Clifford J. Bellone

Proinflammatory Stimuli Induce IKKα-Mediated Phosphorylation of PIAS1 to Restrict Inflammation and Immunity

How inflammatory stimuli signal to the nucleus to restrict inflammation is poorly understood. Protein inhibitor of activated STAT1 (PIAS1), a transcriptional regulator that possesses small ubiquitin-related modifier (SUMO) E3 ligase activity, inhibits immune responses by selectively blocking the binding of NF-kappaB and STAT1 to gene promoters. We report here that PIAS1 becomes rapidly phosphorylated on Ser90 residue in response to various inflammatory stimuli. Mutational studies indicate that Ser90 phosphorylation is required for PIAS1 to repress transcription. Upon TNF treatment, wild-type PIAS1, but not the Ser90A mutant, becomes rapidly associated with the promoters of NF-kappaB target genes. Furthermore, IKKalpha, but not IKKbeta, interacts with PIAS1 in vivo and mediates PIAS1 Ser90 phosphorylation, a process that requires the SUMO ligase activity of PIAS1. Our results identify a signaling pathway in which proinflammatory stimuli activate the IKKalpha-mediated sumoylation-dependent phosphorylation of PIAS1 for the immediate repression of inflammatory gene activation.

RhoA stimulates p27(Kip) degradation through its regulation of cyclin E/CDK2 activity.

RhoA has been identified as an important regulator of cell proliferation. We recently showed that the Ras/RhoA pathway regulates the degradation of p27Kip and the progression of Chinese hamster embryo fibroblasts (IIC9 cells) through G1 into S phase (Weber, J. D., Hu, W., Jefcoat, S. C., Raben, D. M., and Baldassare, J. J. (1997) J. Biol. Chem.272, 32966–32971). In this report, we have demonstrated that, in IIC9 cells, RhoA regulates cyclin E/CDK2 activity, which is required for p27Kip degradation. As previously shown in several fibroblasts cell lines, expression of dominant-negative CDK2 in IIC9 cells blocked serum-induced cyclin E/CDK2 activity and p27Kip degradation. In the absence of serum, expression of constitutively active RhoA(63) resulted in significant stimulation of cyclin E/CDK2 activity and degradation of p27Kip. Cotransfection of dominant-negative CDK2 and RhoA(63) inhibited RhoA(63)-induced cyclin E/CDK2 activity and p27Kipdegradation. In addition, expression of dominant-negative RhoA blocked serum-induced cyclin E/CDK2 activity and p27Kipdegradation. Finally, expression of catalytically active cyclin E/CDK2 rescued the effect of expression of dominant-negative RhoA. Taken together, these data show that RhoA regulates p27Kipdegradation through its regulation of cyclin E/CDK2 activity.

Immunopathogenesis of allergic bronchopulmonary aspergillosis in cystic fibrosis

Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity lung disease mediated by an allergic late-phase inflammatory response to Aspergillus fumigatus antigens. ABPA is characterized by markedly elevated Aspergillus-specific and total IgE levels and eosinophilia, and manifested by wheezing, pulmonary infiltrates, and bronchiectasis and fibrosis, which afflict asthmatic and cystic fibrosis (CF) patients. We propose that ABPA develops in genetically susceptible CF patients due to HLA-DR2 and DR5 restriction, increased sensitivity to IL-4 stimulation, and increased A. fumigatus allergen-specific Th2 CD4+ T-cell-mediated responses. In addition, A. fumigatus proteases play a role in facilitation of antigen transport across the epithelial cell layer by damaging the epithelial integrity and by a direct interaction with epithelial cell surface receptors, resulting in pro-inflammatory cytokine production and corresponding inflammatory responses.

Effect of estrogens on IL-1β promoter activity

Abstract It is well documented that steroid hormones modulate cytokine gene expression. In some tissues estrogens are known to suppress cytokine production while in other tissue types, cytokine expression is enhanced by the hormone. This study was conducted to investigate the regulatory mechanisms which underlie the modulation of the interleukin-1 β (IL-1 β ) gene at the transcription level. To accomplish this, the macrophage cell line RAW264.7, which appeared insensitive to 17 β -estradiol (E 2 ) treatment, was stably transfected with the human estrogen receptor (ER) and an IL-1 β promoter–CAT reporter construct. E 2 markedly enhanced LPS-induced IL-1 β promoter-driven CAT activity in an E 2 dose dependent manner. This responsiveness was estrogen specific since no synergism was observed between LPS and the sex steroids testosterone or progesterone while the estrogen analogue 17 α -estradiol stimulated only at 10 to 100 times the amount required for 17 β -E 2 . Several antiestrogens, H1285, ICI 182 780, and tamoxifen inhibited the estrogen stimulated enhancement of IL-1 β promoter activity in a dose-dependent manner, indicating that this effect was indeed mediated through the ER in a ligand dependent manner. The estrogenic effect appeared to be indirect and time dependent since the addition of E 2 was required hours prior to LPS stimulation; addition of E 2 and LPS at the same time resulted in a greatly reduced estrogenic effect. The estrogen metabolites 17-epiestriol and 16-keto-17 β -E 2 displayed an estrogenic response virtually indistinguishable from E 2 . 4-Hydroxyestradiol displayed activity only at 100-fold the concentration of E 2 while 2-hydroxyestrone showed no activity at any of the concentrations tested. Overall the results demonstrate that E 2 and some metabolites of E 2 synergize with LPS to markedly enhance IL-1 β promoter activity through ER mediated processes.

