Placental proteins as regulators of immunological reactions in pregnancy

The effect of two specific placental proteins, trophoblastic beta 1-glycoprotein (TBG) and chorionic alpha 1-microglobulin (CAG), on the immunological reactions was studied in vitro. TBG in physiological doses suppressed the proliferation of lymphocytes induced by plant mitogens and allogenic cells in the unidirectional mixed cultures, strengthened the effect of concanavalin A upon the induction of cells-suppressors in the culture and, in low concentrations, decreased the percentage of E- and EAC-rosette-forming cells. In none of the tests used CAG was effective. But when studying the effect of TBG and CAG mixture on PHA-induced proliferation of lymphocytes the inhibiting effect of TBG was weakened and, in some cases, completely relieved.     

Comparison of the action of the sera of pregnant women and of trophoblastic beta 1-glycoprotein in a number of immunological tests

The effects of blood sera of pregnant women in the I, II and III trimesters (SPWI, SPWII, SPWIII) were compared with those of purified preparations of trophoblastic beta 1-glycoproteid (TBG) on phytohemagglutinin (PHA) induced proliferative response of lymphocytes of the intact donors and on the production of a factor inhibiting the migration of leucocytes (FIM-L). SPWI and purified TBG preparations in doses corresponding to its level in SPWI were shown to produce similar effects. With the increase of TBG concentration, however, the inhibiting effect of its preparations on the proliferation of lymphocytes increased much more intensively than that of SPWII and SPWIII containing the same dose of this protein. The stimulating effect of TBG on the production of FIM-L increased with the dose as well while SPWII and SPWIII did not exert any effect on the migration of leucocytes. A suggestion is put forward that SPWII and SPWIII contain factors modifying the effect of TBG.     

Effects of trophoblastic beta 1-glycoprotein (TBG) on the functional activity of different cell lines

The effect of TBG on the functional activity of different cell lines, spontaneous and Con A induced proliferation of PBL was studied. If concentration of TBG is higher than 50 mu kg/ml it suppresses the proliferation in many used cell lines, except choriocarcinoma and cancer of uterus. The reliable increasing of spontaneous proliferation of PBL, Jurkat and K-562 cells may be observed if concentration is more lower (0.5-15 mu kg/ml). However proliferation of other cell lines corresponds to control level, and Con A induced proliferation of PBL is inhibited. The effect was more marked at 48, as compared to 24 hours of cell incubation with TBG.     

Extracts of regenerating Planarians regulate proliferation of vertebrate cells

The effects of extracts from intact and regenerating planarians on cell behaviour in culture were studied. The extracts were added to the culture of Chinese hamster fibroblasts and to the primary culture of human lymphocytes. Some extracts contained active agents which influenced the proliferation of fibroblasts increasing or decreasing the mitotic index. The extracts exerted no effect on the mitotic index of lymphocytes. When the extracts were added to the lymphocyte culture together with phytohemagglutinin, which induces the proliferation, the mitotic index somewhat increased. The extracts of regenerating planarians contain factors which activate and inhibit cell proliferation in culture. The active factors stimulated, rather than induced proliferation.     

大鼠肺泡巨噬细胞对人胚肺成纤维细胞增殖的抑制作用

实验采用[^3H]TdR掺入标记法测定微量培养人胚肺成纤维细胞的增殖,观察到健康大鼠的肺泡巨噬细胞（alveolar macrophage,AM)可抑制成纤维细胞增殖。经调理的酵母多糖激活后,AM的抑制作用加强;而经消炎痛处理的AM,抑制作用转为被促进增殖作用所取代;测定AM上清液中前歹腺素E（prostaglandin E,PGE)含量,显示其抑制作用与PGE含量相关。结果提示,AM有抑制作促进肺成纤维细胞增殖的双重作用,正常时以抑制作用占优势;PGE可能是AM产生的主要的肺纤维化抑制因子。     

The requirement for macrophage-lymphocyte interaction in T lymphocyte proliferation induced by generation of aldehydes on cell membranes.

Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents. Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages. Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr. By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation. The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation.     

