AIM, a murine apoptosis inhibitory factor, induces strong and sustained growth inhibition of B lymphocytes in combination with TGF-beta1

Lymphocyte proliferation is stimulated by differential combinations of various cytokines, antigens and adhesion molecules. However, mechanisms of negative regulation in lymphocytes are poorly understood despite their potential importance in controlling the balance of lymphocyte proliferation, particularly at inflammatory sites. We recently reported a novel murine soluble protein, termed AIM, which inhibits apoptosis of a variety of cell types including CD4/CD8 double-positive thymocytes. AIM is secreted specifically by macrophages and belongs to the macrophage scavenger receptor cysteine-rich domain superfamily. Here we show that in addition to the apoptosis-inhibitory effect, AIM induces strong, long-term inhibition of B lymphocyte proliferation in combination with transforming growth factor-beta1 (TGF-beta1 in vitro), resulting in almost complete block of proliferation and immunoglobulin secretion. The function of AIM as a cell growth inhibitor requires pretreatment of B cells with TGF-beta1 which appears to increase expression of the AIM receptor on the B cell surface. Thus B lymphocyte proliferation is dramatically down-regulated by sequential exposition to TGF-beta1 followed by AIM. Like many cytokines, AIM has different functions depending on the types of target cells and the combination with other cytokines.     

Connective tissue growth factor does not affect transforming growth factor-beta 1-induced Smad3 phosphorylation and T lymphocyte proliferation inhibition

Transforming growth factor-beta 1 (TGF-beta(1)) is a key regulator of immune tolerance. TGF-beta(1) controls T lymphocyte activation and is involved in the immunosuppressive function and generation of regulatory T lymphocytes. Connective tissue growth factor (CTGF) has an essential role in the formation of connective tissue and blood vessels. CTGF expression is induced by TGF-beta(1) in several cell types and CTGF mediates several of the downstream actions of TGF-beta(1). Since little is known about the potential synergy between CTGF and TGF-beta(1) in T lymphocyte biology, the purpose of the present study was to determine whether CTGF can modulate TGF-beta(1)-mediated effects on human CD4+ T lymphocytes. Human recombinant CTGF was expressed in HEK293 cells. rCTGF was biologically active demonstrated by induction of proliferation in the endothelial cell line EA hy 926. rCTGF alone did not potentiate or diminish anti-CD3-induced CD4+ T lymphocyte proliferation and did not activate the Smad signaling pathway in CD4+ T lymphocytes. Furthermore, rCTGF did not attenuate TGF-beta(1)-mediated inhibition of CD4+ T lymphocyte proliferation and TGF-beta(1)-induced Smad signaling in CD4+ T lymphocytes. These results indicate that rCTGF had no detectable effects of its own on human CD4+ T lymphocytes and did not potentiate the effects of low amounts of TGF-beta(1) on human CD4+ T lymphocytes. Overall, these data support the hypothesis that CTGF does not act on CD4+ T lymphocytes.     

TGF-beta down-regulates IL-1alpha-induced TLR2 expression in murine hepatocytes

We have previously reported that the proinflammatory cytokine interleukin (IL)-1alpha can up-regulate functional Toll-like receptor 2 (TLR2) expression in primary-cultured murine hepatocytes, and bacterial lipopeptide (BLP) is capable of signaling through TLR2 to induce serum amyloid A (SAA) expression in hepatocytes. In the present study, we investigated the effect of the anti-inflammatory cytokine transforming growth factor-beta (TGF-beta) on TLR2 expression in primary-cultured murine hepatocytes. At the mRNA and protein levels, TGF-beta up-regulated TLR2 expression but inhibited TLR2 expression induced by IL-1alpha at 24 h. BLP-induced SAA promoter activity could be augmented by pretreatment with IL-1alpha but not TGF-beta or the combination of TGF-beta and IL-1alpha. TLR2 promoter activity and nuclear factor (NF)-kappaB activation by IL-1alpha were inhibited by TGF-beta treatment. Pretreatment with TGF-beta strongly suppressed IL-1alpha-induced TLR2 promoter activity and NF-kappaB activation, which was consistent with the down-regulation of type I IL-1 receptor (IL-1RI) mRNA expression. IL-1alpha up-regulated IL-1RI mRNA, but it was inhibited by the treatment with TGF-beta. These results suggest that TGF-beta suppresses the induction of TLR2 expression by IL-1alpha through down-regulation of IL-1RI expression. These results also demonstrate the disparity between IL-1alpha and TGF-beta in regulating TLR2-mediated SAA production in hepatocytes.     

