were involved in the regulation of IL-2R on B cells. After a 2-day incubation with IL-4, expression of IL-2R p55 was markedly induced, but expression of IL2-R p70-75 was profoundly suppressed in a dose-dependent manner. These abilities of IL-4 to promote IL-2R p55 expression and suppress IL-2R p70-75 expression were inhibited by the presence of IFN-gamma. Other cytokines, including IL-1, IL-2, IL-5, and IL-6, had little effect on the expression of these two subunits. These findings suggest that IL-4 is a cytokine modulating B cell response through the regulation of IL-2R.     

Transforming growth factor-beta1 suppresses interleukin-15-mediated interferon-gamma production in human T lymphocytes

One of the most remarkable means by which tumour cells manage to evade recognition and elimination by the immune system is the release of immunosuppressive mediators, such as interleukin (IL)-10 or transforming growth factor-beta (TGF-beta). For antitumour immunotherapies to reach their full potential, cytokine cocktails will have to be custom-tailored to the tumour's individual cytokine microenvironment. One of the components of such a cytokine cocktail may be interleukin (IL)-15, which has demonstrated an excellent stimulatory potential of antitumour immunity. In an in vitro model, we have previously been able to show that the negative effects of IL-10 on IL-15-mediated cytotoxic T-cell activation can be outweighed by the addition of interleukin (IL)-12. The mechanism by which TGF-beta may influence the effect of IL-15 remains poorly understood, however. We have therefore taken our T-cell model further and have studied the effect of TGF-beta on IL-15-mediated interferon-gamma (IFN-gamma) production. In activated, IL-15-stimulated peripheral blood T lymphocytes, TGF-beta suppressed IFN-gamma mRNA and protein levels by approximately 75%. This effect was likewise observed on both CD4+ and CD8+ T cells and, in contrast to the effect of IL-10 in this system, could not be neutralized by the addition of IL-12. Thus, immunotherapy for TGF-beta-producing tumours may benefit from the addition of TGF-neutralizing activity rather than IL-12.     

Evidence that the CD45 phosphatase regulates the activity of the phospholipase C in mouse T lymphocytes.

The importance of the tyrosine phosphatase CD45 in the regulation of lymphocyte activation was first demonstrated using antibodies against the extracellular domain of CD45 in functional assays. More recently it was reported that CD45-negative mutants were nonresponsive to stimulation through the T cell receptor-CD3 complex. We have studied the effect of CD45 cross-linking on the early signals induced by CD3 in mouse T cells. We show that CD45 cross-linking inhibits the increase in inositol phosphates and cytoplasmic Ca2+ induced by cross-linking of CD3. This indicates that CD45 is involved in the regulation of phospholipase C.     

Is schizophrenia caused by excessive production of interleukin-2 and interleukin-2 receptors by gastrointestinal lymphocytes?

Excessive production of interleukin-2 (IL-2) and IL-2 receptors (IL-2R) by gastrointestinal (GI) T-lymphocytes is hypothesized as the cause of schizophrenia. It is based on: 1) IL-2 given to human volunteers can cause all the symptoms of schizophrenia; 2) GI lymphocytes in nonhuman primates produce much more IL-2 and IL-2R when stimulated than peripheral blood lymphocytes; 3) the GI tract is the largest lymphoid 'organ' in the body. The hypothesis appears to: 1) explain the protective effect of rheumatoid arthritis on schizophrenia; 2) make mechanistically plausible the findings on wheat and schizophrenia; 3) be consistent with and explain many of the known immunological abnormalities in schizophrenia.     

Functional CD40 ligand expression on T lymphocytes in the absence of T cell receptor engagement: involvement in interleukin-2-induced interleukin-12 and interferon-γ production

Despite the fact that the great majority of T cells at the site of an inflammatory response are not antigen specific, the mechanisms leading to activation and recruitment of these bystander T cells are poorly understood. We previously reported that soluble (s)CD23 potentiated the interleukin (IL)-2-induced interferon (IFN)-γ production by T cells co-cultured with autologous monocytes in the absence of T cell receptor (TCR) engagement. Our present data demonstrate that the IL-2-induced IFN-γ secretion, in the presence but also in the absence of sCD23, is strictly IL-12 dependent, inasmuch as anti-IL-12 antibody abrogated both responses. Most interestingly, anti-CD40 ligand (CD40L) monoclonal antibody significantly inhibited IL-2-induced IL-12 as well as IFN-γ production. These results suggest that CD40L was expressed on T cells in the absence of TCR engagement. Indeed, purified unstimulated T cells readily expressed CD40L. IL-2 and monocytes did not up-regulate CD40L on resting T cells. It is proposed that low levels of CD40L expression on non-antigen stimulated T cells are sufficient to signal through CD40 molecules on accessory cells and to induce IL-12 secretion, which in turn can synergize with IL-2 for the induction of IFN-γ production, thus contributing to the inflammatory process.     