Effect of environmental estrogens on IL-1β promoter activity in a macrophage cell line

Environmental estrogens or estrogen disrupters have recently received a great deal of attention because of their potential health impact on reproductive tissues. Few, if any, studies have been made on the impact of these compounds on the immune system. We sought to determine the activities of various environmental estrogens on the modulation of the interleukin-1β (IL-1β) gene in a model monocytic cell line, hER+IL-1β-CAT+. This cell line stably transfected with the human estrogen receptor, and an IL-1β promoter construct fused to the CAT reporter gene allows us to monitor the effect of estrogenic compounds on IL-1β promoter activity. 17β-Estradiol (E2) markedly enhanced lipopolysaccharide-(LPS) induced IL-1β promoter-driven CAT activity in a dose-dependent manner. The mycotoxins α-zearalenol and zearalenone both exhibited full agonist activity, but at lower potencies, with EC50 values of 1.8 and 54 nM, respectively, compared with E2 at 0.5 nM. In addition, genistein was a very low-potency agonist, having an EC50 of 1.5 µM. Similar to the E2 response, the slope factors for α-zearalenol, zearalenone, and genistein were close to 3.0, suggesting positive cooperativity in the estrogenic response. The activity of the mycotoxins appeared to be mediated through the estrogen receptor, since both the antiestrogens H1285 and ICI 182,780 effectively inhibited their agonist activity in a dose-dependent manner. Representative environmental estrogenic compounds both from plant and industrial sources were also tested. Unlike the mycoestrogens, none of the compounds, with the exception of genistein, synergized with LPS to enhance IL-1β promoter activity. When tested for antiestrogenic activity, the industrial compound 4-octylphenol was able to antagonize the response to E2; however, the response was three orders of magnitude less potent than H1285. Naringenin, a plant flavonoid, showed little or no ability to antagonize the response to E2. Overall, the results show that some environmental estrogens that display agonist activity in reproductive tissue also have an effect on IL-1 gene expression in hemopoietic-derived tissue.

Antibody Responses to Vaccinia Membrane Proteins after Smallpox Vaccination

BACKGROUND Vaccinia virus (VV) membrane proteins are candidates for orthopoxvirus subunit vaccines and potential targets for therapeutic antibodies. Human antibody responses to these proteins after VV vaccination have not been well characterized. METHODS Pre- and postvaccination (day 26-30) serum specimens from 80 VV vaccine recipients were examined for immunoglobulin G antibodies specific for B5, A33, A27, and L1 by enzyme-linked immunosorbent assay (ELISA). Responses were compared between vaccinia-naive and previously vaccinated (nonnaive) recipients and between nonnaive recipients of undiluted or 1 : 10 diluted vaccine. RESULTS VV vaccination elicited anti-A33 and anti-A27 antibodies in nearly all vaccinia-naive subjects (100% and 93%, respectively). Preexisting antibodies were commonly detected in nonnaive subjects (for anti-B5, 68%; for anti-A33, 59%; for anti-A27, 38%; and for anti-L1, 10%). Anti-B5 antibodies were strongly boosted by undiluted vaccine (geometric mean titer [GMT], 151 vs. 1010 for pre- vs. postvaccination; P<.001), whereas anti-L1 antibody responses were less robust (detection rate, 31%; GMT, 75) in nonnaive subjects. Diluted vaccine elicited antibody responses that were similar to those elicited by undiluted vaccine. CONCLUSIONS Vaccination with VV elicits long-lived specific antibody responses directed against VV membrane proteins that vary by previous vaccination status but not with respect to 10-fold dilution of vaccine. B5, A33, and A27 should be considered for inclusion in future human orthopoxvirus subunit vaccines.

