Immunotherapy for lung metastases of murine renal cell carcinoma: synergy between radiation and cytokine-producing tumor vaccines.

We investigated the combination therapy of local radiation of lung metastasis and vaccination with autologous tumor cells that produced interleukin (IL)-2, interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) using the mouse Renca pulmonary metastasis model. Wild-type Renca (W/Renca) were transfected with pEF-BOS vector incorporating cDNAs for IL-2, IFN-gamma, or GM-CSF to express these cytokines. W/Renca, IL-2-producing Renca (Renca/IL-2), and IFN-gamma-producing Renca (Renca/IFN-gamma) produced subcutaneous tumor at the injection site in eight of eight, one of eight, and two of eight mice, respectively. No tumors were found in the GM-CSF-producing Renca (Renca/GM-CSF) group (zero of eight). Renca/IFN-gamma produced subcutaneous (s.c.) tumors in all Balb/c nude mice, but Renca/IL-2 and Renca/GM-CSF did not. To test the elicitation of antitumor activity, Balb/c mice were injected intravenously with 1 x 10(5) W/Renca on day 0, vaccinated, s.c., with 1 x 10(6) cells each of 5,000 rad preirradiated Renca/IL-2, Renca/IFN-gamma, and Renca/GM-CSF or 3 x 10(6) cells of preirradiated W/Renca on days 1, 7, and 14, and radiated with 300 rad to both lungs on day 5. The animals were killed on day 21 and tumor nodules in the lungs were enumerated. Neither local irradiation alone nor the combination of lung radiation and multiple vaccination with irradiated W/Renca significantly reduced the number of lung tumors. In contrast, the combination of lung radiation and the multiple vaccinations with cytokine-producing Renca cells significantly reduced the number of lung tumors. This regimen was more effective than the multiple vaccinations with cytokine-producing Renca cells alone. These studies demonstrate the efficacy of vaccination with autologous tumor cells expressing these cytokines and sensitization of the tumor target with radiation.     

Reovirus virotherapy overrides tumor antigen presentation evasion and promotes protective antitumor immunity

Tumor-associated immunosuppressive strategies, such as lack of tumor antigen recognition and failure of lymphocyte activation and homing, resist the development of tumor-specific immunity and hamper the immune response-mediated elimination of cancerous cells. In this report, we show that reovirus virotherapy overrides such a tumor immune evasion and establishes clinically meaningful antitumor immunity capable of protecting against subsequent tumor challenge. Reovirus-mediated destruction of tumor cells facilitates the recognition of tumor antigens by promoting the display of otherwise inaccessible tumor-specific immunogenic peptides on the surface of dendritic cells (DC). Furthermore, on exposure to reovirus, DCs produce IL-1α, IL-1β, IL-6, IL-12p40/70, IL-17, CD30L, eotaxin, GM-CSF, KC, MCP-1, MCP-5, M-CSF, MIG, MIP-1α, RANTES, TNF-α, VCAM-1, VSGF, CXCL-16, AXL, and MCP-2; undergo maturation; and migrate into the tumor microenvironment along with CD8 T cells. These reovirus-activated DCs also acquire the capacity to prime tumor antigen-specific transgenic T cells in vitro and intrinsic antitumor T-cell response in vivo. Further, reovirus virotherapy augments the efficacy of DC- or T cell-based anticancer immunotherapies and synergistically enhances the survival in tumor-bearing mice. Most importantly, antitumor cellular immune responses initiated during reovirus oncotherapy protect the host against subsequent tumor challenge in a reovirus-independent but antigen-dependent manner. These reovirus oncotherapy-initiated antitumor immune responses represent an anticancer therapeutic entity that can maintain a long-term cancer-free health even after discontinuation of therapy.     

