Encapsulation of parathyroid cells in hollow fibers: a preliminary report

The purpose of experiments was to evaluate the survival and functioning of human parathyroid cells after encapsulation in hollow fibers (HFs). The polypropylene HFs K600(PP Accurel (Akzo-Nobel, Germany) of inner diameter 0.6 mm, wall thickness 0.2 mm, original or surface modified were used for encapsulation. Production of parathormone (PTH) by encapsulated cells was measured in vitro. HF were filled with parathyroid cell suspension and tightly closed. Encapsulated cells were cultured for 9 or 33 days in RPMI 1640 containing 10% FCS or in Chang's medium. The level of PTH, produced by encapsulated cells was evaluated in the culture medium with radioimmunoassay test (RIA). The assays were performed every 2-4 days. The result of PTH assay was similar in both types of tested media as well as with unmodified and modified HFs, being 2-4 pg/ml of culture medium per 10(3) encapsulated cells. In conclusion, encapsulation in original or modified HFs ensures diffusion of nutrients from culture medium to encapsulated cells and allows for functioning of cells for at least 33 days in vitro.     

Effect of growth and differentiation stimuli on the development of antigen-responsive B cells in fetal liver

The development of the B cell immune repertoire was studied using an in vitro fetal organ culture system. In order to analyze the mechanism by which B cell precursors clonally expand and diversify, fetal lymphoid tissues were incubated in the presence of several factors known to influence B cell differentiation: IL-1, IL-2, WEHI-3 culture supernatant containing IL-3, and a factor from a cyclic neutropenia patient (CNF). By analyzing the effect of exogenous factors on the frequency of antigen-responsive B cells, the ability of the factor to either inhibit or enhance clonal expansion was determined. It was found that the addition of IL-1, WEHI-3 supernatant, or CNF increased the frequency of DNP-responsive B cells suggesting an enhancement of clonal expansion. IL-2, on the other hand, did not alter the frequency of antigen-responsive B cells. The effect of added factors on the kinetics of appearance of phosphorylcholine (PC)-responsive B cells, which are known to be acquired in ontogeny about 2 weeks later than DNP-responsive B cells, was also analyzed. The data indicate that CNF, unlike IL-1, IL-2, and WEHI-3 culture supernatant, results in an earlier appearance of PC-responsive B cells. These results suggest that soluble factors may play a role in the generation of the B cell repertoire.     

Different growth factor requirements for the ex vivo amplification of transplantable human cord blood cells in a NOD/SCID mouse model

The growth factor combination containing early acting cytokines FLT-3 ligand (FL), Stem Cell Factor (SCF) and thrombopoietin (TPO) is able to maintain, for an extended culture period, early stem cells, defined as long-term repopulating NOD/SCID mice (Scid Repopulating Cell-SRC) contained in cord blood (CB). In this culture system, the role of IL-6 and IL-3 has not been clearly established. Using a combination of FL+TPO+SCF with or without IL-6, we were able to form CB CD34+ cells for 30 weeks. The CB CD34+ cells cultured in this system engrafted NOD/SCID mice after 6 weeks of culture; the cells from primary recipients were also able to engraft secondary NOD/SCID mice. When CB CD34+ cells were cultured in the presence of IL-3 in the place of IL-6 we observed an even better expansion of cells and a similar clonogenic progenitor output in the first 8 weeks of culture. However, more primitive LTC-IC output increased up to week 6 with the growth factor combination containing IL-3 and then decreased and disappeared, while with the growth factor combination with or without IL-6 increased up to week 23. Cells cultured for 4 weeks with the 4-factor combination containing IL-3 engrafted NOD/SCID mice less efficiently. Repopulation of NOD/SCID mice was no longer observed when ex vivo expansion was performed for 6 weeks. This study provides some evidence that no differences could be detected in long-term maintenance and even expansion of human primitive cord blood cells cultured with FL+TPO+SCF in the presence or absence of IL-6. Under the culture conditions employed in this study, the presence of IL-3 reduced the repopulating potential of expanded CB CD34+ cells.     

