TNF-Alpha as an Initiator of Allodynia and Anxiety-Like Behaviors in a Preclinical Model of PTSD and Comorbid Pain.

Post-Traumatic Stress Disorder (PTSD) is a debilitating mental health disorder that occurs after exposure to a traumatic event. Patients with comorbid chronic pain experience affective distress, worse quality of life, and poorer responses to treatments for pain or PTSD than those with either condition alone. FDA-approved PTSD treatments are often ineffective analgesics, requiring additional drugs to treat co-morbid symptoms. Therefore, development of new treatment strategies necessitate a better understanding of the pathophysiology of PTSD and comorbid pain. The single prolonged stress (SPS) model of PTSD induces the development of persistent mechanical allodynia and thermal hyperalgesia. Increased Nociceptin/Orphanin FQ (N/OFQ) levels in serum and CSF accompany these exaggerated nociceptive responses, as well as increased serum levels of the pro-inflammatory cytokine tumor necrosis factor (TNF-α). Therefore, the primary goal was to determine the role of TNF-α in the development of SPS-induced allodynia/hyperalgesia and elevated serum and CNS N/OFQ using two approaches: TNF-α synthesis inhibition, and blockade with anti-TNF-α antibody that acts primarily in the periphery. Administration of TNF-α synthesis blocker, thalidomide (THL), immediately after SPS prevented increased TNF-α and development of allodynia and hyperalgesia. The THL effect lasted at least 21 days, well after thalidomide treatment ended (day 5). THL also prevented SPS-induced increases in serum N/OFQ and reversed regional N/OFQ mRNA expression changes in the CNS. Serum TNF-α increases detected at 4 and 24 h post SPS were not accompanied by blood brain barrier disruption. A single injection of anti-TNF-α antibody to male and female rats during the SPS procedure prevented the development of allodynia, hyperalgesia, and elevated serum N/OFQ, and reduced SPS-induced anxiety-like behaviors in males. Anti-TNFα treatment also blocked development of SPS-induced allodynia in females, and blocked increased hypothalamic N/OFQ in males and females. This suggests that a peripheral TNF-α surge is necessary for the initiation of allodynia associated with SPS, as well as the altered central and peripheral N/OFQ that maintains nociceptive sensitivity. Therefore, early alleviation of TNF-α provides new therapeutic options for investigation as future PTSD and co-morbid pain treatments.

Loss of Interleukin-6 Influences Transcriptional Immune Signatures and Alters Bacterial Colonization in the Skin.

The skin functions as a protective barrier to inhibit the entry of foreign pathogens, all the while hosting a diverse milieu of microorganisms. Over time, skin cells, immune cells, cytokines, and microbes interact to integrate the processes of maintaining the skin's physical and immune barrier. In the present study, the basal expression of two immunologically divergent mouse strains C57BL/6 and BALB/c, as well as a strain on the C57 background lacking IL-6, was characterized. Additionally, cutaneous antimicrobial gene expression profiles and skin bacterial microbiome were assessed between strains. Total RNA sequencing was performed on untreated C57BL/6 (control), BALB/c, and IL-6-deficient skin samples and found over 3,400 genes differentially modulated between strains. It was found that each strain modulated its own transcriptional "profile" associated with skin homeostasis and also influenced the overall bacterial colonization as indicated by the differential phyla present on each strain. Together, these data not only provide a comprehensive view of the transcriptional changes in homeostatic skin of different mouse strains but also highlight the possible influence of the strain differences (e.g., Th1/Th2 balance) as well as a role for IL-6 in overall skin immunity and resident microbial populations.

Evaluation of skin irritation following weathered crude oil exposure in two mouse strains.

