小鼠胸腺基质细胞系分泌的趋化因子的分析

利用Boyden小室法和FACS分析法,我们分析了五株小鼠胸腺基质细胞系(MTSC)培养上清液对中性粒细胞,单核巨噬细胞和淋巴细胞的化学趋化因子(Chemokines)活性,及定向迁移的淋巴细胞中B细胞、CD 4~ CD 8~-和CD4~-CD8~ T细胞的比例。结果显示,五株MTSC的培养上清液对上述靶细胞均有不同程度的趋化作用.MTSC细胞分泌趋化因子的情况可分为三类:1.MTEC 1和MTEC 2产生的Chemokine(s)对中性粒细胞和淋巴细胞的趋化作用相对较强;2.MTDC 4分泌的Chemokine(s)主要作用于单核巨噬细胞;3.MTEC 3和MTEC 5分泌的Chemokine(s)对多种类型的靶细胞,包括中性粒细胞、单核巨噬细胞和淋巴细胞,表现的趋化作用没有明显的强弱之分。MTSC-SN对B细胞的趋化活性普遍高于对T细胞的趋化活性,对CD 4~-CK 8~ T细胞的趋化活性高于对CD 4~ CD 8~-T细胞的趋化活性。MTSC-SN中趋化因子的分析,有利于新型chemokines的发现及其生物功能的阐明,并可进一步研究Chemok-ine(s)在T细胞发育中的作用。     

小鼠胸腺上皮细胞系MTEC1产生30kDr MCP—1类趋化因子

A medullary type mouse thymic epithelial cell line MTEC1 can produce chemokines. Crude culture supernant of MTEC1 cells was concentrated by controlled-pore glass beads. One of these chemotactic proteins was isolated from MTEC1 supernatant and purified to homogeneity by heparin-Sepharose affinity chromatography, cation-exchange FPLC and Reverse Phase-HPLC. The chemotactic factor for both lymphocytes and monocytes was identified as a 30 kD protein by SDS-PAGE analysis under reducing conditions. After cleavage of the NH2-terminally blocked protein with formic acid, the amino acid sequence of the internal fragment was analyzed and found to be identical to the amino acid sequence of mouse MCP-1/JE. The protein was hence identified as a glycosylated MCP-1/JE-like chemokine secreted by thymic epithelial cells. The characterization of chemokines produced by thymic stromal cells will benefit the analysis of the mechanism of thymus homing and the finding of new chemokines.     

IL—1诱导小鼠胸腺上皮细胞（MTEC1）克隆分泌化学趋化因子

To analyze the capability of IL-1 and IL-6 in the induction of chemokine (CF) production by mouse thymus epithelial cell (MTEC1) clones, MTEC1 cells were cloned through one cell culture and individual cell clones were established in long term culture referred to as MTEC1-DW clones. The constitutive production of IL-1, IL-6 and CF by MTEC1-DW clones was evaluated and the patterns of the cytokine production determined. Addition of exogenous IL-1 or IL-6 or both to the cultures of those MTEC1-DW clones that are unable to produce CF, and incubated for 2 days, then, to assess the chemotactic activity in the cell culture supernatants (SNs). In the opposite, addition of anti-IL-1 mAb(s) to the cultures of those MTEC1-DW clones that can produce IL-1 and CF to neutralize secreted IL-1 then, to test chemotactic activity in the SNs after 2-day incubation. The results showed that in the MTEC1-DW clones which were unable to constitutively produce IL-1 or CF, addition of IL-1 could induce these cloned cells to produce CF with high chemotactic activity. By constrast, addition of anti-IL-1 mAb(s) to those MTEC1-DW clones that constitutively produce IL-1 and CF could significantly inhibit them to produce CF. IL-6 only exhibited weak activity in the induction of CF production by those cloned cells. Therefore, in the cytokine network regulation, CF production is mainly induced by endogenously produced IL-1 in MTEC1 cells.     

Optimal chemotactic responses of leukemic T cells to stromal cell-derived factor-1 requires the activation of both class IA and IB phosphoinositide 3-kinases

Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 are a multifunctional chemokine/receptor system with essential roles in the development of the immune system and other aspects of embryogenesis, including vascularization and organ development. SDF-1 is also a potent chemoattractant for T cells and has roles in both inflammation and immune homeostasis. Our group has previously demonstrated that phosphoinositide 3-kinase (PI 3-kinase) is activated in SDF-1-stimulated T cells and is indeed required for SDF-1-mediated chemotaxis. In this study Jurkat clones were established, stably expressing dominant negative constructs of class IA and class IB PI 3-kinases under the control of the tetracycline off inducible gene system, to determine the relative roles of these PI 3-kinases in SDF-1 signaling. Our results show that expression of either kinase-dead PI3Kgamma (KD-PI3Kgamma) or Deltap85 (a construct unable to bind class I(A) p110alpha, -beta, or -delta) leads to a partial inhibition of SDF-1-stimulated protein kinase B phosphorylation, but had no effect on SDF-1-induced phosphorylation of the mitogen-activated protein kinase ERK1/2. Functional studies demonstrated that expression of KD-PI3Kgamma markedly inhibited SDF-1-mediated chemotaxis, typically eliciting 40-60% inhibition. Interestingly, the expression of Deltap85 also leads to inhibition of the SDF-1-mediated chemotactic response, albeit to a much lesser extent than achieved with the KD-PI3Kgamma mutant, typically in the range of 20-40% inhibition. Furthermore, the inhibition of chemotaxis by the expression of dominant negative class IA or class IB PI 3-kinases could be enhanced by the presence of the PI 3-kinase inhibitor LY294002. Together, these results demonstrate that optimal chemotactic response of leukemic T cells to SDF-1 requires the activation of both class IA and class IB PI 3-kinases.     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturationin vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed.     

IL-1诱导小鼠胸腺上皮细胞(MTEC1)克隆分泌化学趋化因子

通过将本室所建小鼠胸腺上皮细胞系MTEC1进行克隆,获得由单一细胞来源的12个MTEC1-DW细胞克隆,检测各克隆分泌IL-1,IL-6及CF活性,分析诱导MTECl-DW细胞克隆分泌CF的因素.选择不分泌IL-1及CF的MTEC1-DW克隆,于细胞培养中加入外源IL-1或/及IL-6,分析其细胞培养液中CF活性;选择分泌高活性IL-1及CF的MTEC1-DW克隆,于细胞培养中加入抗IL-1mAb,阻断IL-1活性,分析其细胞培养液中CF活性.结果显示IL-1能诱导MTEC1-DW细胞克隆分泌CF,IL-6的这种作用则很微弱.     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed. Project supported by the National Natural Science Foundation of China (Grant No. 39230320).     

Suppression by 17beta-estradiol of monocyte adhesion to vascular endothelial cells is mediated by estrogen receptors

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.     

Multiple types of cytokines constitutively produced by an established murine thymic epithelial cell line.

An untransformed murine thymic epithelial cell line (MTEC1) has been established. Without exogenous stimulation, the MTEC1 cells constitutively produced multiple types of cytokines, including IL-1, IL-6, IL-7, GM-CSF and chemotactic factor(s). Of which, IL-6, GM-CSF and chemotactic factor(s) were abundant; IL-1, moderate; and IL-7 at low level. MTEC1 cells neither produced detectable IL-3 nor TNF alpha. Thus, the MTEC1 cells may be useful not only in the evaluation of the signals required for thymic selection in vitro, but also useful in the analysis of the endogenous regulation of the autocrine cytokine production cascade.     

CrkL plays a role in SDF-1-induced activation of the Raf-1/MEK/Erk pathway through Ras and Rac to mediate chemotactic signaling in hematopoietic cells

Intracellular signaling mechanisms regulating SDF-1-induced chemotaxis of hematopoietic cells have remained elusive. Here we demonstrate that overexpression of the adaptor molecule CrkL enhances SDF-1-induced chemotaxis of hematopoietic BaF3 and 32Dcl3 cells. Overexpression of CrkL also enhanced SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway as well as that of the small GTPases Ras, Rap1, and Rac, while a dominant negative mutant of Ras or Rac suppressed CrkL-enhanced Erk activation. SDF-1 stimulation induced tyrosine phosphorylation of CrkL, which was inhibited by the Src family kinase inhibitor PP1 or by dominant negative mutants of Lyn, thus indicating that Lyn mediated SDF-1-induced phosphorylation of CrkL. However, inhibition of the Lyn kinase activity failed to affect SDF-1-induced activation of the small GTPases and Erk. On the other hand, SDF-1-induced activation of the Erk signaling pathway as well as chemotaxis was inhibited by overexpression of a CrkL mutant lacking the N-terminal SH3 domain, which mediates interaction with various signaling molecules including guanine nucleotide exchange factors for the Ras and Rho family GTPases. SDF-1-induced chemotaxis was also inhibited by the dominant negative Ras or Rac mutant as well as by the MEK inhibitor PD98059. These results indicate that CrkL mediates SDF-1-induced activation of the Raf-1/MEK/Erk signaling pathway through Ras as well as Rac in hematopoietic cells and, thereby, plays important roles in the induction of chemotactic response.     