Selectivity of hemoregulatory peptide (HP5b) action in culture

A synthetic analog of a hemoregulatory peptide associated with mature human granulocytes (HP5b) has been investigated for inhibitory effects on various cell types in culture as compared to inhibitory action on mouse and human myelopoietic colonies (CFU-gm), which occurs from 1 X 10(-13) to 1 X 10(-6) M in vitro. This includes colony formation by lymphoid T and B cells in capillary cultures, as well as mitogen activation of T, B and NK cells. At higher concentrations, i.e., above 1 X 10(-7) M, an inhibitory effect was found on colony formation. Neither the production of interleukin (IL) 3 by mitogen-activated T cells, nor the proliferation of the IL-3-dependent L/B cell line were affected by the peptide up to 1 X 10(-5) M. A slight inhibitory effect was found above 1 X 10(-9) M on mouse 3T3 fibroblasts. A series of malignant cell lines was also tested. No effect was seen between 1 X 10(-11) and 1 X 10(-7) M on human mammary carcinoma cells in culture. On Ehrlich ascites mouse mammary carcinoma cells a 30% inhibition was seen at 10(-6) M. On a human glioblastoma cell line (GaMg) no effect was seen, and on a rat glioma cell line (BT5C) an inhibitory effect was seen at 1 X 10(-7) M and above. No significant inhibition of cell growth was seen on SC1 mouse lymphoma cells from 1 X 10(-9) to 1 X 10(-5) M during 7 days of culture. The investigated normal and malignant cell types in culture were thus not inhibited in very low concentrations which act on CFU-gm. However, a variable inhibitory effect was found at higher concentrations where the inhibition of myelopoiesis was maximal and at concentrations where the inhibition is released. The hemoregulatory peptide thus seems to be a concentration-dependent selective inhibitor of myelopoiesis. The finding that various malignant cells do not respond at lower concentrations supports the possibility of using the peptide as a protector of normal cells during cancer chemotherapy.     

Gr-1+ myeloid cells lacking T cell protein tyrosine phosphatase inhibit lymphocyte proliferation by an IFN-gamma- and nitric oxide-dependent mechanism

The T cell protein tyrosine phosphatase is involved in the immune system regulation, as evidenced by defective function and development of several hemopoietic cell populations in T cell protein tyrosine phosphatase (TC-PTP)-deficient mice. In particular, B and T cell proliferation is greatly inhibited when total splenocytes are stimulated by LPS or anti-CD3 mAb. To define the functional defect of TC-PTP(-/-) lymphocytes, we isolated T and B cells from the spleen of TC-PTP(-/-) mice. We show that the proliferative response of lymphocytes was greatly increased when cultured as a purified population, indicating that an inhibitory population is present in TC-PTP(-/-) spleen. However, TC-PTP(-/-) lymphocytes have a 2- to 3-fold lower proliferation rate compared with TC-PTP(+/+) lymphocytes, suggesting that, as shown previously in embryonic fibroblasts, TC-PTP is involved in the control of cell cycle in lymphocytes. We have characterized phenotypically and functionally the inhibitory population present in the spleen of TC-PTP(-/-) mice. We show that a Gr-1(+)-enriched cell population isolated from TC-PTP(-/-) mice suppresses the CD3-induced proliferation of T cells in coculture in vitro. The specific inhibition of NO synthesis with N(G)-monomethyl-L-arginine.monoacetate restored splenocyte responses, and there is a strict correlation between NO levels and the degree of suppression. Neutralization of IFN-gamma with specific mAb almost completely abolished the inhibitory activity of Gr-1(+) cells and concomitantly high levels of NO secretion. Moreover, inhibition of lymphocyte proliferative responses required cell-cell contact to achieve sufficient levels of NO. These findings demonstrate an important function of TC-PTP in the induction of the NO pathway that mediates inhibition of T cell proliferation.     

Cellular basis of immunomodulation by cholera toxin in vitro with possible association to the adjuvant function in vivo