Marrow stromal cells for interleukin-2 delivery in cancer immunotherapy

Marrow stromal cells (MSCs) can be easily gene-modified and clonally expanded making them ideal candidates for transgenic cell therapy. However, recent reports suggest that MSCs possess immunosuppressive effects, which may limit their clinical applications. We investigated whether interleukin (IL)-2 gene-modified MSCs can be used to mount an effective immune response against the poorly immunogenic B16 melanoma model. We first show that primary MSCs mixed with B16 cells and injected subcutaneously in syngeneic recipients do not affect tumor growth. On the other hand, IL-2-producing MSCs mixed with B16 cells significantly delayed tumor growth in an IL-2 dose-dependent manner. Furthermore, we observed that matrix-embedded IL-2-producing MSCs injected in the vicinity of preestablished B16 tumors led to absence of tumor growth in 90% of treated mice (p < 0.001). We demonstrated that tumor-bearing mice treated with IL-2-producing MSCs developed CD8-mediated tumor-specific immunity and significantly delayed tumor growth of a B16 cell challenge (p < 0.05). In addition, treatment of cd8-/-, cd4-/- and beige mice revealed that CD8+ and natural killer (NK) cells, but not CD4+ cells, were required to achieve antitumor effect. In conclusion, MSCs can be exploited to deliver IL-2 and generate effective immune responses against melanoma in mice with normal immune systems.     

Combined interleukin 1/interleukin 2 therapy of mice injected with highly metastatic Friend leukemia cells: host antitumor mechanisms and marked effects on established metastases

Peritumoral injection of recombinant human interleukin 1 beta (IL-1 beta) in mice transplanted subcutaneously with Friend erythroleukemia cells (FLC) resulted in a marked increase in survival time and inhibition of metastatic tumor growth in liver and spleen. In contract, IL-2 treatment alone did not significantly inhibit the development of FLC metastases. A synergistic antitumor effect was observed after combined IL-1/IL-2 therapy of these mice. The antitumor action of IL-1/IL-2 treatment was abolished or markedly reduced in mice treated with antibodies to CD4 or CD8 antigens, whereas antibodies to asialo-GM1 were ineffective. A clear-cut increase in the percentage of CD4+ cells was observed in the spleens of cytokine-treated mice on days 17 and 23. On day 23 of cytokine therapy, CD8+ cells were increased in both spleens and lymph nodes. On day 17, infiltrates of host-reactive cells (i.e., lymphocytes, granulocytes, and monocytes) were observed in both spleen and liver from FLC-injected mice treated with IL-1/IL-2, in association with tumor cells. On days 17 and 23, spleen cells and cells recovered from mesenteric lymph nodes of IL-1/IL-2-treated mice exerted a potent antitumor effect as determined by Winn assay experiments. This antitumor activity was abolished by preincubation of spleen cells with anti-CD8 antibody, but not by treatment with antibodies to asialo-GM1; antibodies to CD4 exerted only a slight effect. Combined IL-1/IL-2 therapy was more effective on established (i.e., 6-7-d) FLC tumors than on early (i.e., 1-d) tumor-transplanted mice. IL-1/IL-2 treatments were also highly effective in increasing survival time of mice from which the subcutaneous primary tumors were excised 7 d after FLC injection. These data indicate that in mice injected with FLC, the antitumor effects of IL-1/IL-2 are mediated by CD4+ and CD8+ cells (but not NK cells), and suggest that this combined cytokine treatment may be effective against established metastatic tumors.     

GM-CSF transgene expression in the airway allows aerosolized ovalbumin to induce allergic sensitization in mice.

The purpose of this study was to explore whether repeated exposure to aerosolized ovalbumin (OVA) in the context of local expression of GM-CSF can initiate a Th2-driven, eosinophilic inflammation in the airways. On day -1, Balb/c mice were infected intranasally with an adenovirus construct expressing GM-CSF (Ad/GM-CSF). From day 0 to day 9 mice were exposed daily to an OVA aerosol. Mice exposed to OVA alone did not show any evidence of airway inflammation. Mice receiving both Ad/GM-CSF and aerosolized OVA exhibited marked airway inflammation characterized by eosinophilia and goblet cell hyperplasia. Migration of eosinophils into the airway was preceded by a rise in IL-5 and IL-4. Both IL-5 and class II MHC were critically required to generate airway eosinophilia. After resolution, airway eosinophilia was reconstituted after a single OVA exposure. Flow cytometric analysis of dispersed lung cells revealed an increase in macrophages and dendritic cells expressing B7.1 and B7.2, and expansion of activated (CD69-expressing) CD4 and CD8 T cells in mice exposed to OVA and Ad/GM-CSF. Our data indicate that expression of GM-CSF in the airway compartment increases local antigen presentation capacity, and concomitantly facilitates the development of an antigen-specific, eosinophilic inflammatory response to an otherwise innocuous antigen.     