A potential role for caveolin-1 in estradiol-17beta-induced proliferation of mouse embryonic stem cells: involvement of Src, PI3K/Akt, and MAPKs pathways

Although both estrogen and caveolin have been implicated in many physiological functions, their precise relationship is not completely understood in mouse embryonic stem (ES) cells. Thus, this study was designed to examine the relationship between estradiol-17beta (E(2)) and caveolin-1 in mouse ES cell proliferation. E(2) increased the expression of caveolin-1 and caveolin-2 mRNA and proteins, but pre-treatment with ICI 182,780 [an estrogen receptor (ER) antagonist] inhibited E(2)-induced increase in caveolin-1 and caveolin-2 proteins expression. E(2) also increased phosphorylated levels of caveolin-1, Src, and Akt. Phospho-caveolin-1 was significantly blocked by ICI 182,780 or pyrazolopyrimidine 2 (PP2; a Src-kinase inhibitor). LY 294002 (a PI3K inhibitor) or PD 98059 (an ERK1/2 inhibitor) prevented E(2)-induced increase in caveolin-1 expression and the accompanying [(3)H]-thymidine incorporation. Furthermore, inhibition of caveolin-1 expression using a caveolin-1 siRNA significantly attenuated E(2)-induced up-regulation of proto-oncogenes, cell cycle regulatory proteins, [(3)H]-thymidine incorporation, overall cell number, and percent of the cell population in S phase, while mediating a concomitant increase in the G0/G1 population. In conclusion, E(2) stimulates mouse ES cell proliferation partially through up-regulating caveolin-1 via the Src, PI3K/Akt, ERK1/2 signaling pathways.     

The ubiquitin-related protein PLIC-1 regulates heterotrimeric G protein function through association with Gbetagamma

PLIC-1, a newly described ubiquitin-related protein, inhibited both Jurkat migration toward SDF-1alpha and A431 wound healing, but the closely related PLIC-2 did not. PLIC-1 prevented the SDF-1alpha-induced activation of phospholipase C, decreased ligand-induced internalization of SDF-1alpha receptor CXCR4 and inhibited chemotaxis signaled by a transfected Gi-coupled receptor. However, PLIC-1 had no effect on Gs-mediated adenylyl cyclase activation, and inhibited only the Gbetagamma-dependent component of Gq-initiated increase in [Ca2+]i, which is consistent with selective inhibition of Gbetagamma function. PLIC-1 colocalized with G proteins in lamellae and pseudopods, and precipitated Gbetagamma in pull downs. Interaction with Gbetagamma did not require PLIC-1's ubiquitin-like or ubiquitin-associated domains, and proteasome inhibition had no effect on SDF-1alpha activation of phospholipase C, indicating that PLIC-1's inhibition of Gbetagamma did not result from effects on proteasome function. Thus, PLIC-1 inhibits Gi signaling by direct association with Gbetagamma; because it also interacts with CD47, a modulator of integrin function, it likely has a role integrating adhesion and signaling components of cell migration.     

Regulation of monocyte chemotactic protein-1 expression in human endometrial stromal cells by estrogen and progesterone.

There is a cyclicity in the number of endometrial macrophages that is most likely secondary to changes in steroid hormone levels. One cytokine that controls macrophage migration is monocyte chemotactic protein-1 (MCP-1). In the endometrium, highest levels of MCP-1 are detected perimenstrually, when estrogen levels are low; however, when estrogen levels are high (around the time of ovulation), MCP-1 levels are lowest. We hypothesized that sex steroids may be involved in the regulation of macrophage migration by regulating MCP-1 expression. We investigated the regulation of MCP-1 expression in human endometrial stromal cells by estradiol 17beta (E2) and progestins. We found that MCP-1 mRNA levels decreased in response to E2 (5 x 10(-8) M), with biphasic nadirs at 8 h and 24 h. MCP-1 protein production was also inhibited by E2 in a concentration-dependent manner. Tamoxifen, an anti-estrogen, alone (10(-7) M) did not affect MCP-1 expression, but it reversed the E2-induced inhibition up to 80%. Progesterone (10(-7) M) alone slightly decreased MCP-1 levels, and the combination of E2 and progesterone further decreased them, but that decrease was not different from that observed using E2 treatment alone. In summary, we found that E2 inhibits MCP-1 expression in endometrial stromal cells, and we speculate that E2 may control endometrial macrophage migration by regulating MCP-1 expression.     

Estrogenic compounds suppressed interferon-gamma production in mouse splenocytes through direct cell-cell interaction

Here, we reported the effects of 17beta-estradiol (E2), isoflavone genistein (Gen), and daidzein (Dai) on the production of interferon (IFN)-gamma by splenocytes isolated from C57BL/6N mice. When mouse splenocytes were stimulated with lipopolysaccharide, E2, Gen, and Dai suppressed the production of IFN-gamma. However, when only nonadherent cell populations of splenocytes were tested, none of these estrogenic compounds suppressed IFN-gamma production. This result indicates that IFN-gamma production by nonadherent cell populations of splenocytes treated with estrogens is regulated by adherent cell populations. Moreover, direct cell-cell interaction between both populations was necessary for suppression of IFN-gamma production by nonadherent populations. In addition, E2 conjugated with bovine serum albumin inhibited IFN-gamma production as well as E2. This result suggests that the plasma membrane-associated estrogen receptor plays a prominent role in this suppression mechanism.