Cholera toxin (CT) is a potent oral immunogen that also acts as a strong mucosal adjuvant for immune responses to related as well as unrelated Ag. To elucidate the immunomodulating effects of CT at the cellular level we have examined interactions of CT with APC and with B and T lymphocytes in vitro. CT markedly stimulated the production of IL-1 from APC (mouse peritoneal macrophages or macrophage cell line P388D1) but did not induce Ia-Ag and had marginal, if any, effect in potentiating Ia Ag expression stimulated by rIFN-gamma on these cells. CT had differential effect on T cell proliferation in vitro, usually strongly inhibitory but on Con A-stimulated spleen cells during prolonged (greater than or equal to 5 days) culture or when added on day 4 or later to these cultures up to a two- to three-fold enhancement of proliferation was seen. CT-induced inhibition of T cell proliferation was associated with decreased production of IL-2 and anergy to exogenously added IL-2 despite apparently normal expression of IL-2R. Similar to what was found with T cells LPS-stimulated spleen B cells demonstrated both inhibition and enhancement of proliferation in the presence of CT: in high concentrations (greater than or equal to 10(-8) M) and early in culture (day 3) CT had a strong inhibitory effect on the proliferation of B cells, whereas later (day 6) and/or at lower CT concentrations (10(-9) to 10(-11) M) the proliferation was increased up to 10-fold. The net effect of CT treatment on Ig-production by LPS-stimulated spleen B cells was seen as an enhanced level of IgA and IgG but not IgM in culture supernatants. The differential effects of CT on the cells of the immune system observed in vitro may, singly or in combination, explain the immunostimulatory function of CT.     

PI3K/AKt/mTOR信号通路介导黄芩苷抑制增生性瘢痕组织成纤维细胞的增殖

黄芩苷作为一种黄酮类成分可通过抑制细胞增殖、促进凋亡发挥抗肿瘤作用,但它是否对异常增生的瘢痕具有抑制增生的作用尚不清楚.本研究探讨黄芩苷抑制人增生性瘢痕组织成纤维细胞增殖的分子机制. 采用MTT比色法检测不同浓度的黄芩苷（2.24×10-2 ～ 2.24×102 mmol/L）对体外培养的增生性瘢痕组织成纤维细胞增殖的抑制作用.发现浓度为2.24×100～2.24×102 mmol/L黄芩苷处理组明显抑制增生性瘢痕组织成纤维细胞的增殖（P<0.05）.转染后的荧光素酶报告基因活性检测、RT-PCR及Western印迹分析技术检测其mRNA水平及细胞的帽状依赖翻译的表达.2.24×102 mmol/L黄芩苷处理后,黄芩苷作用组的mRNA水平并无明显差异（P>0.05）;增生性瘢痕成纤维细胞的帽状依赖结构的翻译明显被黄芩苷所抑制.采用Western印迹分析检测被黄芩苷干预的增生性瘢痕组织成纤维细胞的增殖相关的蛋白的表达;m7GTP琼脂糖珠沉淀结合蛋白4E-BP1与eIF4E的变化.发现增殖相关的蛋白mTOR、p70S6K、S6、4EBP1、eIF4E及其上游的AKT表达明显下调（P<0.05）,而PTEN表达明显上调.p-AKT(Ser473)、p-mTOR(Ser2448)、p-S6(Ser235/236)、p-4EBP1(Thr37/ 46)、p-PTEN（T380/S382/383）磷酸化水平下降（P<0.05）.在黄芩苷作用下的增生性成纤维细胞中的游离的4E-BP1明显减少（P<0.05）,而与eIF4E结合的4E-BP1明显增加（P<0.05）黄芩苷诱导游离的4E-BP1与eIF4E结合,从而抑制帽状依赖蛋白翻译.以上结果说明,黄芩苷可通过抑制PI3K/AKT/mTOR信号通路抑制人增生性瘢痕组织成纤维细胞的增殖.     

Electrophysiological and functional effects of sphingosine-1-phosphate in mouse ventricular fibroblasts

The aim of this study was to characterize the effects of sphingosine-1-phosphate (S1P) on cardiac ventricular fibroblasts. Impacts of S1P on fibroblast excitability, cell migration, proliferation and secretion were characterized. The patch-clamp technique in the whole-cell configuration was used to study the S1P-induced current from mouse ventricular fibroblasts. The expression level of the S1P receptor during cell culture duration was evaluated by western-blot. Fibroblast proliferation and migration were quantified using the methylene blue assay and the Boyden chamber technique, respectively. Finally, fibroblast secretion properties were estimated by quantification of the IL-6 and collagen levels using ELISA and SIRCOL collagen assays, respectively. We found that S1P activated SUR2/Kir6.1 channel and that this effect was sensitive to specific inhibition of the S1P receptor of type 3 (S1P3R). In contrast, S1P1R receptor inhibition had no effect. Moreover, the S1P-induced current increased with cell culture duration whereas S1P3R expression level remained constant. The activation of SUR2/Kir6.1 channel by S1P via S1P3R stimulated cell proliferation and decreased IL-6 and collagen secretions. S1P also stimulated fibroblast migration via S1P3R but independently from SUR2/Kir6.1 channel activation. This study demonstrates that S1P, via S1P3R, affects cardiac ventricular fibroblasts function independently or through activation of SUR2/Kir6.1 channel. The latter effect occurs after fibroblasts differentiate into myofibroblasts, opening a new potential therapeutic strategy to modulate fibrosis after cardiac physiopathological injury.     