Petroleum crude oil spills are common and vary in size and scope. Spill response workers throughout the course of remediation are exposed to so-called weathered oil and are known to report diverse health effects, including contact dermatitis. A murine model of repeated exposure to weathered marine crude oil was employed utilizing two strains of mice, C57BL/6 and BALB/c, to investigate the pathology of this irritant and identify the principal hydrocarbon components deposited in skin. Histopathology demonstrated clear signs of irritation in oil-exposed skin from both mouse strains, characterized by prominent epidermal hyperplasia (acanthosis). BALB/c mice exposed to oil demonstrated more pronounced irritation compared with C57BL/6 mice, which was characterized by increased acanthosis as well as increased inflammatory cytokine/chemokine protein expression of IL-1ß, IL-6, CXCL10, CCL2, CCL3, CCL4, and CCL11. A gas chromatography/mass spectrometry method was developed for the identification and quantification of 42 aliphatic and EPA priority aromatic hydrocarbons from full thickness skin samples of C57BL/6 and BALB/c mice exposed to oil samples. Aromatic hydrocarbons were not detected in skin; however, aliphatic hydrocarbons in skin tended to accumulate with carbon numbers greater than C16. These preliminary data and observations suggest that weathered crude oil is a skin irritant and this may be related to specific hydrocarbon components, although immune phenotype appears to impact skin response as well.

IL-6 Negatively Regulates IL-22Rα Expression on Epidermal Keratinocytes: Implications for Irritant Contact Dermatitis.

Irritant Contact Dermatitis (ICD) is characterized by epidermal hyperplasia and inflammatory cytokine release. IL-6 has been shown to be involved in the pathogenesis of ICD; however, the involvement of the IL-22/IL-22Rα axis and its relation to IL-6 in the inflammatory response following irritant exposure are unknown. Using a chemical model of ICD, it was observed that mice with a keratinocyte-specific knockout of IL-6Rα (IL-6Rα Δker) presented with increased inflammation and IL-22Rα and IL-22 protein expression relative to WT following irritant exposure, indicating that IL-6Rα deficiency in epidermal keratinocytes leads to the upregulation of IL-22Rα and its ligand during ICD. Furthermore, it was shown that IL-6 negatively regulates the expression of IL-22Rα on epidermal keratinocytes. This effect is functional as the effects of IL-22 on keratinocyte proliferation and differentiation were markedly reduced when keratinocytes were pretreated with IL-6 prior to IL-22 treatment. These results show that IL-6 modulates the IL-22/IL-22Rα axis in the skin and suggest that this occurrence may be associated with the increased epidermal hyperplasia and exacerbated inflammatory response observed in IL-6Rα Δker mice during ICD.

IL6Rα function in myeloid cells modulates the inflammatory response during irritant contact dermatitis.

Irritant contact dermatitis (ICD) is characterized by epidermal hyperplasia, infiltration of leucocytes into lesional skin and inflammatory cytokine release. The cellular infiltrate during ICD comprises primarily cells of the myeloid lineage. Our group has previously shown that the cytokine IL-6 confers a protective effect to lesional skin during ICD. How IL-6Rα function in myeloid cells is involved in the inflammatory response during ICD is, however, unknown. In the present study, utilizing a chemical model of ICD, it is shown that mice with a myeloid-specific knockout of the IL-6Rα (IL-6RαΔmyeloid ) display an exaggerated inflammatory response to benzalkonium chloride (BKC) and Jet propellant-8 (JP8) fuel, two well-characterized irritants relative to littermate control. Results from immunohistochemical and flow cytometric analyses revealed that IL-6RαΔmyeloid mouse skin displayed increased epidermal hyperplasia and inflammatory monocyte influx into lesional skin but lower numbers of resident macrophages relative to littermate controls after irritant exposure. Multiplex immunoassay revealed significantly higher levels of pro-inflammatory cytokines IL-1α and TNF-α, but reduced expression of chemokine proteins including CCL2-5, CCL7, CCL11, CXCL1 and CXCL10 in IL-6RαΔmyeloid mouse skin relative to littermate control following irritant exposure. These results highlight a previously unknown role of IL-6Rα function in myeloid cells in modulating the inflammatory response and myeloid population dynamics during ICD.

Keratinocyte-specific deletion of the IL-6RΑ exacerbates the inflammatory response during irritant contact dermatitis.