Studies on the committed differentiation of CD34+ hematopoietic progenitor cells]

OBJECTIVE: To elucidate the capacity of different hematopoietic growth factors (HGFs) combinations for inducing CD34+ cells to proliferate and differentiate committedly to granulocytes, erythrocytes, megakaryocytes and dendritic cells (DC). METHODS: CD34+ cells were isolated from umbilical cord blood by using a high-gradient magnetic cell sorting system (MACS), and expanded with HGFs in a liquid culture system. Colony forming cells and antigen expression were studied by colony forming assays and FACS. RESULTS: Different combinations of HGFs, including SCF, IL-3, IL-6, GM-CSF, Epo and Tpo increased BFU-E, CFU-GM, CFU-MK and CD41a+ cells by 14.97 +/- 2.89 fold, 14.46 +/- 3.19 fold, 34.67 +/- 4.62 fold and 17.29 +/- 2.34 fold, respectively. Combination of HGFs allowed generation of a large number of DCs. Cultures of CD34+ cells with the combination of FLT-3 ligand + GM-CSF + IL-4 + TNF-alpha yielded 24.28% +/- 2.14% CD1a+ cells, while the control cultures did not. CONCLUSION: It is possible to induce CD34+ cells to proliferate and differentiate committedly to different lineage of hematopoietic cells and DC.     

Immune effects of escalating doses of granulocyte-macrophage colony-stimulating factor added to a fixed,low-dose,inpatient interleukin-2 regimen: a randomized phase I trial in patients with metastatic melanoma and renal cell carcinoma

Previous studies in cancer patients demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulated the interleukin (IL)-2 receptor on T lymphocytes and monocytes suggesting that subsequently administered IL-2 would produce greater immune effects. The authors treated 21 patients with metastatic renal cell carcinoma and melanoma on a randomized phase I study to test this hypothesis. All 21 patients received a fixed dose of IL-2 (72,000 IU/kg every 8 hours for 5 days) administered intravenously as an inpatient. Patients were randomized to receive IL-2 alone or in combination with GM-CSF at a dose of 125 or 250 mcg/m /d (Sargramostim; Immunex Corporation, WA, U.S.A.) daily for 7 days by subcutaneous injection starting on day 1, the day before IL-2 treatment. The results from this study demonstrated that GM-CSF did not worsen the toxicities produced by IL-2 alone. Grade 3 confusion occurred in four patients, three who received IL-2 alone. No partial or complete tumor responses were seen. Assays of serum soluble IL-2 receptor (sIL2R) and neopterin, measures of T cell and monocyte activation, respectively, demonstrated a significant increase in sIL2R but not neopterin, 24 hours after the first dose of GM-CSF. In combination with IL-2, the higher dose of GM-CSF (250 mcg/m ) produced higher sIL2R levels on days 3 and 7 than the 125-mcg/m dose of GM-CSF or IL-2 alone. Although neopterin levels did not increase after 1 day of GM-CSF, the addition of IL-2 resulted in a significantly increased neopterin level on day 3 at the higher dose of GM-CSF. On day 7, neopterin levels in all three groups were similarly increased over baseline. Ten days after treatment, neopterin levels had returned to normal, but sIL2R levels remained markedly increased (12 fold) over baseline in the higher GM-CSF dose group. The authors conclude that 1) monocyte activation was not significantly enhanced by 1 day of GM-CSF treatment; 2) the 250-mcg/m GM-CSF dose plus IL-2 produced superior T cell activation compared with a lower dose of GM-CSF plus IL-2 or to IL-2 alone; and 3) the combination of GM-CSF and IL-2 was safe and tolerable but was not associated with any clinical responses.     

