Evaluation of Cyclin D1 as a Discriminatory Immunohistochemical Biomarker for Idiopathic Pulmonary Fibrosis.

BACKGROUND: Distinction of idiopathic pulmonary fibrosis (IPF) from other chronic fibrosing interstitial pneumonitides, such as hypersensitivity pneumonitis (HP) and connective tissue diseases, is critical due to varied biological and clinical outcomes. However, their histologic overlaps often pose diagnostic challenges. A recent study suggested an association of herpesvirus saimiri infection with IPF. Productive viral infection is associated with coexpression of pirated mammalian protein cyclin D1, shown to be overexpressed by immunohistochemistry (IHC) in the regenerating alveolar epithelium in IPF but not in normal lungs. We evaluated the diagnostic utility of cyclin D1 to discriminate between IPF and other fibrosing interstitial lung diseases. MATERIALS AND METHODS: A retrospective study of cyclin D1 IHC expression in 27 consecutive cases of chronic fibrosing interstitial lung diseases from 2011 to 2017: 12 usual interstitial pneumonia (UIP) pattern; 5 nonspecific interstitial pneumonia pattern; 3 HP pattern; 7 unclassifiable was performed. Five cases of normal lung obtained from lobectomy specimen for malignancy are included as control. Immunoreactivity was graded semiquantitatively on a scale of 0 to 3. RESULTS: Cyclin D1 staining was uniformly strongly positive in all cases evaluated in the study, particularly in proliferating type II pneumocytes in the region of fibrosing areas. There was no statistical difference in the extent of cyclin D1 expression between UIP and non-UIP groups (2.7 vs. 2.5) and IPF versus non-IPF groups (2.7 vs. 2.4). Cyclin D1 expression is lower in control group compared with UIP groups (1.2 vs. 2.7). CONCLUSIONS: Cyclin D1 is not a specific marker of UIP pattern/IPF. The high expression of cyclin D1 in lung tissue of fibrosing interstitial pneumonitides regardless of etiology most likely correlates with proliferation in type II pneumocytes.

Phase I Trial of Intratumoral Injection of CCL21 Gene-Modified Dendritic Cells in Lung Cancer Elicits Tumor-Specific Immune Responses and CD8+ T-cell Infiltration.

Purpose: A phase I study was conducted to determine safety, clinical efficacy, and antitumor immune responses in patients with advanced non-small cell lung carcinoma (NSCLC) following intratumoral administration of autologous dendritic cells (DC) transduced with an adenoviral (Ad) vector expressing the CCL21 gene (Ad-CCL21-DC). We evaluated safety and tumor antigen-specific immune responses following in situ vaccination (ClinicalTrials.gov: NCT01574222).Experimental Design: Sixteen stage IIIB/IV NSCLC subjects received two vaccinations (1 × 106, 5 × 106, 1 × 107, or 3 × 107 DCs/injection) by CT- or bronchoscopic-guided intratumoral injections (days 0 and 7). Immune responses were assessed by tumor antigen-specific peripheral blood lymphocyte induction of IFNγ in ELISPOT assays. Tumor biopsies were evaluated for CD8+ T cells by IHC and for PD-L1 expression by IHC and real-time PCR (RT-PCR).Results: Twenty-five percent (4/16) of patients had stable disease at day 56. Median survival was 3.9 months. ELISPOT assays revealed 6 of 16 patients had systemic responses against tumor-associated antigens (TAA). Tumor CD8+ T-cell infiltration was induced in 54% of subjects (7/13; 3.4-fold average increase in the number of CD8+ T cells per mm2). Patients with increased CD8+ T cells following vaccination showed significantly increased PD-L1 mRNA expression.Conclusions: Intratumoral vaccination with Ad-CCL21-DC resulted in (i) induction of systemic tumor antigen-specific immune responses; (ii) enhanced tumor CD8+ T-cell infiltration; and (iii) increased tumor PD-L1 expression. Future studies will evaluate the role of combination therapies with PD-1/PD-L1 checkpoint inhibition combined with DC-CCL21 in situ vaccination. Clin Cancer Res; 23(16); 4556-68. ©2017 AACR.

Do mast cells play a pathogenetic role in neurofibromatosis type 1 and ulcerative colitis?

