Effects of transforming growth factor β1 expression in a rat colon carcinoma: growth inhibition, leukocyte infiltration and production of interleukin-10 and tumor necrosis factor α

The cytokine transforming growth factor β-1 (TGFβ1), was transfected into a TGFβ1-negative rat colon carcinoma. The growth of isografts of TGFβ1-expressing tumors was compared to that of vector control transfectants. The TGFβ1 transfectant grew significantly more slowly after intrahepatic isografting than did vector control and wild-type tumors. The TGFβ1-transfected tumor tissue had significantly greater infiltration of both CD4⁺ and CD8⁺ T lymphocytes than did the vector control tumor. The tumor-infiltrating leukocytes (TIL) from TGFβ1-transfected tumor secreted significantly more of the cytokines interleukin-10 (IL-10) and tumor necrosis factor α (TNFα) than did TIL from the vector control tumor. The TGFβ1 transfectant also demonstrated a significantly slower outgrowth in immunodeficient SCID mice, supporting a non-T-lymphocyte-dependent mechanism for the tumor retardation. In SCID mice, the TGFβ1-transfected tumor demonstrated significantly greater infiltration of both granulocytes and macrophages than did the vector control transfectant. We also demonstrated a direct inhibitory effect of rat TNFα on tumor proliferation in vitro. These results suggest that TGFβ1 induces a local secretion of immunomodulating cytokines and that this may influence monocytes, lymphocytes and granulocytes to retard tumor outgrowth. Received: 7 July 1999 / Accepted: 12 August 1999     

Variable expression of tumor necrosis factor α in human malignant melanoma localized by in situ hybridization for mRNA

 Tumor necrosis factor α (TNFα) is a cytokine, produced by lymphocytes and monocytes, with cytotoxic activity against some but not all tumor cell lines. Resistance to the cytolytic effects of TNFα has been reported in cell lines with autocrine TNFα production. The purpose of this study was to investigate whether human primary malignant melanoma and tumor infiltrating lymphocytes produce TNFα in vivo. Optimal conditions for in situ hybridization for TNFα mRNA in paraffin-embedded tissue were established. Analysis of 13 primary malignant melanomas and 3 metastatic lesions with different degrees of immunohistochemical TNFα positivity demonstrated that, in some tumors, both melanoma cells and leukocytes contained TNFα mRNA and protein. These findings demonstrate variable production of TNFα in primary and metastatic melanoma in vivo.The previously described resistance to TNFα cytolytic activity may, therefore, be clinically important. Received: 18 December 1996 / Accepted: 12 June 1997     

Antitumor effects of the combination immunotherapy with interleukin-12 and tumor necrosis factor α in mice

 There is strong evidence that antitumor activity of interleukin-12 (IL-12) in vivo is mediated, in part, through interferon (IFNγ) produced by IL-12-stimulated natural killer and T cells. Since IFNγ and tumor necrosis factor α (TNFα) have been reported to synergize in antitumor effects in a number of models, we decided to examine whether the combined treatment with recombinant mouse IL-12 and recombinant human TNFα would produce similar effects. The efficacy of the combined IL-12/TNFα immunotherapy was evaluated in three tumor models in mice: B16F10 melanoma, Lewis lung (LL/2) carcinoma and L1 sarcoma. Intratumoral daily injections of 1 μg IL-12 in combination with 5 μg TNFα into B16F10-melanoma-bearing mice resulted in a significant retardation of the tumor growth as compared with that in controls and in mice treated with either cytokine alone. Similar effects were obtained using 0.1 μg IL-12 and 5 μg TNFα in LL/2 carcinoma and L1 sarcoma models. Antitumor activity against L1 sarcoma was still preserved when TNFα at a low dose (1 μg) was combined with 0.1 μg IL-12 and applied for a prolonged time. Potentiation of antitumor effects, which was observed in IL-12/TNFα-based immunotherapy, could result from at least three different mechanisms, partly related to stimulation of IFNγ and TNFα production in treated mice: (a) direct cytostatic/cytotoxic effects on tumor cells, (b) induction of antitumor activity of macrophages, and (c) inhibition of blood vessel formation in the tumor. Our studies demonstrate that combination tumor immunotherapy with IL-12 and TNFα may be more effective than single-cytokine treatment, and suggest possible mechanisms by which IL-12 and TNFα may exert potentiated therapeutic effects against locally growing tumors. Received: 17 February 1997 / Accepted: 5 August 1997     