Tumor-induced immunosuppression: a barrier to immunotherapy of large tumors by cytokine-secreting tumor vaccine

An active immunotherapy strategy with cytokine-assisted tumor vaccine, although often effective for small tumor burdens, is much less so for large tumor burdens. This study examines how large tumors might suppress the T cell functions and escape from the immune responses elicited by a granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor vaccine. According to our results, the T cells isolated from the tumor-bearing mice treated late with the vaccine failed to confer protective activity on naive mice against a wild-type tumor challenge, unlike those isolated from the early-treated group. Nevertheless, the antitumor activity of the inactive T cells could be restored on in vitro stimulation. Expression of transforming growth factor beta (TGF-beta) and interleukin 10 (IL-10), the potent immunosuppressive factors, was detected in the parental tumor cell line RLmale 1 (a murine T leukemia cell line), as well as in the tumor region, the levels of which correlated with tumor progression. An in vitro assay of T cell functions revealed that the TGF-beta in the conditioned medium of RLmale 1 cells mainly affected the activation, whereas the IL-1male affected the activation to a lesser extent, but significantly affected the cytolytic activity, of tumor-specific T cells. The immunosuppressive activity of IL-10 was also signified by the findings that administration of the conditioned medium of RLmale 1 cultured in a serum-free medium, in which the TGF-beta activity was then lost while the IL-10 activity still remained, or of recombinant IL-10 to the early-treated group of mice abrogated the known efficacy of tumor vaccine on the small tumors. These data suggested that the efficacy of cytokine-secreting tumor vaccine was blocked by the immunosuppressive factors secreted from the large tumors. The results have important implications for the clinical design of immunotherapeutic strategies for advanced cancer patients.     

The protocol of clinical trial and basic experiments for esophageal cancer using gene transduction

We examined whether antitumor effect could be produced by retrovirally expressed interleukin-2(IL-2) gene, glanulocyte macrophage-colony stimulating factor(GM-CSF) gene, herpes simplex virus-thymidine kinase(HSV-tk) gene and p53 gene in human esophageal cancer cells using nude mice. Loss of tumorigenicity of IL-2 or GM-CSF producing cancer cells were observed. The antitumor effect was also evidenced by the injection of these cells into established tumors of wild-type cells. In suicide gene therapy on esophageal cancer, the growth suppression of esophageal cancer cells transducing HSV-tk gene tumors in nude mice induced by ganciclovir treatment and all the tumors disappeared. The wild-type p53 transduced tumor cells became markedly susceptible to irradiation and anticancer agents. Administration of cisplatine noticeably suppressed the growth of p53 transduced tumors inoculated in nude mice. We established the clinical protocol of gene therapy for esophageal cancer using wild-type p53 gene with adenovirus vector. In this autumn we are going to start this clinical trial.     

Different mitogen-activated protein kinase-dependent cytokine responses in cells of the monocyte lineage

Macrophages release cytokines that may contribute to the chronic inflammation observed in pulmonary conditions such as asthma and chronic obstructive pulmonary disease. Thus, inhibition of macrophage cytokine production may have a therapeutic benefit. Human lung macrophages are a rich source of the proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, and IL-8, that are elevated in the bronchoalveolar lavage and sputum of subjects with respiratory diseases. Cytokine production from both monocytes and macrophages is mediated by mitogen-activated protein kinase (MAPK) pathways. This study compared the effects of a novel p38 MAPK inhibitor, N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl]amino}ethyl)-guanidine (PCG), and an extracellular signal-regulated kinase (ERK) pathway inhibitor, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059), on cytokine release from lipopolysaccharide (LPS)-stimulated human monocytes, monocyte-derived macrophages (MDM), and lung macrophages. Lung macrophages, MDM, and monocytes were stimulated with LPS, and cytokine release was measured by enzyme-linked immunosorbent assay. Immunoblots were performed to confirm p38 and ERK1/2 MAPK expression and activity. PCG inhibited TNF-alpha release more effectively from monocytes compared with MDM or macrophages (maximal inhibition was 99.3 +/- 1.4, 62.7 +/- 4.3, and 58.6 +/- 6.6%, respectively; n = 7-9). PD098059 was less effective at suppressing TNF-alpha release from monocytes compared with MDM and lung macrophages (maximal inhibition was 37.4 +/- 2.8, 70.1 +/- 4.5, and 68.7 +/- 5.1%, respectively; n = 7-9). The pattern of GM-CSF, IL-6, and IL-8 release was comparable with that of TNF-alpha. These data suggest a differential involvement for each of these MAPK pathways in macrophage cytokine production compared with monocytes.     