Osteogenic differentiation of encapsulated rat mesenchymal stem cells inside a rotating microgravity bioreactor: in vitro and in vivo evaluation

The objective of this study is to evaluate the in vitro and in vivo osteogenic potential of rat bone marrow mesenchymal stem cells (BM-MSCs) using chitosan/hydroxyapatite (C/HAp) microbeads as encapsulation matrix under osteoinductive medium and dynamic culture conditions. The degradation characteristics of C/HAp microbeads were evaluated under in vitro and in vivo conditions for 180 days. BM-MSCs were encapsulated in C/HAp microbeads with >?85% viability, and were cultured in a slow turning lateral vessel-type rotating bioreactor simulating microgravity conditions for 28 days, under the effect of osteogenic inducers. MTT assay showed that the metabolic activity of encapsulated cells was preserved >?80% after a week. In vitro experiments confirmed that the encapsulated BM-MSCs differentiated into osteoblastic cells, formed bone-like tissue under osteogenic microgravity bioreactor conditions. Preliminary in vivo study indicated C/HAp microbeads containing BM-MSCs were able to repair the surgically-created small bone defects in the rat femur. BM-MSCs-C/HAp composite microbeads may have potential for modular bone regeneration.     

Long term growth of factor-producing lymphoid and myeloid cells in serum-free medium

The ability to grow lymphoid and myeloid cells in serum-free culture medium allows researchers to analyze the factors and mechanisms required for hemopoietic cell growth and differentiation without the interference of undefined serum components. Therefore, we used a serum-free medium, RITC 55-9 that consisted of modified Dulbecco's MEM supplemented with bovine serum albumin (BSA), transferrin (Tf) and insulin (Ins) to culture human T lymphoid (Mo), murine myelomonocytoid (WEHI-3B) and murine interleukin (IL)-3-dependent (32Dcl/H4) cell lines. Mo was maintained in RITC for more than 8 months and had a mean viability of 59% and the same doubling times as in serum-containing medium (SCM). Under these conditions, Mo cells produced hemopoietic colony-stimulating activity that included production of a basophil/eosinophil differentiation factor of similar content to that produced in SCM. WEHI-3B cells grown for more than 12 months in RITC, or for more than 3 months in RITC without Tf and Ins, had a doubling time of 20 h, whereas cells maintained in protein-free RITC showed a 2-fold increase in doubling time then died within 3 months. The IL-3 production by WEHI-3B cells cultured in RITC was higher than the production by cells grown in SCM. When IL-3 was assayed in 32Dcl/H4 cells that had been maintained in RITC for more than 4 months, a lower response to IL-3 was found, an indication that components other than the BSA, Tf and Ins in fetal calf serum are required for optimal cell growth and differentiation.     

Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages

When cells of mouse myelomonocytic leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h. According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Iad, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages. Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably. Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells. These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.     

Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages

When cells of mouse myelomonocytyc leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (l-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h. According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Ia, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages. Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably. Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells. These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages.     

Infection of immune mast cells by Harvey sarcoma virus: immortalization without loss of requirement for interleukin-3.

Cells from adult mouse spleens were cultured in WEHI-3 cell-conditioned medium, which contains the lymphokine interleukin-3 (IL-3). Under these conditions, cells grow well for 4 to 8 weeks; the cultures contain a variety of cell types for the first 1 to 2 weeks but are subsequently composed largely of immune mast cells. We found that infection of these cultures with Harvey sarcoma virus (HaSV) profoundly enhanced the growth potential of the cells, resulting in the reproducible isolation of long-term cell lines. These HaSV-infected cells appeared to be phenotypically identical to the immune mast cells found in uninfected cultures as determined by biochemical, immunological, and cytological tests. Although the cells expressed protein p21Ha-ras at levels similar to those in HaSV-transformed fibroblasts, they continued to require IL-3 for growth in vitro. Similar IL-3-dependent, long-term mast cell lines were also cultured from the enlarged spleens present in HaSV-infected mice. These results suggest that high-level expression of an activated Ha-ras oncogene enhances growth in these cells, perhaps by stimulating the progression of the cells into S, without affecting differentiation or altering the requirements for normal growth factor.     

Conditional immortalization of mouse myelomonocytic, megakaryocytic and mast cell progenitors by the Hox-2.4 homeobox gene.