IKKγ (NEMO) is involved in the coordination of the AP-1 and NF-κB pathways

Tumor necrosis factor alpha (TNFα) activates the nuclear factor-kappaB (NF-κB) pathway in various cell types, leading to expression of cell survival and inflammatory proteins. One mechanism of cell survival brought about by NF-κB is the inhibition of Activator Protein-1 (AP-1), which when activated, could lead to cell death. However, TNFα can also induce the AP-1 pathway, and the mechanisms by which these two pathways are regulated in response to TNFα are poorly understood. We proposed that Inhibitor of κB Kinase gamma (IKKγ) (which is also known as NF-κB essential modulator, NEMO) plays a key role in integrating and coordinating these two pathways. Our results showed that IKKγ activates the AP-1 pathway, via a mechanism that is dependent on the first leucine zipper (LZ) domain of IKKγ, by interacting with two proteins of the AP-1 complex, c-Jun and c-Fos, and changing the phosphorylation status of c-Jun. Even though IKKγ is required for the activation of NF-κB, we found that it reduced the activity of NF-κB when it was overexpressed. In summary, we demonstrated that transfected IKKγ, while inhibiting the NF-κB pathway, directly interacts with the AP-1 proteins and activates the AP-1 pathway independent of its effects on NF-κB. Our results indicate that IKKγ regulates TNFα signaling by coordinating cell responses mediated by the AP-1 and NF-κB pathways. A. S. Shifera and J. M. Friedman contributed equally to this article. Marshall S. Horwitz—Deceased: This article is dedicated to his loving memory.     

Production of urinary tumour necrosis factors and soluble tumour necrosis factor receptors in bladder cancer patients afterbacillus Calmette-Guerin immunotherapy

Intravesical immunotherapy for bladder cancer is the most effective form of tumour immunotherapy. Following repeated instillations of bacillus Calmette-Guérin (BCG) organisms into the bladder large 0quantities of several cytokines are detected in the urine. These cytokines include interleukins IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumour necrosis factor α (TNFα), interferon γ (IFNγ) and also soluble intercellular adhesion molecule ICAM-1. In the work reported here we simultaneously quantified urinary levels of TNFα, TNFβ, TNF receptor I and TNF receptor II by enzyme-linked immunosorbent assay (ELISA) techniques and compared this with bioactive levels of TNF. This was undertaken with a limited number of patients throughout a course of six instillations of immuno therapy. Sequential instillations of BCG induced secretion of TNFα and TNFβ into urine. These cytokines were not always secreted simultaneously, perhaps suggesting differential regulation of their synthesis. Maximal concentrations of TNFα were 675 pg/ml and TNFβ 47 pg/ml. High levels of both species of soluble TNF receptor were readily identified in urine. Maximal levels of sTNF-RI were 6200 pg/ml (range from 0) and for sTNF-RII 7800 pg/ml (range from 0). Contrasting with earlier published observations concerning cytokine levels, the concentration of soluble receptor did not increase with repeated instillation. In apparent contrast with the ELISA data, very low levels of bioactive TNF were identified by the L929 bioassay (maximum concentration 1 U/ml) despite the elevated concen t ration of immunoreactive TNF. The large concentrations of soluble TNF receptor in patients’ urine samples could account for the apparently low bioactivity as determined by the L929 cytotoxicity assay. The precise nature of the role of TNF in BCG immunotherapy remains undetermined; however, it is thought that proinflammatory cytokines are in part responsible for the clinical efficacy of this therapeutic approach. Whether other cytokines are antogonised by soluble binding proteins remains to be determined. Furthermore, whether TNF is bioactive in the bladder wall and only neutralised in the urine also requires investigation. Received: 24 August 1994 / Accepted: 17 October 1994     

Expression, mitogenic activity and regulation by growth hormone of growth hormone/insulin-like growth factor in Branchiostoma belcheri