Concurrent association of neurofibromatosis type I and ulcerative colitis has been reported in one clinical case (Tavakkoli et al., 2009). Although this association may represent a casual finding, a common pathophysiology is postulated. Mast cells have been implicated in the pathogenesis of both neurofibromatosis type 1 and ulcerative colitis (He, 2004; Yoshida et al., 2010). Mast cells are typically present in neurofibromas microenvironment where they appear to contribute to tumor initiation, progression and angiogenesis (Staser et al., 2010, 2013). Moreover, interaction of mast cells with nerves throughout the gastrointestinal tract has been correlated with progression and maintenance of ulcerative colitis (Stoyanova and Gulubova, 2002). We describe a 14 year-old male with history of neurofibromatosis type 1 and new onset of ulcerative colitis diagnosed on clinical and histological findings. On gross examination the entire colonic mucosa appeared edematous showing a peculiar granular pattern, with focal erythema, shallow ulcers and multiple sessile polyps. Hematoxylin and eosin stained tissue biopsies from the colonic mucosa showed chronic inflammatory bowel disease, severe activity, consistent with chronic ulcerative colitis. Immunohistochemistry stain of the intestinal lesions revealed high expression of Neuron Specific Enolase (NSE) and S100 highlighting the presence of a Schwann cell component. In addition, c-kit/CD117 positive stain indicated a marked increase of mast cells in the lamina propria. This pattern of cellularity in the lamina propria showing increased mast cells and augmented Schwann cell component was absent in the colonic mucosa of a normal control or a patient with ulcerative colitis alone. Our observation supports the evidence of a pathogenetic role of the mast cell in ulcerative colitis associated with neurofibromatosis type 1. Further investigations are warranted to confirm the significance of this correlation as it may impact therapeutic approaches of these pathologies.

IL-27 inhibits epithelial-mesenchymal transition and angiogenic factor production in a STAT1-dominant pathway in human non-small cell lung cancer.

BACKGROUND: Interleukin-27 signaling is mediated by the JAK-STAT pathway via activation of STAT1 and STAT3, which have tumor suppressive and oncogenic activities, respectively. Epithelial-mesenchymal transition (EMT) and angiogenesis are key processes in carcinogenesis. Although IL-27 has been shown to have potent anti-tumor activity in various cancer models, the role of IL-27 in EMT and angiogenesis is poorly understood. In this study, we investigated the role of IL-27 in regulating EMT and angiogenesis through modulation of the STAT pathways in human non-small cell lung carcinoma (NSCLC) cells. METHODS: STAT activation following IL-27 exposure was measured in human NSCLC cell lines. Expression of epithelial (E-cadherin, γ-catenin) and mesenchymal (N-cadherin, vimentin) markers were assessed by Western blot analysis. Production of pro-angiogenic factors (VEGF, IL-8/CXCL8, CXCL5) were examined by ELISA. Cell motility was examined by an in vitro scratch and transwell migration assays. Selective inhibitors of STAT1 (STAT1 siRNAs) and STAT3 (Stattic) were used to determine whether both STAT1 and STAT3 are required for IL-27 mediated inhibition of EMT and secretion of angiogenic factors. RESULTS: Our results demonstrate that IL-27 stimulation in NSCLC resulted in 1) STAT1 and STAT3 activation in a JAK-dependent manner, 2) development of epithelial phenotypes, including a decrease in the expression of a transcriptional repressor for E-cadherin (SNAIL), and mesenchymal marker (vimentin) with a reciprocal increase in the expression of epithelial markers, 3) inhibition of cell migration, and 4) reduced production of pro-angiogenic factors. STAT1 inhibition in IL-27-treated cells reversed the IL-27 effect with resultant increased expression of Snail, vimentin and the pro-angiogenic factors. The inhibition of STAT3 activation had no effect on the development of the epithelial phenotype. CONCLUSION: IL-27 induces mesenchymal to epithelial transition and inhibits the production of pro-angiogenic factors in a STAT1-dominant pathway. These findings highlight the importance of STAT1 in repressing lung carcinogenesis and describe a new anti-tumor mechanism of IL-27.

CCL21 Chemokine Therapy for Lung Cancer.