Role of iron in Nramp1-mediated inhibition of mycobacterial growth

Innate resistance to mycobacterial growth is mediated by a gene, Nramp1. We have previously reported that Nramp1 mRNA from macrophages of Mycobacterium bovis BCG-resistant (Bcgr) mice is more stable than Nramp1 mRNA from macrophages of BCG-susceptible (Bcgs) mice. Based on these observations and on reports that show that the closely related Nramp2 gene is a metal ion transporter, we evaluated the effect of iron on the growth of Mycobacterium avium within macrophages as well as on the stability of Nramp1 mRNA. The addition of iron to macrophages from Bcgs mice resulted in a stimulation of mycobacterial growth. In contrast, iron increased the capacity of macrophages from Bcgr mice to control the growth of M. avium. When we treated recombinant gamma interferon (IFN-gamma)-activated macrophages with iron, we found that iron abrogated the growth inhibitory effect of IFN-gamma-activated macrophages from Bcgs mice but that it did not affect the capacity of macrophages from Bcgr mice to control microbial growth. A more detailed examination of the effect of iron on microbial growth showed that the addition of small quantities of iron to resident macrophages from Bcgr mice stimulated antimicrobial activity within a very narrow dose range. The effect of iron on the growth inhibitory activity of macrophages from Bcgr mice was abrogated by the addition of catalase or mannitol to the culture medium. These results are consistent with an Fe(II)-mediated stimulation of the Fenton/Haber-Weiss reaction and hydroxyl radical-mediated inhibition of mycobacterial growth.     

Activation of TGF-beta/Smad2 signaling is associated with airway remodeling in asthma

BACKGROUND: Transforming growth factor beta (TGF-beta) has been suggested to play an important role in the development of airway remodeling in asthma; this suggestion is based on evidence that expression levels of TGF-beta are correlated with unique parameters of airway remodeling, such as thickness of basement membrane. However, the relevant studies were inconclusive because they were unable to demonstrate active signaling mediated by the cytokine in the airways of asthmatic individuals. OBJECTIVE: We sought to determine whether TGF-beta signaling was active in the airways of asthmatic subjects and, if so, whether it was correlated with clinicopathologic features associated with the development of airway remodeling in asthma. METHODS: We examined the phosphorylation status of Smad2 in bronchial biopsy samples obtained from 40 asthmatic subjects as a marker of active TGF-beta signaling, and we studied its correlation with basement membrane thickness. RESULTS: Expression levels of phosphorylated Smad2 in bronchial biopsy specimens from asthmatic subjects were higher than those in specimens from normal subjects, and they were correlated with basement membrane thickness in asthma. CONCLUSION: The findings provide evidence that TGF-beta signaling was active in asthmatic airways and that the activity was associated with the development of airway remodeling in asthma.     

GABA-mediated inhibition correlates with orientation selectivity in primary visual cortex of cat

Orientation selectivity is an important emergent property of neurons in the primary visual cortex, and inhibition is thought to play an important role in establishing this selectivity. But the relationship between strength of inhibition and orientation selectivity is unclear. To investigate this relationship, we electrophoretically applied the inhibitory transmitter GABA and the GABA(A) antagonist bicuculline on the same individual area 17 neurons in anesthetized cats. Neurons were classified as weakly orientation-selective, moderately orientation-selective, or strongly orientation-selective, according to the values of an orientation bias index. Orientation bias, half-width of the tuning curve at half-height and an orientation-specificity index (orthogonal to optimal ratio) were compared with or without GABA and bicuculline administration. GABA improved orientation selectivity with the greatest effects on weakly orientation-selective cells, smaller effects on moderately orientation-selective cells, and minimal effects on strongly orientation-selective cells; bicuculline diminished orientation selectivity the most on moderately and strongly orientation-selective cells, with minimal effects on weakly orientation-selective cells. We also found that orientation selectivity correlated with the level of neurons' spontaneous activity. These results suggest that the degree of orientation selectivity of an area 17 neuron correlates with its endogenous inhibition strength, and that GABAergic inhibition can bi-directionally regulate orientation selectivity. This correlation indicates that GABA-mediated inhibition plays an important role in establishing sharp orientation selectivity of individual neurons.     