Amelioration of B16F10 melanoma lung metastasis in mice by a combination therapy with indomethacin and interleukin 2 [published erratum appears in J Exp Med 1987 Mar 1;165(3):935]

Our earlier work revealed that PGE-mediated inactivation of NK cells in tumor-bearing mice by host macrophages promoted spontaneous lung metastasis that could be prevented or ameliorated by chronic indomethacin therapy. Since PGE was found to suppress the in vitro development and/or activation of a family of tumoricidal lymphocytes such as CTL, NK, and LAK cells by one or both of two mechanisms, that is to say, a down regulation of IL-2-R and an inhibition of IL-2 production, the present study tested whether a combined therapy with indomethacin and IL-2 was more effective than one with indomethacin or IL-2 alone in ameliorating established experimental lung metastasis. B6 mice injected intravenously with 10(6) highly metastatic B16F10 melanoma cells showed profuse micrometastases in the lungs by day 5, and macrometastases by day 10 which were confluent on day 21. Chronic indomethacin therapy by the oral route (14 micrograms/ml in drinking water) starting on day 0 or day 5, or a single round of IL-2 therapy (25,000 U rIL-2, every 8 h for 5 d on days 10-14) reduced the number of metastatic nodules by two-thirds (from a median of 473 in control mice receiving vehicles alone) by day 21. A single round of IL-2 as above, combined with either protocol of indomethacin therapy, completely or nearly completely irradicated the lung metastases, corroborated by a histological examination. An evaluation of splenic killer cell activity measured with a 4-h 51Cr-release assay against NK-sensitive YAC-1 lymphoma and B16F10 melanoma or NK-resistant thymic lymphoma 9705 targets revealed negligible activity in control tumor-bearing mice, and a good restoration of activity against NK-sensitive targets with either protocols of indomethacin therapy. IL-2 alone or a combination of IL-2 and indomethacin given by either protocol generated strong killer activity against all these targets, most marked with the combination therapy. Splenic killer cell phenotype in normal as well as all treated animals was ASGM1+, Thy-1-, and Lyt-2-. The combination therapy resulted in the strongest mononuclear cell infiltration in the lungs, with areas of young granulation tissue suggestive of repair sites of original metastases.     

Activated neutrophils induce nitric oxide production in Kupffer cells

Neutrophils (PMN) are proposed to contribute to hepatic dysfunction during sepsis. Transmigrating PMN have been demonstrated to adhere to and injure parenchymal cells (hepatocytes); however, the effect of sepsis-activated PMN on hepatic macrophages or Kupffer cells (KC) is poorly characterized. We hypothesize that PMN influence KC inflammatory mediator production, including nitric oxide. Rodent KC were co-cultured with PMN obtained from controls (Norm-PMN) or endotoxemic rats [lipopolysaccharide (LPS)-PMN] for 18 h. After an 18-h incubation, supernatants and cell lysates of the KC were analyzed for nitric oxide (NO) production. Co-cultures with LPS-PMN/KC demonstrated significantly increased production of nitrite and up-regulation of inducible nitric oxide synthase (iNOS) protein compared to KC alone or Norm-PMN/KC co-cultures. Immunohistochemistry revealed preferential iNOS protein staining in the cytoplasm of KC cultured with LPS-PMN compared to controls. Nitrite production in co-cultures of KC and LPS-PMN where cell contact was inhibited by a cell impermeable but diffusable membrane was significantly reduced. These data provide evidence that KC can be stimulated directly by activated PMN for production of NO. Further, they suggest another mechanism by which PMN modulate hepatic function during sepsis.     