Lung cancer remains a challenging health problem with more than 1.1 million deaths worldwide annually. With current therapy, the long term survival for the majority of lung cancer patients remains low, thus new therapeutic strategies are needed. One such strategy would be to develop immune therapy for lung cancer. Immune approaches remain attractive because although surgery, chemotherapy, and radiotherapy alone or in combination produce response rates in all histological types of lung cancer, relapse is frequent. Strategies that harness the immune system to react against tumors can be integrated with existing forms of therapy for optimal responses toward this devastating disease. Both antigen presenting cell (APC) and T cell activities are reduced in the lung tumor microenvironment. In this review we discuss our experience with efforts to restore host APC and T cell activities in the lung cancer microenvironment by intratumoral administration of dendritic cells (DC) expressing the CCR7 receptor ligand CCL21 (secondary lymphoid chemokine, SLC). Based on the results demonstrating that CCL21 is an effective anti cancer agent in the pre-clinical lung tumor model systems, a phase I clinical trial was initiated using intratumoral injection of CCL21 gene modified autologous DC in lung cancer. Results from the trial thus far indicate tolerability, immune enhancement and tumor shrinkage via this approach.

Novel CCL21-vault nanocapsule intratumoral delivery inhibits lung cancer growth.

BACKGROUND: Based on our preclinical findings, we are assessing the efficacy of intratumoral injection of dendritic cells (DC) transduced with an adenoviral vector expressing the secondary lymphoid chemokine (CCL21) gene (Ad-CCL21-DC) in a phase I trial in advanced non-small cell lung cancer (NSCLC). While this approach shows immune enhancement, the preparation of autologous DC for CCL21 genetic modification is cumbersome, expensive and time consuming. We are evaluating a non-DC based approach which utilizes vault nanoparticles for intratumoral CCL21 delivery to mediate antitumor activity in lung cancer. PRINCIPAL FINDINGS: Here we describe that vault nanocapsule platform for CCL21 delivery elicits antitumor activity with inhibition of lung cancer growth. Vault nanocapsule packaged CCL21 (CCL21-vaults) demonstrated functional activity in chemotactic and antigen presenting activity assays. Recombinant vaults impacted chemotactic migration of T cells and this effect was predominantly CCL21 dependent as CCL21 neutralization abrogated the CCL21 mediated enhancement in chemotaxis. Intratumoral administration of CCL21-vaults in mice bearing lung cancer enhanced leukocytic infiltrates (CXCR3(+)T, CCR7(+)T, IFNγ(+)T lymphocytes, DEC205(+) DC), inhibited lung cancer tumor growth and reduced the frequencies of immune suppressive cells [myeloid derived suppressor cells (MDSC), T regulatory cells (Treg), IL-10 T cells]. CCL21-vaults induced systemic antitumor responses by augmenting splenic T cell lytic activity against parental tumor cells. SIGNIFICANCE: This study demonstrates that the vault nanocapsule can efficiently deliver CCL21 to sustain antitumor activity and inhibit lung cancer growth. The vault nanocapsule can serve as an "off the shelf" approach to deliver antitumor cytokines to treat a broad range of malignancies.

PGE(2) contributes to TGF-beta induced T regulatory cell function in human non-small cell lung cancer.

CD4(+)CD25(bright) regulatory T cells (T(reg)) play an important role in cancer-mediated immunosuppression. We and others have previously shown that prostaglandin E2 (PGE(2)) and transforming growth factor beta (TGF-beta) induce CD4(+)CD25(bright)FOXP3(+)T(reg). Based on these studies, we investigated the requirement for PGE(2) in Treg induction by TGF-beta. TGF-beta stimulation of human CD4(+) T cells induced COX-2-dependent production of PGE(2). PGE(2)-neutralizing antibody treatment significantly reduced the suppressive function of TGF-beta-induced T(reg) (TGF-beta-T(reg)) in vitro. TGF-beta concentration measured in the plasma of non-small cell lung cancer (NSCLC) patients directly correlated with the frequency of circulating CD4(+)CD25(bright)FOXP3(+)T cells. Flow cytometry analysis showed increased FOXP3 expression in circulating CD4(+)CD25(+)HLA-DR- cells of lung cancer patients compared to control subjects. Immunohistochemical analysis revealed co-expression of TGF-beta, COX-2, and FOXP3 in serial sections from resected lung tumor tissues. All together these observations suggest interplay between TGF-beta and COX-2 in the induction of T(reg) activities. Interrupting TGF-beta and PGE(2) signaling may be important in therapeutic interventions that aim to limit T(reg)function in lung cancer.

Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in non-small cell lung cancer.

BACKGROUND: Our previous studies have demonstrated that transduction of human dendritic cells (DC) with adenovirus encoding secondary lymphoid chemokine, CCL21, led to secretion of biologically active CCL21 without altering DC phenotype or viability. In addition, intratumoral injections of CCL21-transduced DC into established murine lung tumors resulted in complete regression and protective anti-tumor immunity. These results have provided the rationale to generate a clinical grade adenoviral vector encoding CCL-21 for ex vivo transduction of human DC in order to assess intratumoral administration in late stage human lung cancer. METHODS: In the current study, human monocyte-derived DC were differentiated by exposure to GM-CSF and IL-4 from cryopreserved mononuclear cells obtained from healthy volunteers. Transduction with clinical grade adenoviral vector encoding CCL21 (1167 viral particles per cell) resulted in secretion of CCL21 protein. RESULTS: CCL21 protein production from transduced DC was detected in supernatants (24-72 hours, 3.5-6.7 ng/4-5 x 10(6) cells). DC transduced with the clinical grade adenoviral vector were > 88% viable (n = 16), conserved their phenotype and maintained integral biological activities including dextran uptake, production of immunostimulatory cytokines/chemokines and antigen presentation. Furthermore, supernatant from CCL21-DC induced the chemotaxis of T2 cells in vitro. CONCLUSION: Viable and biologically active clinical grade CCL21 gene-modified DC can be generated from cryopreserved PBMC.

Inflammation and lung carcinogenesis: applying findings in prevention and treatment.

Lung carcinogenesis is a complex process requiring the acquisition of genetic mutations that confer the malignant phenotype as well as epigenetic alterations that may be manipulated in the course of therapy. Inflammatory signals in the lung cancer microenvironment can promote apoptosis resistance, proliferation, invasion, metastasis, and secretion of proangiogenic and immunosuppressive factors. Here, we discuss several prototypical inflammatory mediators controlling the malignant phenotype in lung cancer. Investigation into the detailed molecular mechanisms underlying the tumor-promoting effects of inflammation in lung cancer has revealed novel potential drug targets. Cytokines, growth factors and small-molecule inflammatory mediators released in the developing tumor microenvironment pave the way for epithelial-mesenchymal transition, the shift from a polarized, epithelial phenotype to a highly motile mesenchymal phenotype that becomes dysregulated during tumor invasion. Inflammatory mediators within the tumor microenvironment are derived from neoplastic cells as well as stromal and inflammatory cells; thus, lung cancer develops in a host environment in which the deregulated inflammatory response promotes tumor progression. Inflammation-related metabolic and catabolic enzymes (prostaglandin E(2) synthase, prostaglandin I(2) synthase and 15-hydroxyprostaglandin dehydrogenase), cell-surface receptors (E-type prostaglandin receptors) and transcription factors (ZEB1, SNAIL, PPARs, STATs and NF-kappaB) are differentially expressed in lung cancer cells compared with normal lung epithelial cells and, thus, may contribute to tumor initiation and progression. These newly discovered molecular mechanisms in the pathogenesis of lung cancer provide novel opportunities for targeted therapy and prevention in lung cancer.

Prostaglandin E2 induces FOXP3 gene expression and T regulatory cell function in human CD4+ T cells.