Resistance to Leishmania major infection correlates with the induction of nitric oxide synthase in murine macrophages.

Inbred strains of mice differ considerably in their innate resistance to leishmanial infection. BALB/c mice are highly susceptible to cutaneous leishmaniasis caused by Leishmania major, whereas CBA mice are resistant. We now show that this resistance correlates with the ability of macrophages to synthesize nitric oxide (NO) following activation with interferon-gamma or tumor necrosis factor alpha. Furthermore, the larger amounts of NO generated by resistant macrophages are related to higher levels of NO synthase activity, a difference which is not attributable to the number or the affinity of the receptors for interferon-gamma on these cells. The level of NO synthesis by activated macrophages was also correlated to the resistance in a number of other inbred mouse strains tested; macrophages from the resistant B10.S, C57BL and C3H mice produced significantly higher levels of NO than the macrophages from the susceptible BALB.b and DBA/2 mice.     

Reduced Smad3 protein expression and altered transforming growth factor-beta1-mediated signaling in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)-beta1, it was our goal to examine components of TGF-beta1-mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of protein levels of the TGF-beta1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF-beta1-stimulated levels of luciferase expression using the TGF-beta1-responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF-beta1-stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF-beta1-mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 protein expression in CF epithelium may also influence inflammatory responses. Our data demonstrate that both CF-phenotype cells lacking Smad3 expression, and A549 cells expressing a dominant-negative Smad3, are unable to support TGF-beta1-mediated inhibition of either the interleukin (IL)-8 or the NOS2 promoter. We conclude that a CF-related reduction in Smad3 protein expression selectively alters TGF- beta1-mediated signaling in CF epithelium, potentially contributing to aggressive inflammatory responses.     

TGF-beta 1 inhibition of IFN-gamma-induced signaling and Th1 gene expression in CD4+ T cells is Smad3 independent but MAP kinase dependent

In addition to classic Smad signaling pathways, the pleiotropic immunoregulatory cytokine TGF-beta1 can activate MAP kinases, but a role for TGF-beta1-MAP kinase pathways in T cells has not been defined heretofore. We have shown previously that TGF-beta1 inhibits Th1 development by inhibiting IFN-gamma's induction of T-bet and other Th1 differentiation genes, and that TGF-beta1 inhibits receptor-proximal IFN-gamma-Jak-Stat signaling responses. We now show that these effects of TGF-beta1 are independent of the canonical TGF-beta1 signaling module Smad3, but involve a specific MAP kinase pathway. In primary T cells, TGF-beta1 activated the MEK/ERK and p38 MAP kinase pathways, but not the JNK pathway. Inhibition of the MEK/ERK pathway completely eliminated the inhibitory effects of TGF-beta1 on IFN-gamma responses in T cells, whereas inhibition of the p38 pathway had no effect. Thus, TGF-beta1's inhibition of IFN-gamma signaling in T cells is mediated through a highly specific Smad3 independent, MEK/ERK-dependent signaling pathway.     

Association of decreased phosphorylation of ERK-2 with costimulation of rat T cell activation by MEK-1 inhibitors and TGF-beta1

Transforming growth factor-beta (TGF-beta) is usually known as an immunosuppressive cytokine, but we and others have shown stimulatory effects of TGF-beta on activation of Th2 T-lymphocytes. In the present investigation we have studied the effect of TGF-beta1 on phosphorylation of ERK, a MAP-kinase downstream of the Ras pathway. ERK is phosphorylated by MEK-1 and PD098059 and U0126 are specific inhibitors for this kinase. We demonstrate in the present study that these inhibitors abrogate the inhibitory effect of adh-splc (adherent-spleen cells) on activation of primary rat T-cells and induce a strong costimulatory effect almost as strong as we have previously shown with TGF-beta1. When TGF-beta1 is acting stimulatory on T-cell activation, it decreases phosphorylation of ERK-2 and thereby its activation. To investigate whether TGF-beta1 and MEK-1 inhibitors influence the same pathways, we compared their effects on cytokine profiles associated with SEA-induced rat T cell activation. TGF-beta1 induced IL-10 production, slightly decreased TNF-alpha production and decreased IFN-gamma production. The PD098059 inhibitor decreased both IFN-gamma and TNF-alpha production and together with TGF-beta1, it totally blocked IFN-gamma, TNF-alpha and IL-10 production. Thus TGF-beta1 and PD098059 showed overlapping but not identical effects on the cytokine pattern.     