The murine myelomonocytic cell line WEHI-3B exhibits ectopic expression of the genes encoding the homeobox protein, Hox-2.4, and the myeloid growth factor, interleukin-3 (IL-3). We showed previously that concomitant expression of IL-3 and Hox-2.4 in bone marrow cells induced the development of transplantable growth factor-independent tumours resembling the WEHI-3B tumour. We have now investigated the effect of enforced expression of Hox-2.4 alone. Bone marrow cells were infected with Hox-2.4 retrovirus and then either cultured in agar or transplanted into irradiated mice. In vitro, colonies derived from virus-infected cells readily yielded IL-3-dependent, non-tumorigenic cell lines of the myelomonocytic, megakaryocytic and mast cell lineages. Surprisingly, both the establishment and maintenance of these lines required very high concentrations of IL-3 and reduced levels promoted differentiation. Transplanted mice analysed after 3 months appeared normal but their spleen and bone marrow contained abundant provirus-bearing progenitor cells, from which IL-3-dependent long-term cell lines could readily be established in vitro. Four of 18 animals monitored for up to 12 months eventually developed clonal leukaemia, associated in three cases with IL-3 production. Thus ectopic expression of Hox-2.4 enhances self-renewal of immature myeloid progenitors and progression to a fully malignant state is favoured by somatic mutations conferring autocrine production of IL-3.     

Regulation of the growth rate of mouse fibroblasts by IL-3-activated mouse bone marrow-derived mast cells

When mouse bone marrow-derived mast cells (BMMC) are cocultured with a confluent layer of mouse 3T3 fibroblasts in the presence of WEHI-3-conditioned medium, the mast cells undergo a phenotypic change toward that of a connective tissue mast cell, and the fibroblasts increase their synthesis of globopentaosylceramide. We now demonstrate that fibroblasts lose their contact inhibition and multiply such that by the 2nd and the 4th wk of coculture there are, respectively, approximately four-fold and six-fold more fibroblasts than in the cultures that are not exposed to BMMC. This in vitro increase in the number of fibroblasts is dependent on the number of mast cells (over the range of 6 x 10(4) to 1 x 10(6) BMMC/culture) initially seeded with the fibroblasts and on the concentration of WEHI-3-conditioned medium present during the coculture. That the fibroblasts also multiply in BMMC/fibroblast cocultures exposed to synthetic IL-3 or to purified IL-3 indicates that IL-3 is a component in WEHI-3-conditioned medium that induces mast cells to produce the fibroblast growth factor. The number of fibroblasts does not increase if fibroblasts are exposed to lysates of BMMC, or to BMMC-derived conditioned medium, or if the two cell types are separated from one another during the coculture with a 3-microns filter or a 0.4-microns filter. Thus, IL-3-activated BMMC must be in proximity to fibroblasts to induce them to multiply. Because of their increased numbers per culture dish, total fibroblasts that were cocultured with mast cells synthesized approximately two-fold more 35S-labeled proteoglycans, incorporated approximately 3-fold more [3H] proline into collagenase-sensitive proteins, and had substantially more alpha 2(I) collagen mRNA than fibroblasts that were maintained in the absence of mast cells. These is vitro studies reveal a sequence by which IL-3-activated mast cells may play a role in the induction of fibrosis.     

成年猴雪旺细胞的在体增殖和体外迁移的研究

为了探讨成年猴雪旺氏细胞的在体增殖和体外迁移的能力,我们对用神经结扎术结扎的A组6只3-13岁雄性恒河猴的腓肠神经进行植块培养,部分细胞培养在聚酯纤维上,2-4周后作抗S-100抗体免疫组化染色和电镜观察;B组2只未做结扎的新生猴腓肠神经培养作为对照.结果显示A组雪旺氏细胞平均在培养的第5天从神经段中迁出,年幼者早于成年猴;细胞在纤维上以螺旋状向前迁移;雪旺氏细胞抗S-100蛋白抗体染色阳性;电镜显示,雪旺氏细胞包卷纤维,但是,未见髓鞘形成.B组神经段培养2周仍无雪旺氏细胞迁出.研究表明,结扎神经使其发生瓦勒氏变性,经植块培养、纯化,能够获得可用于移植的成年猴的雪旺氏细胞.     