Naturally occurring CD4+CD25+ regulatory T cells (T reg) are pivotal in suppressing immune responses and maintaining tolerance. The identification of molecules controlling T reg differentiation and function is important in understanding host immune responses in malignancy and autoimmunity. In this study we show that PGE2 enhances the in vitro inhibitory function of human purified CD4+CD25+ T reg cells. Moreover, PGE2 induces a regulatory phenotype in CD4+CD25- T cells. PGE2-treated T cell-mediated inhibition of anti-CD3-stimulated lymphocyte proliferation did not require cell contact. Phenotypic analysis revealed that PGE2 diminished CD25 expression in both CD4+CD25dim T cells and CD4+CD25bright T reg cells. PGE2 exposure induced the T reg cell-specific transcription factor forkhead/winged helix transcription factor gene (FOXP3) in CD4+CD25- T cells and significantly up-regulated its expression in CD4+CD25+ T reg cells. Similarly, 24-h incubation with supernatants from cyclooxygenase-2-overexpressing lung cancer cells that secrete high levels of PGE2 significantly induced FOXP3 in CD4+CD25- T cells. Finally, PGE2 up-regulated FOXP3 at both mRNA and protein levels and enhanced FOXP3 promoter activity. This is the first report indicating that PGE2 can modulate FOXP3 expression and T reg function in human lymphocytes.

Tumor cyclooxygenase-2/prostaglandin E2-dependent promotion of FOXP3 expression and CD4+ CD25+ T regulatory cell activities in lung cancer.

Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E2 underlie an immunosuppressive network that is important in the pathogenesis of non-small cell lung cancer. CD4+ CD25+ T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4+ CD25+ Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE2 induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE2-mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE2 to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE2 suppresses Treg cell activity and enhances antitumor responses.

IL-20, an anti-angiogenic cytokine that inhibits COX-2 expression.

COX-2 overexpression and subsequent PGE(2) production are frequently associated with non-small cell lung cancer and are implicated in tumor-mediated angiogenesis. Here, we report for the first time that IL-20 downregulates COX-2 and PGE(2) in human bronchial epithelial and endothelial cells. Flow cytometry analysis suggests that IL-20-dependent inhibition of COX-2/PGE(2) occurs through the IL-22R1/IL-20R2 dimers. In addition, we report that IL-20 exerts anti-angiogenic effects, inhibiting experimental angiogenesis. IL-20-mediated inhibition of PMA-induced angiogenesis occurs through the COX-2 regulatory pathway. Altogether our findings revealed that IL-20 is a negative modulator of COX-2/PGE(2) and inhibits angiogenesis.

PGE2 confers survivin-dependent apoptosis resistance in human monocyte-derived dendritic cells.

Control of apoptosis is fundamental for dendritic cell (DC) homeostasis. Numerous factors maintain DC viability throughout their lifespan, including inhibitor of apoptosis proteins. Among them, survivin is overexpressed in many human malignancies, but its physiological function in normal cells has not been fully delineated. Prostaglandin E2 (PGE2), also overproduced in several malignancies, has shown to induce proapoptotic and antiapoptotic effects in different cell types, including immune cells. In DC, PGE2 predominantly affects maturation and modulates immune functions. Here, we show that exposure of monocyte-derived DC to PGE2 (10(-5) M) for 72 h significantly increased DC survivin mRNA and protein expression. In contrast, DC, matured with lipopolysaccharide or tumor necrosis factor alpha, did not reveal survivin induction in response to PGE2. Following exposure to apoptotic stimuli, DC treated with PGE2 exhibited an overall increased viability compared with control DC, and this effect was correlated inversely with caspase-3 activation. Moreover, PGE2-treated, survivin-deficient DC demonstrated reduced viability in response to apoptotic stimuli. Further analysis indicated that PGE2 induced DC survivin expression in an E prostanoid (EP)2/EP4 receptor and phosphatidylinositol-3 kinase-dependent manner. These findings suggest that PGE2-dependent regulation of survivin is important in modulating apoptosis resistance in human DC.

Modulation of pulmonary leukotriene B4 production by cyclooxygenase-2 inhibitors and lipopolysaccharide.