Resistance exercise-induced increase in muscle mass correlates with p70S6 kinase phosphorylation in human subjects

The purpose of the present study was to investigate the possible relationship between a change in Thr³⁸⁹ phosphorylation of p70S6 kinase (p70^S6k) after a single resistance training session and an increase in skeletal muscle mass following short-term resistance training. Eight male subjects performed an initial resistance training session in leg press, six sets of 6RM with 2 min between sets. Muscle biopsies were obtained from the vastus lateralis before (T1) and 30 min after the initial training session (T2). Six of these subjects completed a 14-week resistance-training programme, three times per week (nine exercises, six sets, 6RM). A third muscle biopsy was obtained at the end of the 14-week training period (T3). One repetition maximum (1RM) squat, bench press and leg press strength as well as fat-free mass (FFM, with dual energy X-ray absorptiometry) were determined at T1 and T3. The results show that the increase in Thr³⁸⁹ phosphorylation of p70^S6k after the initial training session was closely correlated with the percentage increase in whole body FFM (r = 0.89, P < 0.01), FFM_leg (r = 0.81, P < 0.05), 1RM squat (r = 0.84, P < 0.05), and type IIA muscle fibre cross sectional area (r = 0.82, P < 0.05) after 14 weeks of resistance training. These results may suggest that p70^S6k phosphorylation is involved in the signalling events leading to an increase in protein accretion in human skeletal muscle following resistance training, at least during the initial training period.     

KIR2DS1-mediated activation overrides NKG2A-mediated inhibition in HLA-C C2-negative individuals

NK cell cytotoxicity is controlled through a balance of bothactivating and inhibitory signals. The HLA specificity of alloreactiveNK cells has been previously shown to be controlled by inhibitorykiller immunoglobulin-like receptors (KIRs). Alloreactive NKcells lyse targets that lack the HLA ligand for their inhibitoryKIR. We have characterized in detail an alloreactive NK clonein which the specificity is controlled by an activating receptor,KIR2DS1. Only target cells expressing the HLA-C group 2 (C2)epitope were lysed by this clone and homozygous C2 targets werelysed more strongly than heterozygous C1/C2 targets. Anti-CD158a(KIR2DS1) blocked lysis of targets confirming KIR2DS1 was responsible.Although this NK clone expressed NKG2A, an inhibitory receptorwhose ligand is HLA-E, targets with ligands for both KIR2DS1and NKG2A were lysed by this clone indicating that the KIR2DS1-mediatedactivation signal overrides the NKG2A-mediated inhibitory signal.KIR2DS1 activated NK clones in polyclonally expanded NK culturesfrom a donor that lacked the C2 epitope accounted for 1% ofall NK cells. This study highlights a potential role for NKcells controlled by activating KIR in mediating NK alloreactivity.     

转化生长因子β1及其信号转导分子Smad 2在病变肾小球中的表达及其意义

目的 探讨转化生长因子β1（TGF-β1）及其信号转导分子Smad2在肾小球肾炎中的表达及其意义。方法 以11例肾小球轻微病变为对照组,对各种不同组织学类型的IgA肾病40例、膜性肾炎10例、硬化性肾炎11例的肾穿刺活检标本,分别行原位分子杂交检测肾小球Smad2mRNA的表达和免疫组织化学ABC方法观察TGF-β1、IV型胶原及纤维蛋白在肾小球中的染色强度,并对其检测结果作图像分析处理。结果 除轻微病变型IgA肾病的Smad2mRNA表达外,其他病理类型肾炎中病变肾小球TGF-β1蛋白和Smad2mRNA的表达均高于对照组,且两者在肾小球中的表达与IV型胶原、纤连蛋白在肾小球中的沉积呈正相关。结论 TGF-β1及其信号转导分子Smad2可能参与了病变肾小球细胞外基质的过量积聚,在肾小球硬化过程中起重要作用。     

TLR-2-mediated metabolic reprogramming participates in polyene phosphatidylcholine-mediated inhibition of M1 macrophage polarization

Immunologic Research - This study aimed to investigate whether the classic hepatoprotective drug polyene phosphatidylcholine (PPC) regulates macrophage polarization and explores the potential role...     

