Stephen J. Pollock

A novel anti-inflammatory and pro-resolving role for resolvin D1 in acute cigarette smoke-induced lung inflammation.

Introduction Cigarette smoke is a profound pro-inflammatory stimulus that contributes to acute lung injuries and to chronic lung disease including COPD (emphysema and chronic bronchitis). Until recently, it was assumed that resolution of inflammation was a passive process that occurred once the inflammatory stimulus was removed. It is now recognized that resolution of inflammation is a bioactive process, mediated by specialized lipid mediators, and that normal homeostasis is maintained by a balance between pro-inflammatory and pro-resolving pathways. These novel small lipid mediators, including the resolvins, protectins and maresins, are bioactive products mainly derived from dietary omega-3 and omega-6 polyunsaturated fatty acids (PUFA). We hypothesize that resolvin D1 (RvD1) has potent anti-inflammatory and pro-resolving effects in a model of cigarette smoke-induced lung inflammation. Methods Primary human lung fibroblasts, small airway epithelial cells and blood monocytes were treated with IL-1β or cigarette smoke extract in combination with RvD1 in vitro, production of pro-inflammatory mediators was measured. Mice were exposed to dilute mainstream cigarette smoke and treated with RvD1 either concurrently with smoke or after smoking cessation. The effects on lung inflammation and lung macrophage populations were assessed. Results RvD1 suppressed production of pro-inflammatory mediators by primary human cells in a dose-dependent manner. Treatment of mice with RvD1 concurrently with cigarette smoke exposure significantly reduced neutrophilic lung inflammation and production of pro-inflammatory cytokines, while upregulating the anti-inflammatory cytokine IL-10. RvD1 promoted differentiation of alternatively activated (M2) macrophages and neutrophil efferocytosis. RvD1 also accelerated the resolution of lung inflammation when given after the final smoke exposure. Conclusions RvD1 has potent anti-inflammatory and pro-resolving effects in cells and mice exposed to cigarette smoke. Resolvins have strong potential as a novel therapeutic approach to resolve lung injury caused by smoke and pulmonary toxicants.

Peroxisome proliferator-activated receptor γ and retinoid X receptor transcription factors are released from activated human platelets and shed in microparticles

Peroxisome proliferator-activated receptor gamma (PPARgamma) and its ligands are important regulators of lipid metabolism, inflammation, and diabetes. We previously demonstrated that anucleate human platelets express the transcription factor PPARgamma and that PPARgamma ligands blunt platelet activation. To further understand the nature of PPARgamma in platelets, we determined the platelet PPARgamma isoform(s) and investigated the fate of PPARgamma following platelet activation. Our studies demonstrated that human platelets contain only the PPARgamma1 isoform and after activation with thrombin, TRAP, ADP or collagen PPARgamma is released from internal stores. PPARgamma release was blocked by a cytoskeleton inhibitor, Latrunculin A. Platelet-released PPARgamma was complexed with the retinoid X receptor (RXR) and retained its ability to bind DNA. Interestingly, the released PPARgamma and RXR were microparticle associated and the released PPARgamma/RXR complex retained DNA-binding ability. Additionally, a monocytic cell line, THP-1, is capable of internalizing PMPs. Further investigation following treatment of these cells with the PPARgamma agonist, rosiglitazone and PMPs revealed a possible transcellular mechanism to attenuate THP-1 activation. These new findings are the first to demonstrate transcription factor release from platelets, revealing the complex spectrum of proteins expressed and expelled from platelets, and suggests that platelet PPARgamma has an undiscovered role in human biology.

