Randle M. Gallucci

Discriminators of mouse bladder response to intravesical Bacillus Calmette-Guerin (BCG)

BackgroundIntravesical Bacillus Calmette-Guerin (BCG) is an effective treatment for bladder superficial carcinoma and it is being tested in interstitial cystitis patients, but its precise mechanism of action remains poorly understood. It is not clear whether BCG induces the release of a unique set of cytokines apart from its pro-inflammatory effects. Therefore, we quantified bladder inflammatory responses and alterations in urinary cytokine protein induced by intravesical BCG and compared the results to non-specific pro-inflammatory stimuli (LPS and TNF-α). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder.MethodsC57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-α. Morphometric analyses were conducted in bladders isolated from all groups and urine was collected for multiplex analysis of 18 cytokines. In addition, chromatin immune precipitation combined with real-time polymerase chain reaction assay (CHIP/Q-PCR) was used to test whether intravesical BCG would alter bladder cytokine gene expression.ResultsAcute BCG instillation induced edema which was progressively replaced by an inflammatory infiltrate, composed primarily of neutrophils, in response to weekly administrations. Our morphological analysis suggests that these polymorphonuclear neutrophils are of prime importance for the bladder responses to BCG. Overall, the inflammation induced by BCG was higher than LPS or TNF-α treatment but the major difference observed was the unique granuloma formation in response to BCG. Among the cytokines measured, this study highlighted the importance of IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-17, GM-CSF, KC, and Rantes as discriminators between generalized inflammation and BCG-specific inflammatory responses. CHIP/Q-PCR indicates that acute BCG instillation induced an up-regulation of IL-17A, IL-17B, and IL-17RA, whereas chronic BCG induced IL-17B, IL-17RA, and IL-17RB.ConclusionTo the best of our knowledge, the present work is the first to report that BCG induces an increase in the IL-17 family genes. In addition, BCG induces a unique type of persisting bladder inflammation different from TNF-α, LPS, and, most likely, other classical pro-inflammatory stimuli.

Differential Expression of Liver Interleukin-6 Receptor-?? in Female Versus Male Ethanol-Consuming Rats

BACKGROUND It is well known that women are more susceptible to alcoholic liver disease (ALD) than men, and inflammation is thought to play a major role in alcohol-induced liver injury. Increased circulating levels of the proinflammatory cytokine interleukin (IL)-6 are a marker for serious ALD in humans. However, IL-6 also has protective effects, such as induction of liver regeneration and inhibition of hepatocyte apoptosis. Although the roles of IL-6 in ALD have begun to be established, little is known about the expression of its receptor (IL-6Ralpha) during chronic alcohol administration. METHODS Male and female rats were intragastrically fed ethanol or control isocaloric liquid diet for 2 and 4 weeks. Liver samples were collected, and gene expression was assessed by reverse transcription-polymerase chain reaction and Western blot. RESULTS Herein, we show clear gender differences in alcohol-induced liver IL-6Ralpha expression. Analysis of rat liver samples showed that ethanol consumption significantly increased IL-6Ralpha messenger RNA and protein expression in females as compared with similarly treated males after 2 and 4 weeks. Increased STAT3 phosphorylation in the livers of ethanol-consuming females also indicated greater IL-6Ralpha activation in these animals. Conversely, ethanol-consuming males displayed increased IkappaB messenger RNA and protein expression, which may inhibit IL-6R expression, compared with females. CONCLUSIONS Given the association of inflammation with ethanol-induced liver damage, these data may offer insight into a possible mechanism by which females develop more severe ALD than males.

IL-6 deficiency exacerbates skin inflammation in a murine model of irritant dermatitis

Contact dermatitis is the second most reported occupational injury associated with workers compensation. Inflammatory cytokines are closely involved with the development of dermatitis, and their modulation could exacerbate skin damage, thus contributing to increased irritancy. IL-6 is a pro-inflammatory cytokine paradoxically associated with both skin healing and inflammation. To determine what role this pleiotropic cytokine plays in chemically-induced irritant dermatitis, IL-6 deficient (KO), IL-6 over-expressing transgenic (TgIL6), and corresponding wild-type (WT) mice were exposed to acetone or the irritants JP-8 jet fuel or benzalkonium chloride (BKC) daily for 7 days. Histological analysis of exposed skin was performed, as was tissue mRNA and protein expression patterns of inflammatory cytokines via QPCR and multiplex ELISA. The results indicated that, following JP-8 exposure, IL-6KO mice had greatly increased skin IL-1β, TNFα, CCL2, CCL3, and CXCL1 mRNA and corresponding product protein expression when compared to that of samples from WT counterparts and acetone-exposed control mice. BKC treatment induced the expression of all cytokines examined as compared to acetone, with CCL2 significantly higher in skin from IL-6KO mice. Histological analysis showed that IL-6KO mice displayed significantly more inflammatory cell infiltration as compared to WT and TgIL6 mice in response to jet fuel. Analysis of mRNA for the M2 macrophage marker CD206 indicated a 4-fold decrease in skin of IL-6KO mice treated with either irritant as compared to WT. Taken together, these observations suggest that IL-6 acts in an anti-inflammatory manner during irritant dermatitis, and these effects are dependent on the chemical nature of the irritant.