Our aim has been to characterize the molecular mechanisms regulating the expression of the channel-forming tight-junctional protein claudin-2 in response to the pro-inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated, for example, in active Crohn’s disease. TNFα caused an 89% decrease of the paracellular resistance in colonic HT-29/B6 cells, whereas transcellular resistance was unaltered. The claudin-2 protein level was increased by TNFα without changes in subcellular tight-junctional protein localization as revealed by confocal laser scanning microscopy. Enhanced gene expression was identified as the source of this increase, since claudin-2-specific mRNA and promoter activity was elevated, whereas mRNA stability remained unaltered. Specific inhibitors and phospho-specific antibodies revealed that the increased gene expression of claudin-2 after TNFα treatment was mediated by the phosphatidylinositol-3-kinase pathway. Thus, the up-regulation of claudin-2 by TNFα is attributable to the regulation of the expression of the gene, as a result of which epithelial barrier function is disturbed, for example, during chronic intestinal inflammation. J. Mankertz and M. Amasheh contributed equally to this work. This work was supported by the Deutsche Forschungsgemeinschaft and the Sonnenfeld-Stiftung Berlin.     

Synergy between interleukin-2 and prothymosin α for the increased generation of cytotoxic T lymphocytes against autologous human carcinomas

 Peripheral blood mononuclear cells (PBMC) from cancer patients were cultured in vitro with irradiated autologous tumor cells isolated from malignant effusions (mixed lymphocyte tumor cultures, MLTC) and low-dose (50 IU/ml) recombinant interleukin-2 (IL-2). The combination of IL-2 and prothymosin α (ProTα) resulted in a greater PBMC-induced response to the autologous tumor than that brought about by IL-2 alone. In particular, ProTα specifically enhanced the CD4⁺ T-cell-mediated proliferation against the autologous tumor. CD4⁺ T cells seemed to recognize tumor antigens presented by HLA-DR molecules expressed on the autologous monocytes, since preincubation of the latter with an anti-HLA-DR monoclonal antibody (mAb) abrogated the response. In addition, MLTC set up with IL-2 and ProTα also generated more MHC-class-I-restricted cytotoxic T lymphocytes (CTL) against the autologous tumor than did MLTC set up with IL-2 alone. The MLTC-induced CTL contained high levels of cytoplasmic perforin and their development was strictly dependent on the presence of both autologous CD4⁺ T cells and monocytes. In the absence of either population there was a strong impairment of both proliferative and cytotoxic responses which was not restored by the presence of ProTα. In contrast, when both cell populations were present, ProTα exerted optimal enhancement of CD4⁺ T cell proliferation, which was associated with potentiated CTL responses. Our data emphasize the role of ProTα for the enhancement of IL-2-induced CTL responses against autologous tumor cells. Such responses require collaborative interactions between CD4⁺, CD8⁺ T cells and monocytes as antigen-presenting cells. Our data are relevant for adoptive immunotherapeutic settings utilizing IL-2 and ProTα-induced autologous-tumor-specific CTL. Received: 2 March 2000 / Accepted: 1 June 2000     

Importance of signaling via the IFN-α/β receptor on host cells for the realization of the therapeutic benefits of cyclophosphamide for mice bearing a large MOPC-315 tumor

Here we show that low-dose cyclophosphamide (CY), that depends for its therapeutic effectiveness on the immunopotentiating activity of the drug for T cell-mediated tumor-eradicating immunity, is curative for ~80% of wild-type (WT) mice bearing a large s.c. MOPC-315 tumor, but only for ~10% of IFN-α/βR^−/− mice bearing a large s.c. MOPC-315 tumor. Histopathological examination of the s.c. tumors of such mice on day 4 after the chemotherapy revealed that the low dose of CY led to accumulation of T lymphocytes in both the WT and the IFN-α/βR^−/− mice. However, in the CY treated tumor bearing WT mice the T lymphocytes were present throughout the tumor mass and in direct contact with tumor cells, but in the CY treated tumor bearing IFN-α/βR^−/− mice most of the T lymphocytes remained in blood vessels. In addition to being important for CY-induced transendothelial migration of T lymphocytes into the tumor mass, we show here that signaling via the IFN-α/βR is also important for CY-induced control of metastatic tumor progression in the spleen and liver of the tumor bearing mice. Finally, CY cured tumor bearing WT mice were resistant to a subsequent challenge with MOPC-315 tumor cells, but the few CY cured tumor bearing IFN-α/βR^−/− mice were not. Thus, signaling via the IFN-α/βR on host cells in MOPC-315 tumor bearers is important for CY-induced: (a) transendothelial migration of T lymphocytes into the tumor mass and the eradication of the primary tumor, (b) control of metastatic tumor progression, and (c) resistance to a subsequent tumor challenge. This work was supported by Research Grant 03-19 from the American Cancer Society-Illinois Division.     