Toll-like receptor 2 mediates prostaglandin E(2) production in murine peritoneal macrophages and splenocytes in response to Candida albicans

The involvement of Toll-like receptor 2 (TLR2) and TLR4 in triggering signal transduction pathways leading to prostaglandin E(2) (PGE(2)) production in response to Candida albicans has been studied in cells from wild-type, TLR2-/- and TLR4-/- knockout mice. In vitro PGE(2) production by macrophages challenged with zymosan, yeast or hypha cells was strongly inhibited in TLR2-deficient cells, but not in TLR4-/- cells, as compared to macrophages from wild-type mice. PGE(2) production was dependent on de novo cyclooxygenase-2 (Cox2) synthesis, since unchallenged cells failed to produce PGE(2) and specific Cox2 inhibition during challenge totally blocked PGE(2) production. Similar results were obtained following in vitro challenge of splenocytes from mice intravenously infected with the low-virulent C. albicans PCA2 strain. This indicates that TLR2 is the major receptor that mediates PGE(2) production in response to C. albicans, probably by upregulating Cox2 expression.     

Capacity to interact with T-cell replacing factor correlates with acquisition by B cells of murine differentiation antigen-1

The objective of this study was to determine whether certain membrane markers on B cells are involved with receipt of a T-cell helper signal. The helper factor, derived from an allogeneic supernatant, is antigen non-specific, non-Ia bearing and, thus, akin to T-cell replacing factor (TRF). The markers are murine differentiation antigens (MDAs) that are detected by two types of isogeneic lymphocyte culture (ILC). In Type 1 ILC replication of neonatal thymic cells is caused by MDA-1 and MDA-2 whereas in Type 2 ILC blastogenesis of adult lymph node T cells is triggered by MDA-2. Bone marrow (BM) cells, known to lack both MDAs, were used to reconstitute X-irradiated CBA/J mice and cells that homed to the spleen were examined by ILC at intervals to determine when the markers arose. In addition, purified splenic B cells from the reconstituted mice were exposed in vitro to sheep erythrocytes and TRF to determine their imimmunological capacity. Spleen cells obtained from mice 12 weeks after reconstitution with BM cells were shown to have acquired MDA-1 and to have the capacity, following interaction with TRF, to produce the maximum number of cells synthesizing IgM and IgG antibodies to SRBC. Spleen cells examined 5–6 weeks following reconstitution expressed MDA-2 but were unresponsive to TRF. BM-derived cells matured earlier in the presence of splenic T cells; they expressed MDA-1 and were able to interact with TRF at 5–6 weeks. On the other hand, BM-derived cells that acquired MDA-2 at an earlier, 2 week interval still remained unable to interact with TRF. These correlations of marker appearances with cellular function suggest MDA-1 may be a receptor for TRF.     

Transforming growth factor-beta 1 (TGF-beta 1)- and beta 2-like activities in malignant pleural effusions caused by malignant mesothelioma or primary lung cancer.

We investigated the levels of TGF-beta in malignant pleural effusions (MPE) caused by malignant mesothelioma (MESO) or primary lung cancer. TGF-beta levels in MPE caused by MESO were 283.9 +/- 219.2 pm (mean +/- s.d.) and were three to six times higher than those due to primary lung cancers (P < 0.01 or P < 0.05). We also evaluated TGF-beta 1- and beta 2-like activities in MPE using specific polyclonal antibodies. Although TGF-beta 1-like activity could be detected in all cases, TGF-beta 2-like activities were detected in five of seven in MESO and in a few cases with primary lung cancer. These results demonstrate that the levels of total TGF-beta and TGF-beta 2-like activity may be clinically useful to differentiate MESO from primary lung cancer. Our data also suggest that TGF-beta may help further characterize the clinical features of MESO.