Platelets and Megakaryocytes Contain Functional Nuclear Factor-κB

Objective—To investigate the presence and role of NF-&kgr;B proteins in megakaryocytes and platelets. The nuclear factor-&kgr;B (NF-&kgr;B) transcription factor family is well known for its role in eliciting inflammation and promoting cell survival. We discovered that human megakaryocytes and platelets express the majority of NF-&kgr;B family members, including the regulatory inhibitor-&kgr;B (I-&kgr;B) and I-&kgr; kinase (IKK) molecules. Methods and Results—Anucleate platelets exposed to NF-&kgr;B inhibitors demonstrated impaired fundamental functions involved in repairing vascular injury and thrombus formation. Specifically, NF-&kgr;B inhibition diminished lamellapodia formation, decreased clot retraction times, and reduced thrombus stability. Moreover, inhibition of I-&kgr;B-&agr; phosphorylation (BAY-11-7082) reverted fully spread platelets back to a spheroid morphology. Addition of recombinant IKK-&bgr; or I-&kgr;B-&agr; protein to BAY inhibitor–treated platelets partially restored platelet spreading in I-&kgr;B-&agr; inhibited platelets, and addition of active IKK-&bgr; increased endogenous I-&kgr;B-&agr; phosphorylation levels. Conclusion—These novel findings support a crucial and nonclassical role for the NF-&kgr;B family in modulating platelet function and reveal that platelets are sensitive to NF-&kgr;B inhibitors. As NF-&kgr;B inhibitors are being developed as antiinflammatory and anticancer agents, they may have unintended effects on platelets. On the basis of these data, NF-&kgr;B is also identified as a new target to dampen unwanted platelet activation.

Orbital fibroblasts from patients with thyroid-associated ophthalmopathy overexpress CD40: CD154 hyperinduces IL-6, IL-8, and MCP-1.

PURPOSE Fibroblast diversity represents an emerging concept critical to our understanding of tissue inflammation, repair, and remodeling. Orbital fibroblasts heterogeneously display Thy-1 and exhibit unique phenotypic attributes that may explain the susceptibility of the human orbit to thyroid-associated ophthalmopathy (TAO). In the present study the authors investigated the role of CD40 ligation on macrophage chemoattractant protein-1 (MCP-1), IL-6, and IL-8 expression in fibroblasts from patients with TAO. METHODS Human orbital fibroblasts were cultured from tissues obtained with informed consent from patients with TAO and from patients undergoing surgery for other noninflammatory conditions. The fibroblasts were then examined by flow cytometry, microscopy, and cytokine assays. RESULTS The authors report that orbital fibroblasts from patients with TAO expressed elevated levels of CD40. Surface CD40 could be further upregulated by IFN-gamma in TAO and control fibroblasts. This upregulation was mediated through Jak2 and could be blocked by dexamethasone and AG490, a powerful and specific inhibitor of tyrosine kinase. Treatment with CD154, the ligand for CD40, upregulated the expression of IL-6, IL-8, and MCP-1 in TAO fibroblasts but failed to do so in control cultures. Thy-1(+) fibroblasts displayed higher CD40 levels than did their Thy-1(-) counterparts and were largely responsible for this cytokine production. IL-1beta also induced MCP-1, IL-6, and IL-8 more vigorously in TAO-derived fibroblasts. CONCLUSIONS Characterization of orbital fibroblasts and their differential expression of cytokines and receptors should prove invaluable in understanding the site-specific nature of TAO and the development of specific therapies.

THY1 expression is associated with tumor suppression of human ovarian cancer.

Microcell-mediated transfer of chromosome 11 into the human ovarian cancer cell line SKOV-3 results in suppression of tumorigenicity in severe combined immunodeficiency (SCID) mice. To identify the differentially expressed transcripts associated with suppression of tumorigenicity, cDNA populations from the slow-growing tumorigenic clone 11(H)8-3, tumorigenic clone 11(H)8-4, and parental SKOV-3 cells were subtracted from the nontumorigenic clones, 11(H)7-2 and 11(C)9-8. The subtracted cDNA populations were either cloned, sequenced and searched in GenBank, or analyzed by gene discovery array screening. A cDNA transcript corresponding to the THY1 gene located at chromosome 11q23 approximately q24 was found to be exclusively expressed in the two nontumorigenic cell clones. In contrast, THY1 expression was not detected in SKOV-3, the tumorigenic hybrid clones, or six other tumorigenic ovarian cancer cell lines. Further analysis using immunocytochemistry and quantitative flow cytometry with a Thy-1-specific antibody confirmed the exclusive expression of THY1 at the protein level in the two nontumorigenic clones. Several cell growth and differentiation-related genes, including thrombospondin 1 (THBS1), SPARC [secreted protein, acidic, cysteine-rich (osteonectin)], and fibronectin (FN1) were also found to be upregulated in the nontumorigenic clones; however, these were expressed in the slow-growing tumorigenic clones as well. Expression of these genes was not observed in the parental SKOV-3 cell line and therefore must be regulated by a gene or genes on chromosome 11. Our results suggest that THY1 is a putative tumor suppressor gene for ovarian cancer and that THBS1, SPARC, and FN1 are genes associated with the regulation of in vivo tumor growth rate.