Comparative analysis of casein synthesis during mammary cell differentiation in collagen and mammary gland development in vivo

Substrata upon which epithelial cells are cultured modulate their morphology,growth, and ability to differentiate. Mouse mammary epithelial cells cannot be induced to synthesize caseins, a marker of cell differentiation, when grown on a plastic surface. An analysis was made of the effect of time within a collagen matrix on the ability of normal mammary epithelial cells to be induced to synthesize caseins and that response was compared to mammary gland development in vivo. Primary cultures of mammary cells from unprimed virgin BALB/c mice were embedded in rat-tail collagen gel mixtures and maintained in growth medium. Induction medium containing lactogenic hormones was added at various times. The cells were monitored every 3-7 days over a period of 8 weeks for cell growth, casein synthesis, and ability to grow in vivo in cleared mammary fat pads. Casein accumulation was assayed quantitatively by an ELISA competition assay and qualitatively by the immunoblot procedure using specific antisera prepared against purified mouse caseins. No marked differences in cell numbers and transplantability potential were observed among cells cultured for various times in collagen. Mammary cells grown in collagen for up to 8 weeks retained the capacity to grow in vivo as normal ductal outgrowths. The duration of culture within collagen prior to hormonal stimulation did influence the kinetics of casein synthesis. Cells cultured for 1 week in growth medium did not accumulate detectable levels of casein until after 3 weeks of induction, whereas cells cultured for 2 or 4 weeks responded by accumulating caseins after 2 weeks and 3 days of induction, respectively. While the levels of total caseins that accumulated under optimal conditions of induction in culture approached levels found during lactation in vivo, the relative proportion of specific casein polypeptides synthesized in culture was altered from alpha casein (43K) in favor of the beta casein (30K) species. These results suggest that a period of culture within collagen is required to permit mammary epithelial cells to become responsive for hormone-induced differentiation. It is possible that during growth within the collagen the cells synthesize and deposit extracellular matrix components important in modulating gene expression.     

Interleukin 6 is essential for antibody secretion by human in vivo antigen-induced lymphoblastoid B cells

In vivo immunization of normal subjects with a variety of antigens generates circulating lymphoblastoid (LB) B cells, which in vitro spontaneously secrete significant levels of specific antibody. Since activation and initial differentiation of these cells occurs in vivo, they provide a useful model for the study of the later stages of B cell maturation. In the present study, we investigated the requirement of interleukin 6 (IL-6) for the "spontaneous" in vitro production of IgG-Tet by LB B cells. Addition of IL-6 to cultures of LB B cells in medium supplemented with 10% fetal calf serum failed to increase the levels of IgG-Tet produced in vitro. However, addition of anti-IL-6 antibodies decreased IgG-Tet production as much as 70%, and this inhibition could be reversed by the addition of IL-6. LB B cells cultured in serum-free medium in order to restrict endogenous IL-6 production secreted only low levels of antibody, unless exogenous IL-6 was added. Addition of 2.5 units/ml of IL-6 to serum-free cultures induced an increase in IgG-Tet secretion nearly comparable to that seen in cultures supplied with serum. The magnitude of the increase in IgG-Tet secretion in response to exogenous IL-6 was inversely related to the number of cells in culture, which was due in part to increased endogenous IL-6 production in cultures with higher cell concentrations. Experiments including hydroxyurea in serum-free cultures indicated that IL-6-dependent enhancement of LB B cells' IgG-Tet secretion was not primarily mediated by cell growth. These observations suggest that in vivo generated LB B cells are not totally committed to antibody secretion, and that IL-6 is essential for in vivo antigen-induced LB B cells to reach the antibody-secreting stage.     

Informing future cartilage repair strategies: a comparative study of three different human cell types for cartilage tissue engineering

A major clinical need exists for cartilage repair and regeneration. Despite many different strategies having been pursued, the identification of an optimised cell type and of pre-treatment conditions remains a challenge. This study compares the cartilage-like tissue generated by human bone marrow stromal cells (HBMSCs) and human neonatal and adult chondrocytes cultured on three-dimensional (3D) scaffolds under various conditions in vitro and in vivo with the aim of informing future cartilage repair strategies based upon tissue-engineering approaches. After 3 weeks in vitro culture, all three cell types showed cartilage-like tissue formation on 3D poly (lactide-co-glycolide) acid scaffolds only when cultured in chondrogenic medium. After 6 weeks of chondro-induction, neonatal chondrocyte constructs revealed the most cartilage-like tissue formation with a prominent superficial zone-like layer, a middle zone-like structure and the thinnest fibrous capsule. HBMSC constructs had the thickest fibrous capsule formation. Under basal culture conditions, neonatal articular chondrocytes failed to form any tissue, whereas HBMSCs and adult chondrocytes showed thick fibrous capsule formation at 6 weeks. After in vivo implantation, all groups generated more compact tissues compared with in vitro constructs. Pre-culturing in chondrogenic media for 1 week before implantation reduced fibrous tissue formation in all cell constructs at week 3. After 6 weeks, only the adult chondrocyte group pre-cultured in chondrogenic media was able to maintain a more chondrogenic/less fibrocartilaginous phenotype. Thus, pre-culture under chondrogenic conditions is required to maintain a long-term chondrogenic phenotype, with adult chondrocytes being a more promising cell source than HBMSCs for articular cartilage tissue engineering.     