Irritant Contact Dermatitis (ICD) is the most common occupational skin disorder. During ICD, keratinocytes initiate the inflammatory cascade by producing cytokines including IL-6. This laboratory previously reported that IL-6 deficiency exacerbates skin inflammation during ICD, yet the role of the IL-6Rα in keratinocyte function has yet to be elucidated. To investigate how IL-6Rα function in keratinocytes influences the inflammatory response during ICD, keratinocyte-specific IL-6Rα KO (IL6raΔker) and WT mice were exposed to two well-known occupational irritants; JP-8 jet fuel, and benzalkonium chloride (BKC), or acetone control for three days. Dermatitis lesions were collected and flow cytometric and immunohistochemical analyses revealed that IL6raΔker skin displayed increased populations of CD11b+CD45+ and F4/80+ cells respectively relative to WT. However, IL6raΔker mouse skin contained reduced numbers of γδ T cells relative to WT. Furthermore, IL6raΔker skin expressed increased levels of pro-inflammatory cytokines including IL-1ß, IL-22, and CCL4 but decreased levels of anti-inflammatory cytokines IL-4 and IL-10. These results indicate that epidermal keratinocyte IL-6Rα function modulates epidermal hyperplasia, immune cell infiltration into skin and cytokine expression during ICD and suggests that the previously reported protective effect of IL-6 during ICD might be mediated primarily by keratinocyte derived IL-6Rα.

Interleukin 6 Function in the Skin and Isolated Keratinocytes Is Modulated by Hyperglycemia.

Diabetes currently affects over twenty-five million Americans. Annual health care cost of diabetes exceeds $254 billion and is associated with a distinct set of diabetic complications that include delayed wound healing and diabetic ulcers. Interleukin 6 (IL-6) plays an important role in wound healing and is known to be elevated in the serum of both type I and type II diabetes patients. This study assesses the expression and function of IL-6 in the hyperglycemic epidermis and keratinocyte culture. Streptozotocin-treated mice were wounded six weeks after induction of hyperglycemia. Wound closure, protein, and mRNA expression were assessed up to 13 days of postwounding. Wound closure was delayed 4-5 days in hyperglycemic animals. Hyperglycemic wounds displayed greater IL-6 and IL-6Rα protein expression at 1, 7, and 10 days of postwounding compared to euglycemic control. However, IL-6Rα mRNA expression was reduced at all time points beyond day 1, while IL-6 mRNA expression did not significantly differ at any time point. SOCS3 mRNA expression was higher in the hyperglycemic skin at every time point. Imaging of fluorescent immunohistology also revealed significantly lower expression of SOCS3, but higher nuclear pSTAT3 in the epidermis of the hyperglycemic skin. Primary mouse keratinocytes cultured in high glucose for 7 days displayed 2-fold higher IL-6Rα mRNA and higher rmIL-6-induced nuclear pSTAT3, but lower SOCS3 basal levels compared to normal glucose-cultured cells. Thus, it appears that delayed diabetic skin wound healing is associated with increased induction and expression of IL-6 and its receptor, but its function in epidermal keratinocytes may be impaired.

Associations Between Immune Phenotype and Inflammation in Murine Models of Irritant Contact Dermatitis.

Irritant contact dermatitis (ICD), the most common occupational cutaneous illness, is an acute inflammatory response caused by topical irritant exposure. Multiple factors are associated with the manifestation and severity of ICD and contribute to the lack of effective prophylactic and treatment strategies. To determine the pathomechanism of ICD caused by the irritants, benzalkonium chloride (BKC) and JP-8 jet fuel, 2 mouse strains, C57BL/6 and Balb/c, were assessed due to their differential immune predispositions. Dermatitis lesions were obtained for histological examination, cytokine protein expression analysis, and determination of immune cell infiltration via flow cytometric analysis. Following acute (3-day) BKC exposure C57BL/6 skin displayed increased neutrophils and expression of 19 distinct cytokines, but fewer dendritic cells and lower expression of IL-1α and IL-9 as compared with Balb/c skin. Following prolonged (7-day) exposure to BKC, inflammatory cell populations trended similar to 3-day exposure; however, only 6 distinct cytokines were higher in C57BL/6, whereas Balb/c displayed higher expression of IL-27, 28, and 31. Following acute JP-8 exposure, C57BL/6 skin displayed higher levels of γδ T cell infiltration, G and M-CSF expression, but lower populations of neutrophils, monocytes, and dendritic cells compared with Balb/c skin. As with BKC, skin inflammatory cell populations following 7-day JP-8 exposure trended similar to 3-day exposure. However, C57BL/6 skin displayed higher levels of IL-6 and LIF, whereas Balb/c showed increased IL-1ß, IL-27, G-CSF, TNFα, and 7 additional chemokines. These findings further define the pathology of ICD, partially explain individual variation of ICD, and offer insight into biomarkers for risk assessment.