Genetic Ablation of the Aryl Hydrocarbon Receptor Causes Cigarette Smoke-induced Mitochondrial Dysfunction and Apoptosis

Background: The aryl hydrocarbon receptor (AhR) suppresses lung inflammation and may protect against cigarette smoke-induced apoptosis. Results: Genetic ablation of the AhR increases the sensitivity of lung cells to smoke-induced apoptosis by regulating antioxidant proteins. Conclusion: The AhR regulates pulmonary cell survival. Significance: The AhR control over lung cell survival may be why only some smokers develop chronic lung diseases such as chronic obstructive pulmonary disease. Cigarette smoke is the primary risk factor for chronic obstructive pulmonary disease (COPD). Alterations in the balance between apoptosis and proliferation are involved in the etiology of COPD. Fibroblasts and epithelial cells are sensitive to the oxidative properties of cigarette smoke, and whose loss may precipitate the development of COPD. Fibroblasts express the aryl hydrocarbon receptor (AhR), a transcription factor that attenuates pulmonary inflammation and may also regulate apoptosis. We hypothesized the AhR would prevent apoptosis caused by cigarette smoke. Using genetically deleted in vitro AhR expression models and an established method of cigarette smoke exposure, we report that AhR expression regulates fibroblasts proliferation and prevents morphological features of apoptosis, including membrane blebbing and chromatin condensation caused by cigarette smoke extract (CSE). Absence of AhR expression results in cleavage of PARP, lamin, and caspase-3. Mitochondrial dysfunction, including cytochrome c release, was associated with loss of AhR expression, indicating activation of the intrinsic apoptotic cascade. Heightened sensitivity of AhR-deficient fibroblasts was not the result of alterations in GSH, Nrf2, or HO-1 expression. Instead, AhR−/− cells had significantly less MnSOD and CuZn-SOD expression, enzymes that protects against oxidative stress. The ability of the AhR to suppress apoptosis was not restricted to fibroblasts, as siRNA-mediated knockdown of the AhR in lung epithelial cells also increased sensitivity to smoke-induced apoptosis. Collectively, these results suggest that cigarette smoke induced loss of lung structural support (i.e. fibroblasts, epithelial cells) caused by aberrations in AhR expression may explain why some smokers develop lung diseases such as COPD.

Isolation and Phenotypic Characterization of Lung Fibroblasts

Primary fibroblasts represent a heterogeneous population of cells that can be separated into subsets on the basis of cell surface markers such as Thy-1. Deriving fibroblasts initially involves obtaining tissue explants from tissues such as the lung, heart, cornea, skin, and orbit. The tissue is mechanically dissociated and cells are allowed to proliferate from the fragments. Following establishment of a primary culture of fibroblasts, it is necessary to characterize the new strain of cells to ensure their purity and fibroblastic phenotype using immunofluorescence and immunohistochemistry to detect the presence or absence of cell-specific surface markers. Characterizing the cells as expressing or lacking Thy-1 can also be performed by immunofluorescence in concert with microscopy or by flow cytometry using an anti-human Thy-1 antibody. In addition, fibroblasts may be sorted according to their expression of Thy-1 by fluorescence-activated cell sorting and/or magnetic beading; use of these techniques can yield greater than 99% purity. Once separated, the pure Thy-1 expressing or lacking fibroblast subsets can be propagated. These subsets can then be used for experimentation to determine functional differences between fibroblasts derived from normal and pathological tissue such as scarred lung.