Xaf1 can cooperate with TNFα in the induction of apoptosis, independently of interaction with XIAP

XIAP-associated factor 1 (Xaf1) binds XIAP and re-localizes it to the nucleus, thus inhibiting XIAP activity and enhancing apoptosis [1]. Xaf1 expression is reduced or absent in tumor samples and cell lines suggesting it may function as a tumor suppressor [2–5]. To further study Xaf1 function we generated Xaf1 inducible cells in the osteosarcoma cell line Saos-2. Despite Xaf1 inducing apoptosis that is dramatically enhanced by TNFα we find no evidence for an interaction between Xaf1 and XIAP. Furthermore, Xaf1 expression sensitized XIAP^−/− fibroblasts to TNFα, demonstrating the existence of a novel mechanism of Xaf1 induced apoptosis distinct from antagonizing XIAP. Xaf1 expression promotes cytochrome c release that cannot be blocked by inhibition of caspase activity. This implicates a role for the mitochondrial apoptotic pathway, consistent with the ability of Bcl2 to block Xaf1 induced apoptosis. The data indicate that in Saos2 cells Xaf1 activates the mitochondrial apoptotic pathway to facilitate cytochrome c release, thus amplifying apoptotic signals from death receptors.     

Regulation of cyclin-dependent kinase inhibitor p21<Superscript>WAF1/CIP1</Superscript> by protein kinase Cδ-mediated phosphorylation

Cyclin-dependent kinase (CDK) inhibitor p21^{WAF1/CIP1(-/-)}-null mice have an increased incidence of tumor formation. Here, we demonstrate that p21^WAF1/CIP1 is unstable in HeLa cells treated with siRNA duplexes that target PKCδ. PKCδ phosphorylates p21^WAF1/CIP1 at a serine residue (¹⁴⁶Ser) located in its C-terminal domain. In cells treated with 12-O-tetradecanoylphorbol 13-acetate, the levels of both p21^WAF1/CIP1 and its ¹⁴⁶Ser-phosphorylated form increased significantly. We also show that a substitution, resulting from a single nucleotide polymorphism (SNP) at ¹⁴⁹Asp found in certain cancer patients, strongly compromises PKCδ-mediated phosphorylation at ¹⁴⁶Ser and results in cells that are relatively resistant to TNFα-induced apoptosis. Thus, post-translational phosphorylation of p21^WAF1/CIP1 is important from an apoptotic cell death, and may also have patho-physiological relevance for the development of human cancer.     

Dendritic cells engineered to express CD40L continuously produce IL12 and resist negative signals from Tr1/Th3 dominated tumors

TNFα-matured dendritic cells (DCs) pulsed with tumor antigens are being evaluated as cancer vaccines. It has been shown that DCs produce IL12 during a limited time span and subsequently enter a stage of IL12 exhaustion. If DCs are generated ex vivo, the patient could receive IL12-exhausted DCs which may be detrimental for stimulating anti-tumor Th1 responses. Furthermore, many cancer patients exhibit a cytokine profile skewed toward IL10 and TGFβ. This immunological profile, called the Tr1/Th3 response, is associated with the presence of regulatory T-cells. Tr1/Th3 responses potently inhibit DC maturation, thereby regulating Th1 responses. In the present study, we produced genetically engineered DCs that continuously express Th1-related cytokines such as IL12, and resist negative signals from Tr1/Th3-dominated bladder carcinoma cells. Human immature DCs were genetically engineered by adenoviral vectors to express CD40L, or were treated with TNFα as a positive control for maturation. The expression of different Th1/Th3 and inflammatory cytokines was monitored. IL12 and IFNγ were expressed by CD40L-engineered DCs, while TNFα-matured DCs lacked IFNγ and exhibited low IL12 expression. The addition of recombinant IL10 to genetically engineered DCs did not abolish their Th1 profile. Likewise, coculture with tumor cell lines expressing TGFβ with or without recombinant IL10 did not revert to the engineered DCs. We further demonstrate that the resistance of CD40L-expressing DCs to TGFβ and IL10 may be due to decreased levels of TGFβ and IL10 receptors. Thus, CD40L-engineered DCs are robust Th1-promoting ones that are resistant to Tr1/Th3-signaling via IL10 and TGFβ.     