Use of curcumin to decrease nitric oxide production during the induction of antitumor responses by IL-2

Nitric oxide (NO) synthesis is strongly induced during interleukin (IL)-2 treatment of mice and humans. Although this free radical can act as a cytotoxic effector molecule against cancer cells, immunosuppressive effects have also been suggested. We evaluated the effects of curcumin on IL-2-induced NO synthesis and IL-2-induced antitumor responses in a mouse ascites tumor model. Curcumin inhibited inducible nitric oxide synthase (iNOS) expression and NO production, and thereby enhanced the proliferation and cytotoxic activity of cocultured lymphocytes and macrophages during IL-2 stimulation which we earlier established as an in vitro model of IL-2-induced NO synthesis. Curcumin also decreased apoptosis of cocultured lymphocytes and macrophages during IL-2 stimulation. In contrast, the curcumin-induced changes in proliferation and apoptosis were not observed in cultures of lymphocytes alone, macrophages alone, and cocultured lymphocytes/iNOS-knock out macrophages, all of which produced little nitrite during IL-2 stimulation. In conjunction with IL-2 treatment, oral curcumin administration significantly inhibited IL-2 therapy-induced urinary nitrite/nitrate excretion and iNOS expression of tumor tissues, and further increased the IL-2 therapy-induced prolongation of survival in a murine Meth-A ascites tumor model. Curcumin may be useful as an adjunct to increase the antitumor activity of IL-2 therapy.     

Presence of cytotoxic B220+CD3+CD4-CD8- cells correlates with the therapeutic efficacy of lymphoma treatment with IL-2 and/or IL-12

Cancer treatment with IL-2 and IL-12 is thought to work via enhancement of proliferation and activity of T cells and NK cells. Incubation of cytotoxic T lymphocytes (CTLs) and NK cells with IL-2 and/or IL-12 results in propagation of a distinct cell type called lymphokine-activated killers (LAK) characterized by increased lytic activity against many tumor types. Here we address the question whether cytokine therapy may be efficient in treatment of a LAK-insensitive tumor and, if so, which cell type, other than classic LAK cells, is responsible for tumor cell killing. We used DBA/2 mice bearing metastasized SL2 lymphoma and treated them with locally applied IL-2 and /or IL-12 injections. We showed that IL-12 treatment is efficient, though there is a rather narrow range of effective doses because of toxicity. This toxicity may be alleviated by a single injection of IL-12 before treatment. Next, we showed that IL-12 synergistically enhances the efficacy of local IL-2 treatment. Moreover, our results indicate that the IL-2/IL-12-mediated therapeutic effect is greatest when it is given after establishment of an immune response to a tumor. Finally, we showed the existence of a unique population of lymphoid cells, namely B220+CD3+CD4-CD8-, at the site of tumor growth. These cells become highly cytotoxic to SL2 cells in mice treated with cytokines late (day 10-14) in the course of the immune response, but not in mice treated early (day 3-7), and cytotoxicity of this unique cell population correlates with the success of therapy.     

Infiltration of COX-2-expressing macrophages is a prerequisite for IL-1 beta-induced neovascularization and tumor growth

Inflammatory angiogenesis is a critical process in tumor progression and other diseases. The inflammatory cytokine IL-1beta promotes angiogenesis, tumor growth, and metastasis, but its mechanisms remain unclear. We examined the association between IL-1beta-induced angiogenesis and cell inflammation. IL-1beta induced neovascularization in the mouse cornea at rates comparable to those of VEGF. Neutrophil infiltration occurred on day 2. Macrophage infiltration occurred on days 4 and 6. The anti-Gr-1 Ab-induced depletion of infiltrating neutrophils did not affect IL-1beta- or VEGF-induced angiogenesis. The former was reduced in monocyte chemoattractant protein-1-deficient (MCP-1(-/-)) mice compared with wild-type mice. After day 4, clodronate liposomes, which kill macrophages, reduced IL-1beta-induced angiogenesis and partially inhibited VEGF-induced angiogenesis. Infiltrating macrophages near the IL-1beta-induced neovasculature were COX-2 positive. Lewis lung carcinoma cells expressing IL-1beta (LLC/IL-1beta) developed neovasculature with macrophage infiltration and enhanced tumor growth in wild-type but not MCP-1(-/-) mice. A COX-2 inhibitor reduced tumor growth, angiogenesis, and macrophage infiltration in LLC/IL-1beta. Thus, macrophage involvement might be a prerequisite for IL-1beta-induced neovascularization and tumor progression.     

