Autocrine growth and tumorigenicity of interleukin 2-dependent helper T cells transfected with IL-2 gene

We introduced a mouse IL-2 cDNA expression vector into an IL-2-dependent mouse helper T cell line HT-2. Transfected cells secreted substantial amounts of IL-2, to which they themselves responded by proliferating without further requirement for exogenous IL-2. The proliferation was a direct function of the cell density and was inhibitable by antibodies against IL-2 or IL-2-R, indicating the autocrine nature of the proliferation. Those producing higher amounts of IL-2 were found to be tumorigenic when inoculated into nude mice. The latency period of tumor development correlated inversely with the level of IL-2 secreted. Tumor cells proliferated in vitro in an IL-2 autocrine fashion indistinguishable from that of the inoculated cells. We thus provide evidence that the aberrant activation of the IL-2 autocrine circuit can lead T cells to malignant transformation.     

Cells transfected with human interleukin 6 cDNA acquire binding sites for the hepatitis B virus envelope protein

Earlier studies revealed that human interleukin 6 (IL-6) contains recognition sites for the hepatitis B virus (HBV) envelope (env) protein, and that IL-6 and anti-IL-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for HBV with the preS(21-47) segment of the HBV env protein, encompassing the complementary attachment site for IL-6. This suggested that IL-6 mediates HBV-cell interactions. We report that: (a) Chinese hamster ovary cells transfected with human IL-6 cDNA and Spodoptera frugiperda ovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS(21-47) region of the HBV env protein, indicating that expression of IL-6 on the surface of cells is sufficient to endow them with receptors for HBV. (b) Among peptides covering the entire sequence of human IL-6 and the corresponding antipeptide antibodies, the peptide IL-6[35-66] and anti-IL-6[35-66] most effectively inhibited the interaction between human hepatoma HepG2 cells and the preS(21-47) ligand, suggesting that this region of the human IL-6 sequence encompasses a binding site for the HBV env protein. (c) Studies with replacement set peptides from the preS(21-47) sequence indicated that residues 21-25, 28, 31, 33-35, 39, and 43-45 can be replaced by alanine (serine) residues, while all the other residues are essential for maintaining the cell receptor/IL-6 binding activity. Further delineation of complementary sites on IL-6 and on the HBV env protein may contribute to the design of compounds inhibiting HBV replication.     

Autonomous growth and tumorigenicity induced by P40/interleukin 9 cDNA transfection of a mouse P40-dependent T cell line

To test the transforming potential of deregulated P40/Interleukin 9 expression, we transfected a mouse P40-dependent T cell line with P40 cDNA, and examined the tumorigenicity of the resulting transfectants. When the cells, which grew autonomously in vitro, were injected intraperitoneally or subcutaneously into syngeneic mice, a very high tumor incidence was observed with as few as 10(4) cells per inoculum. Animals died as a result of widespread dissemination of lymphomatous tissue to abdominal and thoracic organs. The same P40-dependent cell line transfected with a control construct did not form tumors even after injection of 10(7) cells. These results indicate that uncontrolled expression of P40 can support T cell proliferation in vivo, and may be a transforming event involved in the development of certain T cell tumors.     

Expression of an exogenous interleukin 6 gene in human Epstein Barr virus B cells confers growth advantage and in vivo tumorigenicity

The biological role of interleukin 6 (IL-6) molecules in human B cell tumorigenesis was studied by using an episomal expression vector, pHEBoSV-IL6, to introduce stably the human IL-6 gene into human Epstein Barr virus (EBV)-transformed B lymphoblasts. The gene was present in the IL-6-transfected cells in a high copy number and was efficiently expressed, resulting in the secretion of consistent levels of IL-6 molecules. The constitutive expression of the IL-6 gene led to an altered pattern of growth and to a malignant phenotype, as shown by clonogenicity in to an altered pattern of growth and to a malignant phenotype, as shown by clonogenicity in soft agar cultures and tumorigenicity in nude mice. These data suggest that the combined action of EBV, which exerts an immortalizing function, and of the growth-promoting activity of IL-6 molecules, can give rise to fully transformed B cell tumors in immunodeficient subjects.     