The role of the THY1 gene in human ovarian cancer suppression based on transfection studies

In our recent studies, the expression of the THY1 gene encoding a 25-28 kDa glycoprotein located at 11q23-q24, was found to be associated with complete tumor suppression of the ovarian cancer cell line SKOV-3 after the transfer of chromosome 11. These studies raised the possibility that THY1 maybe a candidate tumor suppressor gene for ovarian cancer. To investigate this, the complete cDNA sequence for THY1 was cloned and transfected into SKOV-3 ovarian cancer cells. The expression of THY1 in the transfectants was confirmed by Northern blot analysis, immunocytochemistry, and flow cytometry. Both SKOV-3-THY1 and SKOV-3-null cells were inoculated subcutaneously into severe combined immunodeficiency (SCID) mice to determine in vivo tumorigenicity. THY1 transfectants formed tumors, but overall tumor growth rate and tumor size was significantly reduced compared with their null counterparts. To further correlate THY1 expression with tumorigenicity, the THY1 antisense was transfected into the nontumorigenic clone, 11(C)9-8, which resulted in restoration of tumorigenicity. These data indicate that THY1 expression alone cannot suppress tumorigenicity; however, abrogation of THY1 expression from nontumorigenic cells can restore tumorigenesis. Taken together, the data suggest that THY1 is necessary but not sufficient to suppress ovarian tumorigenicity. Therefore, THY1 can be designated as a putative tumor suppressor gene for human ovarian cancer.

Site-specific mesenchymal control of inflammatory pain to yeast challenge in vulvodynia-afflicted and pain-free women.

Abstract Fibroblast strains were derived from 2 regions of the lower genital tract of localized provoked vulvodynia (LPV) cases and pain-free controls. Sixteen strains were derived from 4 cases and 4 controls, age and race matched, after presampling mechanical pain threshold assessments. Strains were challenged with 6 separate stimuli: live yeast species (Candida albicans, Candida glabrata, Candida tropicalis, and Saccharomyces cerevisiae), yeast extract (zymosan), or inactive vehicle. Production of prostaglandin E2 (PGE2) and interleukin 6 (IL-6) were proinflammatory response measures. Highest IL-6 and PGE2 occurred with vestibular strains after C albicans, C glabrata, and zymosan challenges, resulting in the ability to significantly predict IL-6 and PGE2 production by genital tract location. After C albicans and C glabrata challenge of all 16 fibroblast strains, adjusting for dual sampling of subjects, PGE2 and IL-6 production significantly predicted the presampling pain threshold from the genital tract site of sampling. At the same location of pain assessment and fibroblast sampling, in situ immunohistochemical (IHC)(+) fibroblasts for IL-6 and Cox-2 were quantified microscopically. The correlation between IL-6 production and IL-6 IHC(+) was statistically significant; however, biological significance is unknown because of the small number of IHC(+) IL-6 fibroblasts identified. A low fibroblast IL-6 IHC(+) count may result from most IL-6 produced by fibroblasts existing in a secreted extracellular state. Enhanced, site-specific, innate immune responsiveness to yeast pathogens by fibroblasts may be an early step in LPV pathogenesis. Fibroblast strain testing may offer an attractive and objective marker of LPV pathology in women with vulvodynia of inflammatory origin.

Anticancer Role of PPARγ Agonists in Hematological Malignancies Found in the Vasculature, Marrow, and Eyes

The use of targeted cancer therapies in combination with conventional chemotherapeutic agents and/or radiation treatment has increased overall survival of cancer patients. However, longer survival is accompanied by increased incidence of comorbidities due, in part, to drug side effects and toxicities. It is well accepted that inflammation and tumorigenesis are linked. Because peroxisome proliferator-activated receptor (PPAR)-γ agonists are potent mediators of anti-inflammatory responses, it was a logical extension to examine the role of PPARγ agonists in the treatment and prevention of cancer. This paper has two objectives: first to highlight the potential uses for PPARγ agonists in anticancer therapy with special emphasis on their role when used as adjuvant or combined therapy in the treatment of hematological malignancies found in the vasculature, marrow, and eyes, and second, to review the potential role PPARγ and/or its ligands may have in modulating cancer-associated angiogenesis and tumor-stromal microenvironment crosstalk in bone marrow.