PURPOSE: Emerging data continue to link carcinogenesis to inflammatory events involving the eicosanoid metabolic pathways. We therefore evaluated the effects of cyclooxygenase (COX)-2 inhibition on leukotriene (LT) B(4) synthesis in the lungs of active smokers, as part of a pilot lung cancer chemoprevention study with celecoxib (Celebrex), an oral COX-2 inhibitor. EXPERIMENTAL DESIGN: Bronchoalveolar lavage was performed before celecoxib treatment and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from ex-smoker control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. RESULTS: Treatment with oral celecoxib only modestly increased LTB(4) levels in bronchoalveolar lavage, without increasing the mRNA transcription of 5-lipoxygenase (5-LOX) or 5-LOX-activating protein in AMs, whereas the acute calcium ionophore-stimulated LTB(4) production from smokers' AMs was markedly increased by 10.6-fold. In addition, smokers' AMs were twice as responsive in producing LTB(4) when exposed to lipopolysaccharide compared with ex-smokers' AMs. Concomitant COX-2 inhibition with SC58236, however, did not significantly impact these changes, whereas the 5-LOX inhibitor Zileuton blocked the generation of LTB(4) in a dose-responsive manner. Finally, cycloheximide increased the production of LTB(4) under all conditions, suggesting a shunting phenomenon and/or the presence of pathway inhibitors. CONCLUSIONS: Our findings suggest that whereas oral celecoxib is capable of modulating LTB(4) production in the lung microenvironment, under physiologic conditions, this effect is probably not functionally significant.

Prostaglandin E2-dependent enhancement of tissue inhibitors of metalloproteinases-1 production limits dendritic cell migration through extracellular matrix.

Dendritic cell (DC) migration is crucial for the initiation of immune responses. The balance between metalloproteinases (MMP) and tissue inhibitors of metalloproteinases (TIMP) has been shown to modulate DC migration. PGE2, which is overproduced in a wide variety of human malignancies, has been implicated in MMP and TIMP regulation in various cells, including monocytes. In the present study, we hypothesized that tumor-derived PGE2 would affect DC migratory capacity through the extracellular matrix (ECM) by altering MMP and TIMP balance. Treatment of monocyte-derived immature DC with exogenous PGE2 induced TIMP-1 secretion but not MMP-9 production and was correlated with reduced DC migration through ECM. Because recombinant TIMP-1 replicated PGE2 inhibition of DC migration while anti-TIMP-1 neutralizing Ab reversed it, we conclude that PGE2-mediated induction of TIMP-1 was responsible for the reduced migration of PGE2-treated DC. Similarly, DC cultured for 48 h in supernatants from cyclooxygenase-2 overexpressing lung cancer cells that secrete high levels of PGE2, exhibited decreased migration through ECM. Finally, analysis of E prostanoid receptor expression and their selective inhibition revealed that the enhanced TIMP-1 secretion in PGE2-treated DC was mediated predominantly by the E prostanoid receptor 2. These findings indicate that PGE2-dependent enhancement of TIMP-1 production causes reduced migration of DC through ECM.

Celecoxib modulates the capacity for prostaglandin E2 and interleukin-10 production in alveolar macrophages from active smokers.

PURPOSE: Preclinical data suggest that the cyclooxygenase (COX)-2/prostaglandin (PG) E2 signaling pathway plays an essential role in conferring the malignant phenotype in non-small cell lung cancer. We hypothesized that treatment with oral celecoxib, a selective COX-2 inhibitor, would favorably alter biomarkers of lung cancer risk. This study evaluated the feasibility of COX-2 inhibition as a form of chemoprevention for lung cancer. EXPERIMENTAL DESIGN: Heavy active smokers were enrolled into a pilot study and treated with celecoxib. Bronchoscopy with bronchoalveolar lavage was performed both before and after 1 month of celecoxib treatment to recover alveolar macrophages (AMs) and lining fluid for study. After harvest, AMs were immediately stimulated in vitro with the calcium ionophore A23187. AMs obtained from smokers before treatment and from nonsmoking control subjects were also cultured overnight with SC58236, a selective COX-2 inhibitor, with or without lipopolysaccharide stimulation. RESULTS: Treatment with celecoxib significantly reduced calcium ionophore-stimulated PGE2 production from AMs recovered from smokers. AMs recovered from smokers, but not nonsmokers, were primed to produce high levels of PGE2 and interleukin (IL-10) when stimulated with lipopolysaccharide, and SC58236 significantly abrogated the production of these factors. Moreover, both plasma and bronchoalveolar lavage fluid obtained from treated subjects significantly reduced the production of PGE2 that resulted when a lung cancer cell line, A549, was stimulated with IL-1beta or A23187. CONCLUSIONS: Our findings suggest that oral celecoxib is capable of inhibiting the overproduction of PGE2, as well as modulating the production of IL-10 in the lung microenvironment in individuals at risk for lung cancer.