Tumor cells transfected with a bacterial heat-shock gene lose tumorigenicity and induce protection against tumors

The gene encoding a highly immunogenic mycobacterial heat-shock protein (hsp65) was transfected into the murine macrophage tumor cell line J774. The resulting hsp65-expressing cells (J774-hsp65) were no longer able to produce tumors in syngeneic mice. This loss of tumorigenicity was not mediated through T cells since the transfected cells did not produce tumors in athymic mice. If mice are first immunized with the J774-hsp65 cells and then challenged with the parent J774 cells, the mice do not develop tumors, indicating that the presence of the mycobacterial hsp65 protein greatly enhances immunological recognition of unique structures expressed by the parent tumor cells. This is further confirmed by the demonstration in vitro of T cells derived from J774-hsp65-immunized mice that are cytotoxic for the parent J774 cells. The results provide the basis for a novel strategy for enhancing the immunological recognition and decreasing the tumorigenicity of transformed cells.     

Dendritic cells induce T lymphocytes to release B cell-stimulating factors by an interleukin 2-dependent mechanism

Dendritic cells (DC) are essential accessory cells for T-dependent antibody responses in culture (1). We have outlined a three-stage mechanism to explain the capacity of DC to stimulate primary antibody responses to heterologous erythrocytes. First, DC induced T cells to produce and to become responsive to interleukin 2 (IL-2). This stage corresponded to the syngeneic mixed leukocyte reaction (2) and involved the clustering of DC and T cells into discrete aggregates. Isolated clusters, representing 5-10% of the culture, were critical for IL-2 release and the production of IL-2-responsive T blasts. In the second stage, IL-2 directly triggered the responsive T cells to release B cell helper factors. This role for IL-2 was documented with a rabbit anti-IL- 2 reagent, purified IL-2, and T cells that had been rendered IL-2 responsive by an initial co-culture with DC. T cell growth was not required for IL-2-mediated helper factor release, since irradiated and untreated responders produced similar levels of factor and did so within 3 h of the addition of IL-2. In the final stage, helper factors stimulated the development of antibody-secreting cells from purified B lymphocytes. The helper factors were not H-2 restricted, but for both sheep and horse erythrocytes, the response to factors was antigen dependent and specific. The IL-2 that was present in the DC/T cell- conditioned medium did not act on B cells, since helper activity was neither neutralized nor absorbed by our anti-IL-2 reagent. We conclude that the ability of the DC to induce IL-2 release and responsiveness underlies its capacity to trigger both T and B lymphocyte reactions.     

Development of an interleukin (IL) 6 receptor antagonist that inhibits IL-6-dependent growth of human myeloma cells

The pleiotropic cytokine interleukin 6 (IL-6) plays a role in the pathogenesis of various diseases, such as multiple myeloma, autoimmune and inflammatory diseases and osteoporosis. Therefore, specific inhibitors of IL-6 may have clinical applications. We previously succeeded in developing receptor antagonists of IL-6 that antagonized wild-type IL-6 activity on the human Epstein-Barr virus (EBV)- transformed B cell line CESS and the human hepatoma cell line HepG2. However, these proteins still had agonistic activity on the human myeloma cell line XG-1. We here report the construction of a novel mutant protein of IL-6 in which two different mutations are combined that individually disrupt the association of the IL-6/IL-6 receptor (R) alpha complex with the signaltransducing "beta" chain, gp130, but leave the binding of IL-6 to IL-6R alpha intact. The resulting mutant protein (with substitutions of residues Gln160 to Glu, Thr163 to Pro, and replacement of human residues Lys42-Ala57 with the corresponding residues of mouse IL-6) was inactive on XG-1 cells and weakly antagonized wild-type IL-6 activity on these cells. By introducing two additional substitutions (Phe171Leu, Ser177Arg), the affinity of the mutant protein for IL-6R alpha was increased fivefold, rendering it capable of completely inhibiting wild-type IL-6 activity on XG-1 cells. Moreover, this mutant also antagonized the activity of IL-6, but not that of leukemia inhibitory factor, oncostatin M, or GM-CSF on the human erythroleukemia cell line TF-1, demonstrating its specificity for IL-6. These data demonstrate the feasibility of developing specific IL- 6R antagonists. The availability of such antagonists may offer an approach to specifically inhibit IL-6 activity in vivo.     