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-ß receptor I and II (TGF-ßRI and II) function in epidermal keratinocytes.

It been shown that IL-6 modulates TGF-ß1 expression in fibroblasts, however, what role IL-6 plays concerning TGF-ßR 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-ß receptors in skin. Skin from WT, IL-6 over-expressing mice and IL-6 treated keratinocyte cultures was analysed for TGF-ßRI and TGF-ßRII 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-ßRII expression in the epidermis. IL-6 treatment of keratinocytes induced TGF-ßRI and II expression and augmented TGF-ß1-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-ßRI and II expression and signal transduction via trafficking of the receptor to lipid raft pools.

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-1ß, 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.

Estrogen suppresses heptatic IκB expression during short-term alcohol exposure.

OBJECTIVE: To assess the effects of sex steroids on hepatic inflammatory pathways in short-term chronically ethanol-fed rats. METHODS: Ovariectomized female Wistar rats (8-12 weeks old, n = 8 per treatment group) were implanted with osmotic pumps releasing 17ß-estradiol (20 µg/24 h) or testosterone (25 µg/24 h) and fed liquid diets with or without ethanol (8 % w/v) for two weeks. Hepatic expression of IκBα/ß, TNF-α, and IL-6 mRNA was examined by real-time PCR. Liver (nuclear) NFκB, IκBα and ß, IL-6, and IL-6Rα protein expression was examined by enzyme-linked immunosorbent assay (ELISA) or Western blot. RESULTS: Estrogen alone induced greater steatosis, NFκB translocation, TNF-α mRNA, as well as IL-6, and IL-6R protein. Alcohol consumption along with estrogen treatment further increased steatosis, NFκB translocation, TNF-α mRNA, and IL-6 protein. Conversely, neither estrogen nor ethanol consumption induced IκBα or IκBß mRNA or protein expression, while testosterone robustly induced these inhibitory proteins regardless of treatment. CONCLUSIONS: Estrogen exposure enhances alcohol-induced liver inflammation, and the anti-inflammatory effects of testosterone in the liver might be related to induction of IκB. Elevated inflammation in response to estrogen may overwhelm the regenerative influence of IL-6 in liver, leading to increased steatosis and greater liver damage.

Succession planning in US pharmacy schools.

The deans, associate and assistant deans, and department chairs of a college or school of pharmacy retain historic memories of the institution and share the responsibility for day-to-day operation, sustainability, and future planning. Between the anticipated retirement of baby boomers who are senior administrative faculty members and the steady increase in number of colleges and schools of pharmacy, the academy is facing a shortage of qualified successors. Succession planning involves planning for the effective transition of personnel in leadership positions within an organization. This paper describes the subject of succession planning at a sample population of AACP institutions by obtaining perspectives on the subject from the deans of these institutions via standardized interview instruments. The instruments were utilized with 15 deans; all interview data were blinded and analyzed using analyst triangulation. The majority of deans responded that some level of succession planning was desirable and even necessary; however, none claimed to have a formal succession planning structure in place at his or her home institution. Although widely accepted and well-recognized in the corporate and military sectors, succession planning within pharmacy schools and colleges is neither universally documented nor implemented. Differences exist within the administrative structure of these non-academic and academic institutions that may preclude a uniform succession planning format. While the evidence presented suggests that succession planning is needed within the academy, a concerted effort must be made towards implementing its practice.

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, alpha(1)-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 alpha(1)-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 alpha(2)-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.

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

BACKGROUND: Intravesical 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-alpha). We went further to determine whether BCG treatment alters cytokine gene expression in the urinary bladder. METHODS: C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-alpha. 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. RESULTS: Acute 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-alpha 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-1beta, 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. CONCLUSION: To 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-alpha, LPS, and, most likely, other classical pro-inflammatory stimuli.

IL-6 modulates alpha-smooth muscle actin expression in dermal fibroblasts from IL-6-deficient mice.