Poxvirus interleukin-4 expression overcomes inherent resistance and vaccine-induced immunity: Pathogenesis, prophylaxis and antiviral therapy

In 2001, Jackson et al. reported that murine IL-4 expression by a recombinant ectromelia virus caused enhanced morbidity and lethality in resistant C57BL/6 mice as well as overcame protective immune memory responses. To achieve a more thorough understanding of this phenomenon and to assess a variety of countermeasures, we constructed a series of ECTV recombinants encoding murine IL-4 under the control of promoters of different strengths and temporal regulation. We showed that the ECTV-IL-4 recombinant expressing the highest level of IL-4 was uniformly lethal for C57BL/6 mice even when previously immunized. The lethality of the ECTV-IL-4 recombinants resulted from virus-expressed IL-4 signaling through the IL-4 receptor but was not due to IL-4 toxicity. A number of treatment approaches were evaluated against the most virulent IL-4 encoding virus. The most efficacious therapy was a combination of two antiviral drugs (CMX001(®) and ST-246(®)) that have different mechanisms of action.

Autoimmune Gastritis Mediated by CD4+ T Cells Promotes the Development of Gastric Cancer

Chronic inflammation is a major risk factor for cancer, including gastric cancers and other gastrointestinal cancers. For example, chronic inflammation caused by autoimmune gastritis (AIG) is associated with an increased risk of gastric polyps, gastric carcinoid tumors, and possibly adenocarcinomas. In this study, we characterized the progression of gastric cancer in a novel mouse model of AIG. In this model, disease was caused by CD4(+) T cells expressing a transgenic T-cell receptor specific for a peptide from the H(+)/K(+) ATPase proton pump, a protein expressed by parietal cells in the stomach. AIG caused epithelial cell aberrations that mimicked most of those seen in progression of human gastric cancers, including chronic gastritis followed by oxyntic atrophy, mucous neck cell hyperplasia, spasmolytic polypeptide-expressing metaplasia, dysplasia, and ultimately gastric intraepithelial neoplasias. Our work provides the first direct evidence that AIG supports the development of gastric neoplasia and provides a useful model to study how inflammation drives gastric cancer.

The new ACAM2000™ vaccine and other therapies to control orthopoxvirus outbreaks and bioterror attacks

Quarantine, case tracing and population vaccination facilitated the global eradication, in 1980, of variola virus, the causative agent of smallpox. The vaccines used during the eradication period, including Dryvax®, the smallpox vaccine used in the USA, were live vaccinia and cowpoxvirus-based vaccines, which induced long-lasting and cross-protective immunity against variola and other related poxviruses. These vaccine viruses were produced by serial propagation in domesticated animals. The drawbacks to such serially propagated live viral vaccines include the level of postvaccination local and systemic reactions and contraindications to their use in immunocompromised individuals, individuals with certain skin and cardiac diseases, and pregnant women. In the latter stages of the population-based smallpox vaccination campaign, research began with ways to improve safety and modernizing the manufacture of vaccinia vaccines; however, with the eradication of variola this work stopped. Outbreaks of monkeypoxvirus in humans and the bioterrorist threat of monkeypox and variola virus renewed the need for improved vaccinia vaccines. ACAM2000™ is one of the new generation of smallpox vaccines. It is produced in cell culture from a clonally purified master seed stock of vaccinia derived from the New York City Board of Health strain of vaccinia. The clonally purified master seed assures a more homogeneous vaccine without the inherent mutations associated with serial propagation and the cell culture limits adventitious and bacterial contamination in vaccine production. In preclinical and clinical trials, ACAM2000 demonstrated an immunogenicity and safety profile similar to that of Dryvax.

Regulation of IL-1 gene expression: Differential responsiveness of murine macrophage lines

In order to begin to define the mechanisms by which lipopolysaccharide (LPS) regulates IL-1 gene expression, we have examined IL-1 RNA levels, the transcription rate of the IL-1 genes, and IL-1 mRNA stabilities in P388D1/C, RAW264.7, and murine peritoneal exudate cells (PEC). These experiments showed that total cellular IL-1 RNA levels and IL-1 transcription rates were dramatically upregulated in all three cell types. In all cases, IL-1 alpha and IL-1 beta cellular RNA levels and gene transcription rates were regulated in parallel. However, the profiles of IL-1 gene activation during the 24 h after LPS treatment differed in these three cell types. Additionally, culture in the presence of actinomycin D (Act D) showed differential stabilities of the IL-1 alpha and IL-1 beta RNAs in these cells. In peritoneal exudate cells, the half-lives (t1/2) of the IL-1 alpha and IL-1 beta RNAs were each > 8 h. In RAW264.7 cells, the stability of the IL-1 beta RNA was greater than the IL-1 alpha RNA (t1/2 > 8 h and approximately 6 h, respectively). In P388D1/C cells, the t1/2s of the IL-1 alpha and beta RNAs varied depending on the time of addition of actinomycin D. This and other data suggest that components of the IL-1 RNA catabolic pathway are labile and sensitive to treatment with actinomycin D. Together these data indicate that the two IL-1 genes show a diverse regulatory repertoire, even within related mononuclear phagocytic cells.