Impairment of natural killer functions by interleukin 6 increases lymphoblastoid cell tumorigenicity in athymic mice.

Expression of the human IL-6 gene in EBV-immortalized normal human B lymphocytes following retroviral-mediated transduction rendered these cells highly tumorigenic in athymic mice. The tumors were lymphomas composed of the originally inoculated human lymphoblastoid cells. Co-injection of IL-6 expressing EBV-immortalized cells with IL-6 nonexpressing control cells resulted in increased tumorigenicity of the IL-6 nonexpressing cells. The lymphoblastoid cells expressing IL-6 were indistinguishable from parental cell lines in morphology and in a variety of cell surface characteristics, and did not exhibit growth advantage over parental cell lines in vitro, such that increased tumorigenicity is unlikely to depend upon a direct oncogenic effect of IL-6 on the B cells. Rather, at high concentrations, IL-6 markedly inhibits human lymphoblastoid cell killing by IL-2-activated murine splenocytes in vitro, suggesting that IL-6-related tumorigenicity might depend upon IL-6 inhibiting cytotoxicity at the tumor site. Thus, production of IL-6 by tumor cells that results in natural killer cell dysfunctions illustrates a novel mechanism of tumor cell escape from immune surveillance.     

Interleukin 5 and interleukin 2 cooperate with interleukin 4 to induce IgG1 secretion from anti-Ig-treated B cells

We have analyzed requirements for IL-4-induced secretion of IgG1 from anti-Ig-activated B cells. Activated B cell blasts prepared by culture of high density B cells with anti-Ig failed to secrete IgG1 upon subsequent culture with LPS and IL-4. However, IL-4 markedly suppressed IgM secretion in the same cultures. Addition of a mixture of T cell-derived lymphokines or rIL-5 to LPS-stimulated anti-Ig blasts restored IL-4-stimulated IgG1 secretion; rIL-2 further enhanced the response to IL-4 + rIL-5. These results suggest that IL-4, IL-5, and IL-2 cooperate in the regulation of B lymphocyte Ig isotype expression.     

Human appendix B cells naturally express receptors for and respond to interleukin 6 with selective IgA1 and IgA2 synthesis

Past studies have shown that freshly isolated human B cells from peripheral blood and tonsils do not express IL-6 receptors (IL-6R); however, mitogen or antigen activation of these B cells induces IL-6R and responsiveness to IL-6. In this study, we have shown that a high proportion of B cells enzymatically dissociated from human appendix, a gut-associated lymphoreticular tissue (GALT), expresses the IL-6R, and that recombinant human IL-6 induces significant increases in the number of Ig-producing cells. The recombinant human IL-6-induced increase in Ig-producing cells is restricted to the IgA isotype. Further, IgA2 is the major subclass; however, significant numbers of IgA1 producing cells are also seen. In contrast, human tonsillar and peripheral blood B cells express low levels of IL-6R, and exogenous IL-6 does not increase numbers of Ig-producing cells. When PBMC or tonsillar cells are stimulated with PWM, the former display an equal distribution of IgA1 and IgA2 secreting cells, while tonsillar B cells are mainly of the IgA1 subclass. The distribution of surface Ig-positive (sIg+) B cells in the appendix B cell population is sIgA+ greater than sIgG+ greater than sIgM+, and the sIgA+ B cells express higher levels of IL-6R when compared with sIgG+ or sIgM+ B cells. These studies show that human appendix contains B cell subsets that constitutively express IL-6R, and that a high proportion of these cells are committed to the IgA isotype. Furthermore, higher numbers of IL-6 responsive IgA2 B cells are present in the human appendix as compared to tonsils or PBMC.     