IL-6 deficient (IL-6KO) mice display significantly delayed cutaneous wound closure. Myofibroblasts are the primary mediators of wound closure, and alpha-smooth muscle actin (alpha-SMA) is a marker of fibroblast differentiation to the myofibroblast phenotype. Wounds from IL-6KO, and wild-type mice were collected up to 6 days following wounding. Expression of alpha-SMA mRNA was found to be increased in wounds of IL-6KO mice up to 48 hours post wounding, but decreased below wild-type levels by 72 hours. Recombinant IL-6 treatment of IL-6KO dermal fibroblasts showed an induction of alpha-SMA mRNA and protein peaking at 1 ng/ml cytokine, but declining at higher concentrations. Actinomycin-D treatment of fibroblast cultures indicated that recombinant mouse IL-6 (rmIL-6) induction of alpha-SMA mRNA appeared to be primarily transcriptionally regulated, and extracellular signal-regulated kinase 1/2 kinase, but not signal transducers and activators of transcription 3 was readily phosphorylated in rmIL-6 treated IL-6KO fibroblasts. A dose-response increase in the mRNA expression of the IL-6R signaling inhibitor protein suppressors of cytokine signaling (SOCS) 3 was also noted in rmIL-6-treated IL-6KO fibroblasts. These data indicate that alpha-SMA expression is dysregulated in IL-6KO mice. The expression of alpha-SMA induced by rmIL-6 in fibroblasts from IL-6KO mice appears to be transcriptionally modulated, dependent on JAK1 kinase, and possibly downregulated as a result of increased SOCS3 expression.

JP-8 jet fuel exposure induces inflammatory cytokines in rat skin.

The Department of Defense (DoD) has identified that one of the main complaints of personnel exposed to JP-8 jet fuel is irritant dermatitis. The purpose of this investigation is to describe the JP-8-induced inflammatory cytokine response in skin. JP-8 jet fuel or acetone control (300 microl) was applied to the denuded skin of rats once a day for 7 days. Skin samples from the exposed area were collected 2 and 24 h after the final exposure. Histological examination of skin biopsies showed neutrophilic inflammatory infiltrate. Reverse transcription-polymerase chain reaction (RT-PCR) was performed utilizing skin total RNA to examine the expression of various inflammatory cytokines. The CXC chemokine GROalpha was significantly upregulated at both time points, whereas GRObeta was only increased 2 h post final exposure. The CC chemokines MCP-1, Mip-1alpha, and eotaxin were induced at both time points, whereas Mip-1beta was induced only 24 h post exposure. Interleukins-1beta and -6 (IL-1beta and IL-6) mRNAs were significantly induced at both time points, while TNFalpha was not significantly different from control. Enzyme-linked immunosorbent assay (ELISA) of skin protein confirmed that MCP-1, TNFalpha, and IL-1beta were modulated as indicated by PCR analysis. However, skin IL-6 protein content was not increased 2 h post exposure, whereas it was significantly upregulated by jet fuel after 24 h. Data from the present study indicate that repeated (7 days) JP-8 exposure induces numerous proinflammatory cytokines in skin. The increased expression of these cytokines and chemokines may lead to increased inflammatory infiltrate in exposed skin, resulting in JP-8-induced irritant dermatitis.

Differential expression of liver interleukin-6 receptor-alpha 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.

Interleukin 6 indirectly induces keratinocyte migration.

IL-6-deficient transgenic mice (IL-6 KO) display significantly delayed cutaneous wound healing. To further elucidate the role of IL-6 in skin wound healing, epidermal keratinocyte and dermal fibroblast cells were isolated from neonatal IL-6 KO mice and treated with rmIL-6. It was found that rmIL-6 alone did not significantly modulate the proliferation or migration of cultured IL-6 KO keratinocytes. rmIL-6, however, significantly induced the migration of IL-6 KO keratinocytes (up to 5-fold) when co-cultured with dermal fibroblasts. Culture supernatants from IL-6-treated fibroblasts were also found to induce the migration of keratinocytes to a similar degree. Genomics analysis of treated fibroblasts indicated that rmIL-6 does not induce any known soluble keratinocyte migratory factors. rmIL-6 treatment of fibroblast, however, induced a rapid and sustained phosphorylation of STAT3 protein. These data indicate that IL-6 could influence wound healing by inducing keratinocyte migration through the production of a soluble fibroblast-derived factor, and its activity may be associated with STAT3 activation.