Charles S. Tannenbaum

Tissue-specific expression of murine IP-10 mRNA following systemic treatment with interferon γ

We have examined the tissue distribution of 10‐kd inflammatory protein (IP‐10) mRNA expression in C57Bl/6 mice injected intravenously (i.v.) with various inflammatory stimuli. IP‐10 mRNA was strongly induced by interferon‐γ (IFN‐γ) in liver and kidney but only poorly in skin, heart, and lung. IFN‐γ had nearly equivalent access to these tissues as indicated by the distribution of radiolabeled recombinant IFN‐γ 1 h after injection. The time course of IP‐10 mRNA appearance was rapid and transient in both liver and kidney; maximal expression in the liver (2 h) preceded that in the kidney (3 h) and declined rapidly thereafter in both tissues. Expression of IP‐10 mRNA in the liver and kidney was highly sensitive to IFN‐γ treatment; nearly maximal stimulation occurred with injection of 500 U of IFN‐γ per mouse. Comparable stimulation of IP‐10 mRNA expression in splenic macrophages required 10,000 U of IFN‐γ administered i.v., indicating that liver and kidney responses are 10‐ to 20‐fold more sensitive. IP‐10 mRNA expression in both tissues was not restricted to stimulation by IFN‐γ but was also seen with injection of lipopolysaccharide (LPS) (25 μg/mouse) or IFN‐β (100,000 U/mouse). Two other members of the IP‐10 gene family, KC (gro) and JE (MCP‐1), were expressed at lower levels under similar treatment conditions. Analysis of IP‐10 mRNA distribution in the liver and kidney by in situ hybridization indicated that expression in both tissues was most prominent in the reticuloendothelial cell system, particularly in the endothelial lining of the microvascular circulation. Although the function of the IP‐10 gene product has not been defined, these results suggest that it may play an important role in the response of both the liver and kidney to systemic inflammation.

Tumor-Induced Oxidative Stress Perturbs Nuclear Factor-κB Activity-Augmenting Tumor Necrosis Factor-α–Mediated T-Cell Death: Protection by Curcumin

Cancer patients often exhibit loss of proper cell-mediated immunity and reduced effector T-cell population in the circulation. Thymus is a major site of T-cell maturation, and tumors induce thymic atrophy to evade cellular immune response. Here, we report severe thymic hypocellularity along with decreased thymic integrity in tumor bearer. In an effort to delineate the mechanisms behind such thymic atrophy, we observed that tumor-induced oxidative stress played a critical role, as it perturbed nuclear factor-kappaB (NF-kappaB) activity. Tumor-induced oxidative stress increased cytosolic IkappaBalpha retention and inhibited NF-kappaB nuclear translocation in thymic T cells. These NF-kappaB-perturbed cells became vulnerable to tumor-secreted tumor necrosis factor (TNF)-alpha (TNF-alpha)-mediated apoptosis through the activation of TNF receptor-associated protein death domain-associated Fas-associated protein death domain and caspase-8. Interestingly, TNF-alpha-depleted tumor supernatants, either by antibody neutralization or by TNF-alpha-small interfering RNA transfection of tumor cells, were unable to kill T cell effectively. When T cells were overexpressed with NF-kappaB, the cells became resistant to tumor-induced apoptosis. In contrast, when degradation-defective IkappaBalpha (IkappaBalpha super-repressor) was introduced into T cells, the cells became more vulnerable, indicating that inhibition of NF-kappaB is the reason behind such tumor/TNF-alpha-mediated apoptosis. Curcumin could prevent tumor-induced thymic atrophy by restoring the activity of NF-kappaB. Further investigations suggest that neutralization of tumor-induced oxidative stress and restoration of NF-kappaB activity along with the reeducation of the TNF-alpha signaling pathway can be the mechanism behind curcumin-mediated thymic protection. Thus, our results suggest that unlike many other anticancer agents, curcumin is not only devoid of immunosuppressive effects but also acts as immunorestorer in tumor-bearing host.

Degradation of NF-κB in T Cells by Gangliosides Expressed on Renal Cell Carcinomas

T cells from cancer patients are often functionally impaired, which imposes a barrier to effective immunotherapy. Most pronounced are the alterations characterizing tumor-infiltrating T cells, which in renal cell carcinomas includes defective NF-κB activation and a heightened sensitivity to apoptosis. Coculture experiments revealed that renal tumor cell lines induced a time-dependent decrease in RelA(p65) and p50 protein levels within both Jurkat T cells and peripheral blood T lymphocytes that coincided with the onset of apoptosis. The degradation of RelA/p50 is critical for SK-RC-45-induced apoptosis because overexpression of RelA in Jurkat cells protects against cell death. The loss of RelA/p50 coincided with a decrease in expression of the NF-κB regulated antiapoptotic protein Bcl-xL at both the protein and mRNA level. The disappearance of RelA/p50 protein was mediated by a caspase-dependent pathway because pretreatment of T lymphocytes with a pan caspase inhibitor before coculture with SK-RC-45 blocked RelA and p50 degradation. SK-RC-45 gangliosides appear to mediate this degradative pathway, as blocking ganglioside synthesis in SK-RC-45 cells with the glucosylceramide synthase inhibitor, PPPP, protected T cells from tumor cell-induced RelA degradation and apoptosis. The ability of the Bcl-2 transgene to protect Jurkat cells from RelA degradation, caspase activation, and apoptosis implicates the mitochondria in these SK-RC-45 ganglioside-mediated effects.