Prevention of arthritis by interleukin 10-producing B cells

In this study we have shown that activation of arthritogenic splenocytes with antigen and agonistic anti-CD40 gives raise to a B cell population that produce high levels of interleukin (IL)-10 and low levels of interferon (IFN)-gamma. Transfer of these B cells into DBA/1-TcR-beta-Tg mice, immunized with bovine collagen (CII) emulsified in complete Freund's adjuvant inhibited T helper type 1 differentiation, prevented arthritis development, and was also effective in ameliorating established disease. IL-10 is essential for the regulatory function of this subset of B cells, as the B cells population isolated from IL-10 knockout mice failed to mediate this protective function. Furthermore, B cells isolated from arthritogenic splenocytes treated in vitro with anti-IL-10/anti-IL-10R were unable to protect recipient mice from developing arthritis. Our results suggest a new role of a subset of B cells in controlling T cell differentiation and autoimmune disorders.     

Stable expression of cDNA encoding the human interleukin 2 receptor in eukaryotic cells

Human interleukin 2 (IL-2) receptor cDNA derived from HUT 102B2 cells was stably expressed in murine L cells. These L cell transfectants (a) displayed surface receptors of the aberrant size of the IL-2 receptors on HUT 102B2 cells, (b) did not respond to exogenous IL-2 with augmented proliferation, and (c) expressed low affinity but not high affinity receptors for IL-2.     

转染反义VEGFl21 cDNA恢复K562细胞β1整合素的活化功能

为了探索血管内皮细胞生长因子 (VEGF)在K5 6 2细胞 β1整合素活化功能中的作用 ,利用转染正义 (S)、反义 (As)VEGF12 1cDNA及空载体 (V ,pcDNA3 )的不同K5 6 2细胞株 ,通过流式细胞术检测其粘附分子VLA 4(CD4 9d/CD2 9)和VLA 5 (CD4 9e/CD2 9)表达及 β1整合素的可活化性。结果显示 :K5 6 2 /V ,K5 6 2 /S和K5 6 2 /As细胞 β1整合素VLA 4 (CD4 9d/CD2 9)与VLA 5 (CD4 9e/CD2 9)的表达率均在 92 %以上 ,但经 β1整合素活化剂 8A2抗体激活后 ,K5 6 2 /As细胞可活化表位 9EG7表达率较K5 6 2 /V细胞明显提高 (75 .6± 10 .5vs 4 1.2± 2 .1% ,P <0 .0 1)。结论 :转染反义VEGF12 1cDNA能增加K5 6 2细胞 β1整合素的可活化性     

Establishment of two interleukin 6 (B cell stimulatory factor 2/interferon beta 2)-dependent human bone marrow-derived myeloma cell lines

Two IL-6-dependent human multiple myeloma cell lines, ILKM2 and ILKM3, were established from the bone marrow of patients with IgG-K multiple myeloma. Both cell lines had the typical morphology and immunocytochemical features of myeloma cells. The surface phenotype of both cell lines was PCA-1+, OKT10+, CD10(J-5)-, CD19(B4)-, CD20(B1)-, CD21(B2)-, and OKIa-1-. A monoclonal cytoplasmic Ig, IgG-K or K L chain, was positive in ILKM2 or ILKM3, respectively. EBV nuclear antigen was negative in both cell lines. They proliferated in the presence of macrophages or macrophage-derived factors (MDF). Among the recombinant cytokines examined, IL-6 most strongly augmented the growth of both cell lines. The anti-IL-6 antibody completely inhibited the IL-6-dependent growth and almost completely inhibited the MDF- or purified MDF-dependent growth of both cell lines, ILKM2 and ILKM3 are now being maintained in the culture medium containing 2 ng/ml rIL-6. These results suggest that IL-6 produced by macrophages may play an important role in the growth of myeloma cells in vivo and that macrophages or IL-6 can be used for establishing human myeloma cell lines.     