Serum Profiling of Rat Dermal Exposure to JP-8 Fuel Reveals an Acute-Phase Response

ABSTRACT Dermal exposure to JP-8 petroleum jet fuel leads to toxicological responses in humans and rodents. Serum profiling is a molecular analysis of changes in the levels of serum proteins and other molecules in response to changes in physiology. This present study utilizes serum profiling approaches to examine biomolecular changes in the sera of rats exposed to dermal applications of JP-8 (jet propulsion fuel-8). Using gel electrophoresis and electrospray ionization (ESI) mass spectrometry (MS), levels of serum proteins as well as low-mass constituents were found to change after dermal exposures to JP-8. The serum protein levels altered included the acute-phase response proteins haptoglobin, ceruloplasmin, α1-inhibitor III, and apolipoprotein A-IV. Haptoglobin levels increased after a 1-day JP-8 dermal exposure and continued to increase through 7 days of exposure. Ceruloplasmin levels increased after 5 days of exposure. Serum α1-inhibitor III was reduced after a 1-day exposure and the depletion continued after 7 days of exposure. Apolipoprotein A-IV increased after a 1-day exposure and then returned to basal levels after 3- and 5-day exposures of JP-8. Levels of the acute-phase protein α2-macroglobulin were found to not vary over these time course studies. Using ESI-MS analysis directly on the sera from rats exposed to dermal JP-8, low-mass sera constituents were found to correlate with control (acetone) or JP-8 exposure.

Stress induced in heart and other tissues by rat dermal exposure to JP-8 fuel

Limited information is available regarding the development of systemic organ stress by dermal exposure to JP-8 fuel. In this study, the systemic stress potential of this fuel is evaluated in a rat model subjected to dermal applications of JP-8 for 7 days at 300 μl per day. Tissue histology indicated that JP-8 induces morphological alterations that suggest that tissue stress in the heart is more substantial than stress in the kidney and liver. Immunoblot analysis of tissues revealed increased levels of the inducible heat shock protein 70 (HSP70) in the heart, kidney, and liver after this dermal JP-8 exposure. This exposure also leads to increased levels of heme oxygenase-1 (HO-1/HSP3) in the liver. Additionally during this exposure, a negative regulator of inflammation, IκBα (inhibitor of NF-κB), was increased in the liver, slightly increased in the kidney, and not increased in the heart. Two regions of the rat brain were also examined and HSP70 and IκBα were increased in the cerebellum but not significantly increased in the cortex. This study indicates dermal JP-8 exposure causes systemic alterations that are associated with cytoprotective activities (e.g., in the liver) as well as potentially toxic mechanisms (heart and kidney).

Transcriptional profiling of irritant contact dermatitis (ICD) in a mouse model identifies specific patterns of gene expression and immune-regulation

BACKGROUND Irritant contact dermatitis (ICD) is a cutaneous inflammatory response to a variety of triggers that requires no sensitization and accounts for up to 80% of occupational dermatitis cases. IL-6 has been alternately associated with both allergic and irritant dermatitis and is closely linked to skin wound healing, therefore making it an ideal candidate to investigate in the mechanism of ICD. RESULTS Despite being a well-known pro-inflammatory cytokine, IL-6 deficient (IL-6KO) mice show much more severe ICD than controls. Transcriptome analysis was employed to examine irritant-exposed and control skin samples from C57BL/6 and IL-6KO mice. Over 1900 transcripts were found differentially modulated between C57 (1184 total) and IL-6KO (802 total) mice with the magnitude of expression significantly disparate. Overall gene ontology revealed metabolic and cellular enriched functional processes but numerous pro-inflammatory and immune associated genes (Cxcl2, Cxcl3, Cxcl5, Acod, Hamp, c-Lectins, for example), keratin associated genes (Krt6b and various Krtaps), and members of the Sprr and Lce family, which promote skin barrier integrity and keratinocyte functions, were also differentially modulated. CONCLUSIONS The altered expression of these genes may provide a potential mechanism to explain the increased ICD severity in IL-6-deficient mice. Overall, this study offers new insight into the pathogenesis of ICD, indicates new mediators/biomarkers that may influence the variability of responses to irritants and provides potential targets for therapeutic development.

Interleukin (IL)-6 modulates transforming growth factor-β receptor I and II (TGF-βRI and II) function in epidermal keratinocytes

It been shown that IL‐6 modulates TGF‐β1 expression in fibroblasts, however, what role IL‐6 plays concerning TGF‐βR expression and function in skin is unknown. Therefore, the aim of this study was to investigate the mechanism by which IL‐6 might modulates TGF‐β receptors in skin. Skin from WT, IL‐6 over‐expressing mice and IL‐6 treated keratinocyte cultures was analysed for TGF‐βRI and TGF‐βRII expression via histology, PCR and flow cytometry. Receptor function was assessed by cell migration, bromodeoxyuridine (BrdU) proliferation assays, and Smad7 expression and Smad2/3 phosphorylation. Receptor localization within the membrane was determined by co‐immunoprecipitation. IL‐6 overexpression and treatment increased TGF‐βRII expression in the epidermis. IL‐6 treatment of keratinocytes induced TGF‐βRI and II expression and augmented TGF‐β1‐induced function as demonstrated through increased migration and decreased proliferation. Additionally, IL‐6 treatment of keratinocytes altered receptor activity as indicated by altered Smad2/3 phosphorylation and increased Smad7 and membrane localization. These results suggest that IL‐6 regulates keratinocyte function by modulating TGF‐βRI and II expression and signal transduction via trafficking of the receptor to lipid raft pools.