Spontaneous release of interleukin-2 by lung T lymphocytes in active pulmonary sarcoidosis

We investigated the possible role of interleukin-2, a T-cell product that stimulates the clonal increase of responsive T lymphocytes, in the pathogenesis of pulmonary sarcoidosis. We obtained mononuclear effector cells from the lungs of 10 patients with sarcoidosis and high-intensity alveolitis, 17 patients with sarcoidosis and low-intensity alveolitis, 3 patients with idiopathic pulmonary fibrosis, and 10 normal controls. Lung cells from the group with sarcoidosis and low-intensity alveolitis, from the group with idiopathic pulmonary fibrosis, and from the controls produced insignificant amounts of interleukin-2. However, lung cells from 9 of 10 patients with sarcoidosis and high-intensity alveolitis spontaneously released interleukin-2, and in a proportion that correlated with the proportion of T cells in the lung washings (P less than 0.01). Blood T cells from the same patients did not release interleukin-2. To determine whether release of interleukin-2 by the lung T cells had a biologic effect in vivo, we measured T-lymphocyte replication in the lungs of patients and controls. The lung T lymphocytes replicated at a rate that was several times higher in the patients with sarcoidosis and high-intensity alveolitis than in the other patient groups or the controls (P less than 0.01). These observations suggest that the release of interleukin-2 by lung T cells has a central role in increasing the numbers of lung T cells in active pulmonary sarcoidosis.     

IL-7及其受体在T淋巴细胞发育中的作用及对运动免疫的意义

运动训练显著影响机体的免疫功能,T淋巴细胞亚群的平衡是维持免疫系统内部环境稳定的一个中心环节。IL-7／IL-7受体（IL-7R）在促进淋巴细胞,尤其是T淋巴细胞产生、分化和发育成熟的过程中发挥重要作用。从了解IL-7／IL-7R的生物学特性、信号通路及其在T细胞发育、生存、扩增和记忆性T细胞发育中的重要作用,从细胞免疫产生的源头来分析IL-7／IL-7R运动影响免疫功能的机理,可为改善机体免疫功能提供新的思路。     

IL-7及其受体在T淋巴细胞发育中的作用及对运动免疫的意义

运动训练显著影响机体的免疫功能,T淋巴细胞亚群的平衡是维持免疫系统内部环境稳定的一个中心环节.IL-7/IL-7受体(IL-7R)在促进淋巴细胞,尤其是T淋巴细胞产生、分化和发育成熟的过程中发挥重要作用.从了解IL-7/IL-7 R的生物学特性、信号通路及其在T细胞发育、生存、扩增和记忆性T细胞发育中的重要作用,从细胞免疫产生的源头来分析IL-7/IL-7R运动影响免疫功能的机理,可为改善机体免疫功能提供新的思路.     

Liver of MRL/lpr mice contain interleukin-4-producing lymphocytes and accessory cells that support the proliferation of Th2 helper T lymphocyte clones.

Hepatic nonparenchymal cells (NPC) from mice of nonautoimmune strains support the proliferation of only Th1 and not Th2 helper T lymphocyte (HTL) clones. Because of the multiple systemic and liver-specific immune defects in the autoimmune MRL/lpr mouse strain, we have explored the possibility that hepatic accessory cells from MRL/lpr mice are capable of stimulating the proliferation of Th2 HTL. We report here that hepatic NPC from MRL/lpr and C3H/lpr female mice older than 8 weeks, in contrast to hepatic NPC from MRL/++ and C3H/HeN strains, are able to support in vitro the proliferation of both Th1 and Th2 CD4 clones. Additionally, hepatic lymphocytes (HL) from MRL/lpr mice can be stimulated to produce interleukin (IL)-4 to a much higher degree than HL from the nonautoimmune strains. These results suggest that the activation of Th2 cells by hepatic NPC and production of IL-4 by HL may contribute to the immunologic aberrations in the MRL/lpr mouse strain.     

Characterization of the immunoregulatory properties of thymosin alpha 1 on interleukin-2 production and interleukin-2 receptor expression in normal human lymphocytes

Thymosin alpha 1 (T alpha 1) and thymosin fraction 5 (TF5) have been shown to induce lymphocyte maturation and differentiation as well as to modulate mature immune responses to antigens and mitogens. The present study focused on the characterization of the mechanisms involved in T alpha 1 and TF5 enhancement of phytohemagglutinin (PHA)-induced interleukin-2 (IL-2) secretion and interleukin-2 receptor (IL-2R) expression in human mononuclear cells. We provide evidence that TF5 and T alpha 1 modulate an early event(s) during lymphocyte activation by mitogens. A short preincubation period (30 min) of non-adherent cells with thymosins, followed by extensive washing and subsequent exposure to PHA, was sufficient to enhance the production of IL-2 and the expression of IL-2R induced by the mitogen. Furthermore, the concomitant addition of PHA and thymosin during the preincubation period is not necessary for the enhancing effects to occur. We have also studied the role of macrophages on thymosin modulation of these responses. Results presented here indicate that macrophages are not essential for the interaction of thymosins with T-cells. However, macrophages are an absolute requirement during the exposure to the mitogen after preincubation with thymosins for the manifestation of TF5- and T alpha 1-mediated enhancing effects on IL-2 production and IL-2R expression. Human recombinant interleukin-1 beta (rIL-1 beta) was able to replace this macrophage requirement, indicating that production of IL-1 by these cells is a critical event in thymosin modulation of the IL-2 system. Two-color flow cytometric analysis and experiments involving the use of highly purified helper/inducer (Th, CD4+) and cytotoxic/suppressor (Tc, CD8+) T-cell populations indicated that both, Th and Tc cell populations are targets of thymosin activity. These studies provide additional evidence that thymosins play an important role in the modulation of the normal immune response and begin to define the mechanisms underlying T alpha 1 immunoregulatory properties.