Interleukins and IgA synthesis. Human and murine interleukin 6 induce high rate IgA secretion in IgA-committed B cells

Freshly isolated murine PP B cells were cultured with 10 different cytokines, including IL-1 alpha, IL-2, IL-4, IL-5, IL-6, IL-7, IFN-gamma, TNF-alpha, and TGF-beta, to investigate a possible role for these cytokines in induction of Ig synthesis. Of interest was the finding that only IL-5 and both mouse recombinant (mr) and human recombinant (hr) IL-6 enhanced IgA synthesis. The effect was greater with either mrIL-6 or hrIL-6 than with mrIL-5. IL-6 induced cycling mIgA+ PP B cells to secrete high levels of IgA (approximately 7-fold increase over control). Of importance was the finding that mrIL-6 had little effect on secretion of IgM or IgG by PP B cell cultures. hrIL-6 also increased IgA secretion by PP B cells and this enhancement was abolished by a goat anti-hrIL-6 antiserum. mrIL-6 did not cause B cell proliferation but induced a sharp increase in numbers of B cells secreting IgA. Isotype-switching was not a mechanism for this marked increase in IgA synthesis since mIgA- PP B cells were not induced to secrete IgA by mrIL-6. From these studies we conclude that IL-6 plays an important role in promoting the terminal differentiation of PP B cells to IgA-secreting plasma cells.     

Expression of interleukin 2 receptors on activated human B cells

Using anti-Tac, a monoclonal anti-interleukin 2 (IL-2) receptor antibody, we have explored the possibility that certain activated B cells display receptors for IL-2. Resting normal B cells and unselected B cell lines established from normal individuals were Tac antigen negative. In contrast, the cell surface Tac antigen expression was demonstrable on 6 of 10 B cell lines from patients with Burkitt's lymphoma, 5 of 6 B cell lines derived from patients with HTLV-I- associated adult T cell leukemia (including all four that had integrated HTLV-I into their genome), and on certain normal B cells activated with pokeweed mitogen. Furthermore, cloned Epstein-Barr virus- transformed B cell lines derived from Tac-positive normal B cells continued to express the Tac antigen in long-term cultures and manifested high affinity IL-2 receptors identified in binding studies with purified radiolabeled IL-2. The line 5B4 developed in the present study could be induced with purified JURKAT-derived or recombinant IL-2 to express a larger number of IL-2 receptors. Furthermore, the addition of IL-2 to the 5B4 B cell line augmented IgM synthesis, which could be blocked by the addition of anti-Tac. The size of the IL-2 receptors expressed on the cloned normal B cell lines was similar (53,000-57,000 daltons) to that of receptors on phytohemagglutinin-stimulated T cell lymphoblasts. Thus, certain malignant and activated normal B cells display the Tac antigen and manifest high affinity receptors for IL-2. These data suggest that IL-2 may play a role in the differentiation of activated B cells into immunoglobulin-synthesizing and -secreting cells.     

Proliferating or interleukin 1-activated human vascular smooth muscle cells secrete copious interleukin 6.

The cells that make up blood vessel walls appear to participate actively in local immune and inflammatory responses, as well as in certain vascular diseases. We tested here whether smooth muscle cells (SMC) can produce the important inflammatory mediator IL6. Unstimulated SMC in vitro elaborated 5 X 10(3) pg recIL6/24h (i.e., biological activity equivalent to 5 X 10(3) pg recombinant IL6 (recIL6), as determined in B9-assay with a recIL6 standard). Several pathophysiologically relevant factors augmented IL6 release from SMC including 10 micrograms LPS/ml (10(4) pg recIL6), 10 ng tumor necrosis factor/ml (4 X 10(4) pg recIL6), and most notably 10 ng IL1/ml (greater than or equal to 3.2 X 10(5) pg recIL6). Production of IL6 activity corresponded to IL6 mRNA accumulation and de novo synthesis. SMC released newly synthesized IL6 rapidly, as little metabolically labeled material remained cell-associated. In supernatants of IL1-stimulated SMC, IL6 accounted for as much as 4% of the secreted proteins. In normal vessels SMC seldom divide, but SMC proliferation can occur in hypertension or during atherogenesis. We therefore tested the relationship between IL6 production and SMC proliferation in response to platelet-derived growth factor (PDGF) in vitro. Quiescent SMC released scant IL6 activity, whereas PDGF (1-100 ng/ml) produced concentration-dependent and coordinate enhancement of SMC proliferation and IL6 release (linear regression of growth vs. IL6 release yielded r greater than 0.9). IL6 itself neither stimulated nor inhibited SMC growth or IL6 production. Intact medial strips studied in short-term organoid culture produced large quantities of IL6, similar to the results obtained with cultured SMC. These findings illustrate a new function of vascular SMC by which these cells might participate in local immunoregulation and in the pathogenesis of various important vascular diseases as well as in inflammatory responses generally.     