The regulation of TACE catalytic function by its prodomain

Aim To study the function of the prodomain of ADAM17 (TACE) and to develop an approach for interfering with inflammation processes. Method The expression plasmids of the TACE ectodomain (T1300), prodomain (T591), signal peptide and prodomain (T648), full length (T2472), and the turncated TACE without prodomain (T57-T1824) were constructed and designated as pET-28a-T300, pET-28a-T591, pIRES2-EGFP-648, pEGFP-N1-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824, respectively. After Ni²⁺-NTA resin-affinity chromatography, the recombinant T591 and T1300 proteins were obtained and assayed by western blotting and circular dichroism. The experiment was carried out on THP1 cell lines stimulated by LPS in vitro. The inhibition of recombinant protein T591 to TACE activity was detected by ELISA and immunohistochemical detection. The expression plasmids (pIRES2-EGFP-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824) were used to transfect the U937 cells. HeLa cells were also transfected with pEGFP-N1-T648. The transfected U937 cells were then stimulated by LPS and the effect of expression plasmids on TNF-α secretion was detected by ELISA and flow cytometry (FCM). Results The recombinant prodomain protein inhibited 57% of the TNF-α secretion and mediated an accumulation of TNF-α on the surface of THP1 cells. An intense green fluorescence was seen in the membranes of HeLa cells transfected with pEGFP-N1-T648. The plasmid pIRES2-EGFP-T648 inhibited TNF-α secretion by 61.09% and mediated an accumulation of mTNF-α on the surface of the U937 cells. The secretion of sTNF-α and the level of the mTNF-α in the pIRES2-EGFP-T57-T1824 transfected cells gave no difference when compared with the pIRES2-EGFP transfected cells. Also the secretion of sTNF-α from the cells transfected by the plasmid pIRES2-EGFP-T2472 increased, while the level of mTNF-α decreased, compared with the pIRES2-EGFP-transfected cells. Conclusion The prodomain has dual effects and might be useful in the molecular design of an anti-inflammatory drug.     

Macrophage inflammatory protein (MIP)1α and MIP1β differentially regulate release of inflammatory cytokines and generation of tumoricidal monocytes in malignancy

The C–C chemokines, macrophage inflammatory protein (MIP)1α and MIP1β are potent chemoattractants for the monocytes, which form an important component of the stroma of tumor tissue and may regulate tumor growth and associated inflammation. We examined the role of MIP1α and MIP1β in inducing the release of inflammatory cytokines and the generation of tumoricidal monocytes from the peripheral blood monocytes (PBM) of healthy women and patients with carcinoma of breast (CaBr). Interleukin-1 (IL-1) and tumor necrosis factor (TNF) α release by the PBM was markedly stimulated by MIP1α in CaBr patients, but only marginally so in healthy women. In contrast, MIP1β stimulated the release of these cytokines by the PBM of healthy women, but failed to do so in CaBr patients. MIP1α, but not MIP1β, synergized with LPS in inducing the release of IL-1 from the PBM of both healthy women and CaBr patients. Both MIP1α and MIP1β augmented respiratory bursts in PBM and generated tumoricidal PBM that killed T24 cells, MIP1α being more effective in CaBr patients and MIP1β in healthy women. IFN-γ co-stimulated and IL-4 suppressed MIP1α and β-induced cytotoxicity in PBM. The synergy of IFN-γ was more marked with MIP1α than with MIP1β. The differential effects of MIP1α and MIP1β on the PBM of healthy women and CaBr patients co-related with the levels of expression of CCR1 and CCR5 in these monocytes. The expression of CCR5 was higher than that of CCR1 in the PBM of healthy women and the PBM of the CaBr patients showed overexpression of CCR1 and downregulation of CCR5.     

Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro

Purpose  The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-α 2B secreting recombinant (r) BCG (rBCG-IFN-α). We demonstrated that rBCG-IFN-α augmented T helper type 1 (Th1) cytokine IFN-γ production by PBMC. In this study, we further investigated whether rBCG-IFN-α could also enhance PBMC cytotoxicity toward bladder cancer cells. Materials and methods  PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-α or control MV261 BCG, and used as effector cells in ⁵¹Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-α as well as endogenously expressed IFN-γ and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2. To determine the role of natural killer (NK) and CD8⁺ T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in ⁵¹Cr-release assays. Results  Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-α further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-γ and IL-2 by PBMC was observed after rBCG-IFN-α stimulation. Blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during rBCG-IFN-α stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8⁺ T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant. Conclusions  rBCG-IFN-α is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.     

Peripheral natural killer cell activity and intraperitoneal soluble p55 tumor necrosis factor receptor in patients with malignant ascites: two possible indicators for response to intraperitoneal combined tumor necrosis factor α and interferon γ treatment

 Tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are important immunomodulators. They are capable of acting in a synergistic manner on tumor cells in vitro and in vivo. In a clinical phase I study 13 patients with malignant ascites due to abdominal spread of different primary tumors received intraperitoneally (i. p.) TNFα and IFNγ once weekly over 3 – 8 weeks in order to evaluate the effect of locoregionally administered TNFα/IFNγ on ascites formation. Therefore some peripheral and local immunological functional parameters of peripheral blood and malignant ascites were investigated. Mononuclear lymphocytes and natural killer (NK) cell activity of peripheral blood and ascites, TNF-inhibitory activity, soluble p55 and p75 TNF receptors, and prostaglandin E₂ values in ascites were measured immediately before and 24 h after each TNFα/IFNγ infusion. Peripheral mononuclear lymphocytes and NK activity decreased significantly 24 h after i. p. TNFα/IFNγ application. However, over the entire treatment schedule, peripheral NK activity in all responders showed a continuous increase, when compared to pre TNFα/IFNγ treatment levels. In contrast, NK activity in non-responders constantly decreased. In contrast to non-responders, TNF-inhibitory activity and soluble p55 TNF receptor levels, determined in ascites, decreased in responders. Taken together, our findings suggest, that successful locoregional i. p. TNFα/IFNγ therapy induces systemic immunological reactions possibly after saturation of soluble p55 TNF receptors in ascites, which leads to an increase of peripheral NK activity. Received: 28 September 1995 / Accepted: 16 November 1995     

Interactions between IL-32 and tumor necrosis factor alpha contribute to the exacerbation of immune-inflammatory diseases

IL-32 is a newly described cytokine in the human found to be an in vitro inducer of tumor necrosis factor alpha (TNFα). We examined the in vivo relationship between IL-32 and TNFα, and the pathologic role of IL-32 in the TNFα-related diseases – arthritis and colitis. We demonstrated by quantitative PCR assay that IL-32 mRNA was expressed in the lymphoid tissues, and in stimulated peripheral T cells, monocytes, and B cells. Activated T cells were important for IL-32 mRNA expression in monocytes and B cells. Interestingly, TNFα reciprocally induced IL-32 mRNA expression in T cells, monocyte-derived dendritic cells, and synovial fibroblasts. Moreover, IL-32 mRNA expression was prominent in the synovial tissues of rheumatoid arthritis patients, especially in synovial-infiltrated lymphocytes by in situ hybridization. To examine the in vivo relationship of IL-32 and TNFα, we prepared an overexpression model mouse of human IL-32β (BM-hIL-32) by bone marrow transplantation. Splenocytes of BM-hIL-32 mice showed increased expression and secretion of TNFα, IL-1β, and IL-6 especially in response to lipopolysaccharide stimulation. Moreover, serum TNFα concentration showed a clear increase in BM-hIL-32 mice. Cell-sorting analysis of splenocytes showed that the expression of TNFα was increased in resting F4/80⁺ macrophages, and the expression of TNFα, IL-1β and IL-6 was increased in lipopolysaccharide-stimulated F4/80⁺ macrophages and CD11c⁺ dendritic cells. In fact, BM-hIL-32 mice showed exacerbation of collagen-antibody-induced arthritis and trinitrobenzen sulfonic acid-induced colitis. In addition, the transfer of hIL-32β-producing CD4⁺ T cells significantly exacerbated collagen-induced arthritis, and a TNFα blockade cancelled the exacerbating effects of hIL-32β. We therefore conclude that IL-32 is closely associated with TNFα, and contributes to the exacerbation of TNFα-related inflammatory arthritis and colitis.     