A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes

We previously reported the isolation and characterization of cDNA clones encoding novel lipopolysac‐ charide (LPS)‐inducible mRNAs from murine peritoneal macrophages. We now present the complete coding sequence of a cDNA previously termed D3. Analysis of multiple clones from a murine macrophage cDNA library provided a complete cDNA sequence of approximately 1.6 kb. The corresponding RNA contains a single open reading frame encoding a hydrophilic protein composed of 425 amino acids and is characterized by a region including three perfect and two imperfect repeats of a seven‐ amino‐acid sequence. Based on nucleotide and deduced amino acid sequence, this mRNA is a new member of a previously described multigene cluster of interferon‐ inducible genes termed the Mouse 200 series genes. This new sequence most closely resembles gene 204 because both D3 and 204 genes have segments containing the seven‐amino‐acid repeat sequence. The Mouse 202 and 204 genes, however, have an approximately 200‐amino‐ acid carboxyl‐terminal domain that is absent in the LPS‐ inducible macrophage‐derived cDNA. In addition, D3, 202, and 204 can all be distinguished from one another by virtue of unique 3′ noncoding regions 200‐300 base pairs in length. The D3 unique sequence is largely restricted to the smallest of the three size classes of this gene family expressed in macrophages and is not detected in interferon‐ or platelet‐derived growth factor‐stimulated fibroblasts. Overall, three separate mRNAs have now been described, each of which has three or more of a possible seven nucleotide sequence domains. Although the functions) of the members of this gene family remains unknown, the multiple forms inducible by diverse stimuli and their restricted cell type expression suggest diverse and important physiologic roles for their products in inflammation.

CXC Chemokine Ligand 9/Monokine Induced by IFN-γ Production by Tumor Cells Is Critical for T Cell-Mediated Suppression of Cutaneous Tumors

The role of tumor-produced chemokines in the growth of malignancies remains poorly understood. We retrieved an in vivo growing MCA205 fibrosarcoma and isolated tumor cell clones that produce both CXCL9/monokine induced by IFN-γ (Mig) and CXCL10/IFN-γ-inducible protein 10 following stimulation with IFN-γ and clones that produce IFN-γ-inducible protein 10 but not Mig. The Mig-deficient variants grew more aggressively as cutaneous tumors in wild-type mice than the Mig-producing tumor cells. The growth of Mig-expressing, but not Mig-deficient, tumor cells was suppressed by NK and T cell activity. Transduction of Mig-negative variants to generate constitutive tumor cell production of Mig resulted in T cell-dependent rejection of the tumors and in induction of protective tumor-specific CD8+ T cell responses to Mig-deficient tumors. The results indicate a critical role for tumor-derived Mig in T cell-mediated responses to cutaneous fibrosarcomas and suggest the loss of Mig expression as a mechanism used by tumor cells to evade these responses.

GM1 and Tumor Necrosis Factor-α, Overexpressed in Renal Cell Carcinoma, Synergize to Induce T-Cell Apoptosis

The ability to induce T-cell apoptosis is one mechanism by which tumors evade the immune system, although the molecules involved remain controversial. We found that renal cell carcinoma (RCC)-induced T-cell apoptosis was inhibited by >50% when cocultures were performed with ganglioside-depleted tumor cells, caspase-8-negative lymphocytes, or anti-tumor necrosis factor-alpha (TNFalpha) antibodies, suggesting that tumor gangliosides synergize with signals delivered through TNFalpha death receptors to mediate T-cell killing. The synergy between tumor-derived TNFalpha and the RCC-overexpressed ganglioside GM1 for killing resting T cells is corroborated by studies using purified GM1 and rTNF alpha, which indicate that a 48-hour pretreatment with the ganglioside optimally sensitizes the lymphocytes to a TNFalpha-induced apoptotic death. However, activated T cells, which synthesize TNFalpha themselves, can be killed by exogenous GM1 alone. RelA-overexpressing lymphocytes are protected from GM1 plus TNFalpha-mediated apoptosis, a finding consistent with our previous studies indicating that gangliosides inhibit nuclear factor-kappaB activation. These results are clinically relevant because, similar to T-cells cocultured with GM1-overexpressing RCC lines, T cells isolated from the peripheral blood of patients with metastatic RCC are also heavily coated with that tumor-shed ganglioside. This population of patient cells, unlike T cells isolated from normal donors, is highly susceptible to apoptosis induced by rTNF alpha or by metastatic patient sera, which contain elevated levels of the cytokine. This report thus extends our previous studies by demonstrating that tumor-derived TNFalpha enhances RCC apoptogenicity not only by inducing ganglioside synthesis but also by initiating receptor-dependent apoptosis in T cells in which the nuclear factor-kappaB activation pathway has been inhibited by GM1.

The early competence genes JE and KC are differentially regulated in murine peritoneal macrophages in response to lipopolysaccharide.

Treatment of murine peritoneal macrophages with bacterial lipopolysaccharide (LPS) has been previously documented to induce accumulation of mRNA for the early or competence genes JE and KC; the data further suggested that multiple pathways existed for the transduction of the LPS signal, since induction of mRNA for JE was related to breakdown of polyphosphoinositides while induction of KC was not (Introna et al. 1987 J. Immunol. 138, 3891). This study provides analysis of the regulation of the expression of these genes by using the nuclear transcription assay. We present evidence that LPS enhanced transcriptional activity of the KC gene, but not of the JE gene. By contrast, serum stimulation of quiescent BALB/c-3T3 fibroblasts induced transcription of the JE and KC genes. The data imply that expression of the KC gene in LPS-treated macrophages is regulated transcriptionally, while that of the JE gene is regulated post-transcriptionally. Furthermore, there appear to be two mechanistic pathways for the induction of JE mRNA depending upon the stimulus and upon the cell type: one involving transcriptional and one post-transcriptional control.