丝裂霉素诱导的肝癌细胞凋亡致敏树突状细胞

目的:建立丝裂霉素诱导癌细胞凋亡致敏从人外周血诱导扩增的树突状细胞的方法。设计:以癌细胞凋亡致敏树突状细胞为观察对象的随机对照实验。单位:解放军第三军医大学大坪医院野战外科研究所,解放军军事医学科学院放射医学研究所。材料:实验于1998-04/1999-05在解放军军事医学科学院放射医学研究所完成。细胞株为QBC939胆管癌细胞株,药物为抗肿瘤药物丝裂霉素。方法:从正常人外周血分离获得单核细胞,加入50μg/L重组人粒细胞集落刺激因子,1000U/mL白细胞介素4,隔天一次,共4次,培养第3天,加入丝裂霉素诱导凋亡的胆管癌细胞,再继续体外培养4d后,用树突细胞富集柱收集树突状细胞。主要观察指标:①培养的树突状细胞的鉴定。②树突状细胞和坏死胆管癌细胞以及正常培养的胆管癌细胞共培养,观察其吞噬凋亡小体负载抗原。③检测不同密度的树突状细胞(1×103/孔,5×103/孔,1×104/孔)的免疫刺激活性及负载抗原后的免疫刺激活性,以单核细胞作对照组。结果:①培养、扩增得到的树突状细胞高表达共刺激分子B7和CDla,表面具有典型不规则突起。②丝裂霉素诱导癌细胞凋亡形成的凋亡小体被树突状细胞捕捉、吞噬。③负载抗原的树突状细胞其激发同种异体T淋巴细胞增殖的能力进一步增强。结论:丝裂霉素诱导癌细胞凋亡可以致敏重组人粒细胞集落刺激因子加重组人白细胞介素4从人外周血单核细胞诱导、扩增出的树突状细胞,树突状细胞可以有效提呈凋亡胆管癌细胞的抗原,可望成为有效的肿瘤抗原致敏树突状细胞的新途径。     

Kupffer cell-depleted rats have a diminished acute-phase response following major liver resection

Partial hepatectomy (PH)-induced Kupffer cell (KC) activation results in a rapid release of cytokines inducing the acute-phase response (APR). This study was done to evaluate the role of Kupffer cells (KCs) in the course of the APR following PH and a consecutive endotoxin challenge. KC depletion was performed in rats by i.v. administration of 1 mL liposome-encapsulated dichloromethylene diphosohonate (Cl2MDP). Control rats received 1 mL NaCl 0.9%. Forty-eight hours later, PH was performed. At 24 h after PH, rats were randomized to receive either 1 mL NaCl 0.9% (saline) or 50 microg/kg LPS i.v. in 1 mL. Animals were sacrificed at 4 h after LPS or saline infusion. The APR was determined by measuring hepatic gene expression of alpha 2-macroglobulin, alpha 1-acid glycoprotein, and IL-6 and expression of hepatic albumin. The APR was significantly depressed in KC-depleted rats. Despite increased IL-6 mRNA synthesis in response to low-dose LPS, no enhancement of acute-phase protein synthesis (APP) was found in KC-depleted rats. Hepatic failure was most profound in KC-depleted rats, as indicated by elevated plasma levels of liver transaminases and ammonia. We conclude that after PH, KC function in the remnant liver is important for the acute-phase reaction and reduces endotoxin-induced hepatocyte damage.     