Limited importance of CD40/CD40L interaction in the B7-dependent generation of anti-MOPC-315 cytotoxic T lymphocyte activity by tumor bearer splenic cells stimulated in vitro in the presence of tumor necrosis factor

 We have previously illustrated the importance of B7-2 expression for the enhanced generation of cytotoxic T lymphocyte (CTL) activity by stimulation cultures of tumor bearer splenic cells to which tumor necrosis factor α (TNFα) has been added. Here we show that the B7-1 molecule is also important for CTL generation by such stimulation cultures, although to a much lesser extent than the B7-2 molecule. In addition, we show the importance of CD40/CD40L interaction for the expression of the B7-2 molecule, but not the B7-1 molecule, by tumor bearer splenic cells stimulated in vitro in the presence of TNF. The CD40/CD40L interaction is also shown to be important for the generation of CTL activity by tumor bearer splenic cells stimulated in vitro in the presence of exogenous TNF. However, the CD40/CD40L interaction is less important for the generation of enhanced CTL activity than for the expression of an elevated level of B7-2. Specifically, blockade of CD40/CD40L interaction, which reduced the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the presence of TNF to the level of B7-2 expressed by tumor bearer splenic cells stimulated in vitro in the absence of exogenous TNF, failed to reduce the level of CTL generated to the level generated by tumor bearer splenic cells stimulated in the absence of exogenous TNF. Finally, blockade of CD40/CD40L interaction was inferior to blockade of B7-2/CD28 interaction in inhibiting the generation of CTL activity by tumor bearer splenic cells stimulated in the presence of exogenous TNF. Thus, although CD40/CD40L interaction is important for the generation of enhanced CTL activity by stimulation cultures of tumor bearer splenic cells to which TNF has been added, TNF also mediates its potentiating effect for CTL generation by such stimulation cultures via other mechanisms that are independent of CD40/CD40L interaction but dependent on B7-2 expression. Received: 31 December 1997 / Accepted: 27 March 1998     

Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways

A sequence of intermittent interruptions of oxygen supply (i.e., postconditioning, Postcon) at reoxygenation reduces oxidant-induced cardiomyocyte loss. This study tested the hypothesis that prevention of cardiomyocyte apoptosis by Postcon is mediated by mitogen-activated protein kinases pathways. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Relative to hypoxia alone, reoxygenation stimulated expression of JNKs and p38 kinases, corresponding to increased activity of JNKs (phospho-c-Jun) and p38 (phospho-ATF2). The level of TNFα in cell lysates, activity of cytosolic caspases-8, -3, expression of Bax and the number of apoptotic cardiomyocytes were increased while expression of Bcl-2 was decreased with reoxygenation. Consistent with an attenuation in generation of superoxide anions detected by lucigenin-enhanced chemiluminescence at early period of reoxygenation, treatment of cardiomyocytes with Postcon further reduced expression and activity of JNKs and p38 kinases, level of TNFα, the frequency of apoptotic cells and expression of Bax. However, the inhibitory effects of Postcon on these changes were lost when its application was delayed by 5 min after the start of reoxygenation. Addition of a JNK/p38 stimulator, anisomycin into cardiomyocytes at the beginning of reoxygenation eliminated protection by Postcon. These data suggest that 1) hypoxia/reoxygenation elicits cardiomyocyte apoptosis in conjunction with expression and activation of JNK and p38 kinases, release of TNFα, activation of caspases, and an increase in imbalance of pro-/anti-apoptotic proteins; 2) Postcon attenuates cardiomyocyte apoptosis, potentially mediated by inhibiting JNKs/p-38 signaling pathways and reducing TNFα release and caspase expression.     

Prediction of mutant activity and its application in molecular design of tumor necrosis factor-a

Two models for prediction of the activity and stability of site-directed mutagenesis on tumor necrosis factor-α are established. The models are based on straightforward structural considerations, which do not require the elaboration of sitedirected mutagenesis on the protein core and the hydrophobic surface area by analyzing the pmperties of the mutated amino acid residues. The reliabilities of the models have been tested by analyzing the mutants of tumor necrosis factor-α (TNF-α) whose two leucine residues (L29, L157) were mutated. Based on these models, a TNFα mutant with high activity was created by molecular design. Project supported by the Chinese National High Technology Development Program.