骨髓间质干细胞向大鼠损伤心肌组织的迁移

实验旨在动态观察骨髓间充质干细胞（mesenchymal stem cells,MSCs）向不同微环境下心肌组织的迁移特点,明确组织损伤在干细胞迁移中的作用,为提高干细胞治疗的靶向性和高效性奠定初步试验基础。分离纯化雄性Sprague-Dawley（SD）大鼠的骨髓MSCs,输注入雌性SD大鼠。实验分为4组：正常大鼠＋MSCs移植组,假手术＋MSCs移植组,心肌缺血＋MSCs移植组,心肌缺血对照组（心肌缺血＋培养基移植）。结扎冠状动脉前降支制造心肌缺血模型,将相等数量的雄性MSCs经尾静脉注射移植入前3组雌性大鼠体内,对照组注射等体积培养基,分别于移植后1周及8周取心脏组织标本,采用荧光原位杂交方法（fluorescence in situ hybridization,FISH）检测大鼠Y染色体雄性鉴别基因sty片段的表达,用透射电镜观察大鼠心肌组织超微结构改变。结果发现,移植后1周和8周,正常大鼠移植组和对照组大鼠的心肌组织中均未见sry基因的表达,但假手术移植组和心肌缺血移植组的心肌组织中均可见sty基因的表达,心肌缺血移植组的Y染色体sty基因阳性细胞数量在两个时间点均显著高于假手术移植组（P〈0．01）。分别比较心肌缺血移植组和假手术组在移植后1周和8周的Y染色体sry基因阳性细胞的数量,两个时间点无明显差异。心肌组织的超微结构观察发现心肌缺血移植组大鼠的心肌梗死周边区域可见一些细胞,其形态类似于体外培养的MSCs。研究结果提示MSCs具有向损伤心肌组织迁移的特性,迁移的高峰期可能在组织损伤1周左右,组织损伤及其程度在干细胞迁移中起重要作用。     

Regulation of B lymphocyte responses to IL-4 and IFN-gamma by activation through Ly-6A/E molecules

The Ly-6 family of cell surface molecules has previously been shown to participate in T cell activation. We show that Ly-6A/E proteins also modulated the response of normal B lymphocytes in three separate in vitro assays. First, unfractionated or small resting B cells proliferated when cultured with IFN-gamma, IL-4, and an anti-Ly-6A/E mAb. Second, this anti-Ly-6A/E mAb restored B cell proliferation responses that were inhibited when coculturing the B cells in IFN-gamma, IL-4, and anti-IgM. Third, anti-Ly-6A/E specifically up-regulated the cell surface expression of its own Ag, and this response was dependent upon co-stimulation with IFN-gamma. Mixing of T and B cells in culture suggested that T cells did not contribute substantially to the B cell proliferative response. Moreover, up-regulation of Ly-6A/E was observed for one B cell lymphoma, WEHI-231. Therefore, it appeared that modulation of B cell function by anti-Ly-6A/E was due to a direct effect of the mAb binding to the B cells. Taken together, these data suggest Ly-6A/E proteins are functional on B cells and may play a regulatory role in B cell activation.     