Diphtheria toxin-murine granulocyte-macrophage colony-stimulating factor-induced hepatotoxicity is mediated by Kupffer cells

DT388GMCSF, a fusion toxin composed of the NH2-terminal region of diphtheria toxin (DT) fused to human granulocyte-macrophage colony-stimulating factor (GMCSF) has shown efficacy in the treatment of acute myeloid leukemia. However, the primary dose-limiting side effect is liver toxicity. We have reproduced liver toxicity in rats using the rodent cell-tropic DT-murine GMCSF (DT390mGMCSF). Serum aspartate aminotransferase and alanine aminotransferase were elevated 15- and 4-fold, respectively, in DT390mGMCSF-treated rats relative to controls. Histologic analysis revealed hepatocyte swelling; however, this did not lead to hepatic necrosis or overt histopathologic changes in the liver. Immunohistochemical staining showed apoptotic cells in the sinusoids, and depletion of cells expressing the monocyte/macrophage markers, ED1 and ED2, indicating that Kupffer cells (KC) are targets of DT390mGMCSF. In contrast, sinusoidal endothelial cells seemed intact. In vitro, DT390mGMCSF was directly cytotoxic to primary KC but not hepatocytes. Two related fusion toxins, DT388GMCSF, which targets the human GMCSF receptor, and DT390mIL-3, which targets the rodent IL-3 receptor, induced a less than 2-fold elevation in serum transaminases and did not deplete KC in vivo. In addition, DTU2mGMCSF, a modified form of DT390mGMCSF with enhanced tumor cell specificity, was not hepatotoxic and was significantly less toxic to KC in vivo and in vitro. These results show that DT390mGMCSF causes liver toxicity by targeting KC, and establish a model for studying how this leads to hepatocyte injury. Furthermore, alternative fusion toxins with potentially reduced hepatotoxicity are presented.     

Differential effect of anti-TNF-alpha antibody on proinflammatory cytokine release by Kupffer cells following liver ischemia and reperfusion.

To study the role of tumor necrosis factor-alpha (TNF-alpha) for induction of the proinflammatory cytokine cascade after liver ischemia and reperfusion (I/R), rats were injected intraperitoneally with anti-TNF-alpha monoclonal antibodies (mAb) or placebo (IgG1) 30 min prior to global hepatic ischemia. Blood levels of TNF-alpha, interleukin (IL)-1alpha and -6 were determined. In addition, Kupffer cells (KC) were harvested after 60 min of reperfusion and spontaneous cytokine release was measured. Sham-operated animals were used as controls. Levels of proinflammatory cytokines in serum and KC supernatants were detected using specific bioassays and ELISA. Liver I/R resulted in increased (p < .01) serum levels of TNF-alpha, IL-1alpha, and IL-6, which was associated with an enhanced (p < .05) release of these cytokines by KC. In vivo pretreatment with anti-TNF-alpha mAb led to complete neutralization of TNF-alpha serum levels and decreased (p < .01) IL-6 levels (-62%). Moreover, anti-TNF-alpha mAb markedly (p < .05) decreased the release of TNF-alpha (-69%) and IL-6 (-56%) by KC, while IL-1alpha was not affected. These data indicate that TNF-alpha produced early after liver I/R triggers both its own secretion as well as IL-6 release by KC during reperfusion while the release of IL-1alpha occurs independent from TNF-alpha.     

Intratumoral poly-N-acetyl glucosamine-based polymer matrix provokes a prolonged local inflammatory response that, when combined with IL-2, induces regression of malignant mesothelioma in a murine model

The authors have previously shown that cytokines delivered directly into malignant mesothelioma (MM) tumors can retard tumor growth and mediate tumor regression under certain conditions. In this report the authors compared the efficacy of serial intratumoral injections of three cytokines, GM-CSF, IL-12, and IL-2, to their sustained release using a single injection in a poly-N-acetyl glucosamine gel. IL-2 combined with the polymer gel gave optimal antitumor results when MM tumors were accessible as either subcutaneous deposits or as masses spread throughout the peritoneal cavity. The gel acted not only as a slow-release cytokine depot but also as a trigger for inflammation and recruited several immune cell types to the gel/tumor interface; when combined with IL-2 (but not with GM-CSF or IL-12), it acted as a selective reservoir for infiltrating CD8+ T cells. Hence, the IL-2/gel may provide a microenvironment that allows intratumoral T cells to proliferate and retain their cytolytic functions as they encounter their cognate antigens expressed by tumor cells.