Type V collagen selectively inhibits human endothelial cell proliferation

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.     

Crucial role of fibroblasts in regulating epidermal morphogenesis

Epidermis reconstructed on de-epidermized dermis (DED) was used to investigate whether fibroblasts can substitute growth factors needed for generation of a fully differentiated epidermis. For this purpose, a centrifugal seeding method was developed to reproducibly incorporate different fibroblast numbers into DED. Using (immuno)histochemical techniques, we could demonstrate that in the absence of fibroblasts the formed epidermis consisted only of two to three viable cell layers with a very thin stratum corneum layer. However, in the presence of fibroblasts keratinocyte proliferation and migration was stimulated and epidermal morphology markedly improved. The stimulatory effect of fibroblasts showed a biphasic character: keratinocyte proliferation increased in the initial phase but decreased in later stages of cell culture. After 3 weeks culture at the air-liquid interface, the proliferation index decreased irrespective of the number of fibroblasts present within the dermal matrix to levels observed also in native epidermis. Keratin 10 was localized in all viable suprabasal cell layers irrespective of the absence or presence of fibroblasts. Keratin 6 was downregulated with increasing numbers of fibroblasts, and keratins 16 and 17 were absent in fibroblast-populated matrices. The expression of involucrin or transglutaminase 1 showed a similar pattern as for the keratins. Irrespective of the number of fibroblasts incorporated into DED, the expression of alpha(3), alpha(6), beta(1), and beta(4) integrin subunits was upregulated. In fibroblast-free DED matrices normalization of epidermal differentiation was only achieved when the culture medium was supplemented by keratinocyte growth factor. The results of this study indicate that normalization of epidermal differentiation can be achieved using a non-contractile dermal matrix populated with fibroblasts.     

LFA-1/ICAM-1在ConA诱导的小鼠胸腺细胞活化中的作用

用抗LFA-1/ICAM-1粘附分子单克隆抗体和ConA联合刺激小鼠胸腺细胞,初步研究了该膜分子在经TCR/CD3介导的胸腺细胞活化信号传导以及胸腺细胞亚群选择中的作用。在ConA刺激系统中,抗ALFA-1/ICAM-1单抗均能抑制胸腺细胞的增殖应答,且以抗LFA-1单抗的作用更为显著;而在PMA加钙离子载体A23187刺激体系中,抗LFA-1单抗却表现出明显的促活化效应。当加入IL-2 时,抗LFA——1/ICAM-1单抗便不能抑制ConA刺激的胸腺细胞活化。此外,抗体对已活化的胸腺母细胞增殖也无影响。FACS分析的结果还显示,抗LFA-1单抗可明显降低CD4~-CD8~ 胸腺细胞亚群的比例,而抗ICAM-1单抗对此无影响。表明胸腺细胞表面粘附分子LFA-1具有直接参与TCR/CD3途径介导的跨膜信号传导的功能,并对CD4~-CD8~ 胸腺细胞亚群的功能分化与成熟可能起重要作用。     

人参皂甙Rb1,Rg1,Re和Rh1对体外培养细胞增殖的影响

本研究应用体外培养的人胚肺成纤维细胞(2BS)和人宫颈癌细胞(Hela)为实验模型。在培养液中分别加入人参根总皂甙(SRG)和人参皂甙Rb_1、Rg_1、Re、Rh_1孵育3～7d后,用MTT法和细胞计数法测定细胞的增殖。MTT测定结果表明,Rb_1、Rg_1、Re、Rh_1可以促进衰老细胞的增殖,但对未衰老的细胞增殖没有显著影响,对Hela细胞的增殖有抑制作用。细胞计数法测定结果表明,总皂甙和4种皂甙单体都可以降低Hela细胞的群体增殖率和克隆生长率,其中Re、Rh_1作用显著。同时,增加衰老细胞的群体增殖率和克隆生长率,其中Rb_1和Rg_1作用显著。     