Sequential development of interleukin 2-dependent effector and regulatory T cells in response to endogenous systemic antigen

Transfer of naive antigen-specific CD4(+) T cells into lymphopenic mice that express an endogenous antigen as a systemic, secreted protein results in severe autoimmunity resembling graft-versus-host disease. T cells that respond to this endogenous antigen develop into effector cells that cause the disease. Recovery from this disease is associated with the subsequent generation of FoxP3(+)CD25(+) regulatory cells in the periphery. Both pathogenic effector cells and protective regulatory cells develop from the same antigen-specific T cell population after activation, and their generation may occur in parallel or sequentially. Interleukin (IL)-2 plays a dual role in this systemic T cell reaction. In the absence of IL-2, the acute disease is mild because of reduced T cell effector function, but a chronic and progressive disease develops late and is associated with a failure to generate FoxP3(+) regulatory T (T reg) cells in the periphery. Thus, a peripheral T cell reaction to a systemic antigen goes through a phase of effector cell-mediated pathology followed by T reg cell-mediated recovery, and both require the growth factor IL-2.     

创伤患者血清白细胞介素33和可溶性抑癌蛋白2水平与创伤的程度及预后关系

目的：研究创伤患者血清中白细胞介素33（interleukin 33, IL-33）及其受体可溶性抑癌蛋白2（soluble suppression of tumorigenicity, sST2）水平的变化,并分析两者与患者的创伤严重度及预后间的关系。方法：根据创伤严重度评分(injury severity score, ISS)将67例患者分为轻度创伤组（ISS<20分）和重度创伤组（ISS≥20分）,并选同期30名健康体检者作为健康对照组,分别于创伤后4 h、24 h、72 h、7 d等时间点抽取外周静脉血,应用酶联免疫吸附试验检测血清IL-33和sST2水平,分别比较3组间各时间点的血清IL-33和sST2水平,并比较28 d内患者的病死率;同时比较存活者与死亡者各时间点血清IL-33和sST2水平,应用受试者工作特征（receiver operating characteristic, ROC）曲线选取最佳临界值,评估创伤后血清IL-33和sST2水平预测患者死亡的价值。结果：重度创伤患者创伤后4 h、24 h血清IL-33和sST2水平均高于轻度创伤患者[IL-33,（38.75±28.43） pg/mL比（19.62±9.98） pg/mL,（42.31±37.37） pg/mL比（23.47±13.42） pg/mL,（P<0.05）;sST2,（6.50±3.74） ng/mL比（4.89±2.40） ng/mL,（8.35±2.69） ng/mL比（6.78±3.22） ng/mL,（P<0.05）];死亡患者创伤后24 h至7 d的血清IL-33和sST2水平显著高于存活患者（P<0.01）,创伤后24 h血清IL-33和sST2水平的ROC曲线下面积分别为0.799（P=0.003）和0.751（P=0.012）,最佳临界值分别为15.27 pg/mL和10.99 ng/mL,其预测死亡的灵敏度分别为100%和70%,特异度分别为64%和83%。结论：创伤患者的血清IL-33、sST2水平升高与创伤的程度重及近期预后不良相关,创伤后24 h血清IL-33 和sST2水平可能作为预测创伤患者近期预后的生物标志物。