人树突状细胞的大量培养及鉴定

摘要 目的：探讨人树突状细胞体外大量培养及鉴定方法。方法：采用免疫磁珠法分离纯化CD34⁺干细胞;采用含有TPO、SCF、Flt3L和IL-3的扩增培养基培养1周,以及含有SCF、Flt3L、GM-CSF和IL-4的分化培养基培养2-3周,获得CD34⁺细胞来源树突状细胞。采用普通光学显微镜观察细胞形态,牛鲍氏血细胞计数板进行细胞计数,荧光抗体标记、流式细胞仪检测细胞纯度和细胞表面共刺激分子的表达情况。结果：以含有TPO、SCF、Flt3L和IL-3的培养基扩展培养一周,及含有SCF、Flt3L、GM-CSF和IL-4的培养基诱导分化3周,可获得大量悬浮细胞;细胞数目扩增倍数约达50倍;普通光学显微镜下可见悬浮细胞有明显的树突状凸起;流式细胞术检测结果显示悬浮细胞中CD141和CD11c双阳性细胞（等同于单核细胞来源树突状细胞）比例达30%,此群细胞高表达HLA-DR和CD209,低表达共刺激分子CD80和CD86;细胞寿命较短,40天时培养体系中悬浮细胞和CD34⁺细胞来源树突状细胞数目急剧减少。结论：采用多细胞因子联合刺激可获得大量的树突状细胞,为树突状细胞的特性及功能学研究奠定了基础。     

IL-15-mediated induction of LFA-1 is a late step required for cytotoxic differentiation of human NK cells from CD34+Lin- bone marrow cells

Optimal differentiation of cytotoxic NK cells is important to provide protective innate immunity to patients after bone marrow transplantation. In vitro differentiation of CD56(+)CD3(-) NK cells takes weeks and is supported by several cytokines, including IL-2, IL-7, and IL-15, and thus can be useful for immunotherapy. However, IL-2 therapy is problematic in vivo, and NK cells differentiated in vitro with only IL-7 lack cytotoxicity. We assessed whether human NK cells initially differentiated in vitro from CD34(+)Lin(-) bone marrow cells with IL-7 could acquire cytotoxicity after exposure to additional cytokines and what changes promoted cytotoxicity. The cells cultured with IL-7 already had granzyme B as well as perforin, as previously reported, the proteins of cytotoxic granules. The cells also lacked LFA-1. After 1 wk of secondary culture with either IL-2 or IL-15, but not with IL-12 or IL-18, the IL-7-cultured cells acquired cytotoxicity. IL-2 or IL-15 also induced LFA-1. Ab to the LFA-1 subunits CD11a and CD18 blocked lysis by the NK cells, indicating that the new LFA-1 correlated with, and was essential for, the cytotoxic function of the in vitro generated cells. The LFA-1 also participated in target cell binding by the in vitro differentiated cells. In this study, we demonstrated a new function for IL-15, the induction of LFA-1 in NK progenitor cells, and that IL-15 does more than merely support NK progenitor cell proliferation. The efficacy after only 1 wk of IL-15 administration is a positive practical feature that may apply to human therapy.     

Tumor-promoting phorbol esters stimulate the proliferation of interleukin-3 dependent cells

To determine whether activation of protein kinase C is involved in the proliferation of interleukin-3 (IL-3) -dependent cells, we examined the effect of tumor-promoting phorbol esters on the in vitro proliferation of the IL-3-dependent cell lines FD and DA-1. The viability of FD and DA-1 cells cultured for 24 hours in 100 nM phorbol myristate acetate (PMA) and 10% FCS was similar to that of cells cultured in 25% WEHI-3 conditioned medium as a source of IL-3, and 10% FCS. FD cells failed to proliferate in concentrations of FCS of up to 50%, while DA-1 cell proliferation was not markedly influenced by FCS. By contrast, PMA promoted the proliferation of FD and DA-1 cells in the absence of FCS and enhanced their proliferation in the presence of 10% FCS, 60- and 20-fold, respectively. Stimulation of proliferation was achieved with as little as 10 nM PMA and was maximal at 100 nM PMA. Low concentrations (0.05-0.1%) of WEHI-3 CM promoted the proliferative response of FD and DA-1 cells to PMA, but at concentrations of WEHI-3 CM greater than 0.8%, no further increment in proliferation was obtained with PMA. As little as 1/2 hour of exposure to phorbol esters was sufficient to cause translocation of protein kinase C from the cytosol to the membranes of DA-1 cells, and 1 hour of exposure to phorbol esters was sufficient to stimulate DNA synthesis. A protein kinase C inhibitor, H-7, at a concentration of 10 microM inhibited phorbol ester-induced stimulation of DA-1 cell proliferation. When DA-1 cells were exposed to the calcium ionophore A23187 in addition to both a phorbol ester and IL-3, their proliferation was enhanced over that stimulated by only the phorbol ester and IL-3. The data indicate that stimulation of proliferation of IL-3-dependent cells involves the activation of protein kinase C.