Antibodies to nonpolymorphic determinants of the Thy-1 molecule inhibit T cell proliferation

The effect of anti-Thy-1 monoclonal antibodies on murine mixed lymphocyte reactions and concanavalin A-induced mitogenesis were investigated. It is demonstrated that rat antibodies against nonpolymorphic determinants of the murine Thy-1 antigen inhibited cell proliferation in the absence of complement. In contrast, antibodies against polymorphic determinants of Thy-1 had no effect on T cell activation. Inhibition of T cell proliferation did not depend on the isotype of the blocking antibody, because both IgM and IgG antibodies against monomorphic determinants were inhibitory, whereas IgM or IgG antibodies against allotypic determinants were inactive. In addition, the blocking activity could not be attributed to the xenogeneic (rat) origin of the antibodies to nonpolymorphic Thy-1 determinants, because rat anti-Thy-1.2 antibodies had no effect on cell activation. Thus, the efficacy of anti-Thy-1 antibodies as T cell inhibitors was determined by the antibody specificity. The suppressive mechanism of anti-Thy-1 antibodies was effective throughout the entire course of mixed lymphocyte reactions. Addition of antibodies at any time point during the first 90 hr of a 120-hr mixed lymphocyte culture resulted in significant suppression of the proliferative response. However, in some cases an early enhancement preceded suppression of the response. The modulation of proliferative responses by anti-Thy-1 did not result from a nonspecific mitogenic effect of the antibodies on T lymphocytes, because no effects were observed when antibodies were added to responder cells alone. These results suggest that the Thy-1 molecule, or a molecule that is located on the cell membrane in close proximity to the Thy-1 antigen, is involved in the activation of T lymphocytes.     

Salivary gland extract from Ixodes ricinus tick polarizes the cytokine profile toward Th2 and suppresses proliferation of T lymphocytes in human PBMC culture.

Tick salivary gland extract (SGE) was previously shown to inhibit murine T cell proliferation. In mice, SGE has an inhibitory effect on Th1 and a stimulatory effect on Th2 cytokine elaboration. In the present study, tick-mediated immunomodulation of human T cell proliferation and cytokine elaboration was analyzed using human peripheral blood mononuclear cells (PBMCs) stimulated with concanavalin A (Con A) or lipopolysaccharide (LPS). Using flow cytometry, tick saliva-induced changes were investigated in human mononuclear cell subpopulations. SGE from Ixodes ricinus dose-dependently inhibited human T cell proliferation. This finding supports the flow cytometry data, showing that the percentage of Con A-activated HLA-DR-CD3+ T lymphocytes and CD4+ CD8+ double-positive T cells decreased after SGE treatment. SGE significantly inhibited the in vitro production of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) secreted by Th1 lymphocytes. In contrast, the elaboration of IL-4, IL-6, and IL-10 secreted by Th2 lymphocytes was significantly stimulated by I. ricinus SGE. Similarly, the production of both IL-1alpha and IL-1beta was significantly stimulated after SGE treatment. These data indicate that the tick-induced immunomodulatory events in humans are similar to those previously described in a murine model.     

Endogenous fragment of hemoglobin, neokyotorphin, as cell growth factor

It is shown that neokyotorphin (the -globin fragment 137–141) stimulates proliferation of normal cells (murine embryonic fibroblasts, red bone marrow and spleen cells) and tumor cells (murine melanoma and transformed fibroblasts L929) in the absence or in the presence of fetal bovine serum. In contrast to serum deprivation conditions, the ability to potentiate L929 cell growth in the presence of fetal serum is strongly cell density dependent. The peptide also enhances the viability of L929 cells, murine embryonic fibroblasts and of the primary cultures of murine red bone marrow cells and splenocytes under serum-deprivation conditions for at least 72 h. The results of flow cytometry analysis suggest that the effect of neokyotorphin on survival of L929 cells in serum-free culture medium is due to maintenance of cell proliferation in the absence of growth factors. Along with cell cycle progression the peptide induces reversible reduction of L929 cell size.