All-trans retinoic acid modulates the balance of matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 in patients with emphysema.

STUDY OBJECTIVE: The balance between proteases and antiproteases plays an essential role in the pathogenesis of emphysema. This study was designed to evaluate the impact of all-trans retinoic acid (ATRA) on the balance of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in patients with emphysema. DESIGN AND SETTING: As part of a clinical study, ATRA was administered to 20 patients with emphysema for 12 weeks and evaluated for its effects on plasma levels of MMP-9 and TIMP-1. Plasma MMP-9 levels were also measured in a separate cohort of patients with emphysema and matched control subjects to evaluate the relationship of circulating enzyme levels to lung disease. To further investigate the effects of ATRA on protease activity within the lung microenvironment, alveolar macrophages (AM) recovered from the lungs of active smokers with COPD were cultured with ATRA in vitro. MEASUREMENTS AND RESULTS: Administration of ATRA to patients with emphysema produced a 45 +/- 14% reduction (mean +/- SEM) in plasma MMP-9 by enzyme-linked immunosorbent assay and a similar reduction in MMP-9 enzyme activity, while having little effect on TIMP-1 levels. Baseline MMP-9 levels were higher in patients with emphysema compared to nonsmoking control subjects, suggesting a relationship between plasma levels and the presence of lung disease. In vitro, concentrations of ATRA similar to those achieved in the plasma of study subjects significantly reduced both the production and enzyme activity of MMP-9 by AM. In the same experiments, TIMP-1 levels increased significantly, resulting in a marked reduction in the MMP-9/TIMP-1 molar ratio. CONCLUSION: We conclude that ATRA can modulate protease/antiprotease balance in a manner that may impact on disease pathogenesis.

Overexpression of CCL-21/secondary lymphoid tissue chemokine in human dendritic cells augments chemotactic activities for lymphocytes and antigen presenting cells.

BACKGROUND: Ex vivo generated dendritic cells (DC) genetically modified to express secondary lymphoid tissue chemokine (CCL-21/SLC) have been shown to stimulate potent antitumor responses in murine models. When injected intratumorally, CCL-21 colocalizes DC and lymphocyte effector cells at the tumor site. This may improve tumor antigen presentation and T cell activation by utilizing the tumor as an in vivo source of antigen for DC. In order to develop DC-based cancer therapies for intratumoral injection that could promote tumor antigen uptake and presentation in situ, we constructed and characterized an adenoviral vector that expresses human CCL-21 (AdCCL-21). RESULTS: Human monocyte derived DC were cultured in GM-CSF and IL-4 for 6 days. Following AdCCL-21 transduction, CCL-21 protein production was assessed by ELISA on day 8. DC transduced with AdCCL-21 at multiplicities of infection (MOIs) of 50:1 or 100:1 produced up to 210 +/- 9 ng/ml and 278 +/- 6.5 ng/ml /106 cells/48 hours, respectively. Following transduction, an immature DC phenotype was maintained and an upregulation of the costimulatory molecule, CD86 was noted. In addition, supernatant from AdCCL-21-DC caused significant chemotaxis of peripheral blood lymphocytes and mature DC. CONCLUSIONS: These studies demonstrate that AdCCL-21-DC generate functional levels of CCL-21 without adversely altering DC phenotype. These findings strengthen the rationale for further investigation of AdCCL-21-DC as a DC-based therapy in cancer treatment.

Tumor cyclooxygenase 2-dependent suppression of dendritic cell function.

Dendritic cells (DCs) serve as professional antigen-presenting cells and are pivotal in the host immune response to tumor antigens. To define the pathways limiting DC function in the tumor microenvironment, we assessed the impact of tumor cyclooxygenase (COX)-2 expression on DC activities. Bone marrow-derived DCs were cultured in either tumor supernatant (TSN) or TSN from COX-2-inhibited tumors. After culture, DCs were pulsed with tumor-specific peptides, and their ability to generate antitumor immune responses was assessed following injection into established murine lung cancer. In vitro, DC phenotype, alloreactivity, antigen processing and presentation, and interleukin (IL)-10 and IL-12 secretion were evaluated. DCs cultured in TSN failed to generate antitumor immune responses and caused immunosuppressive effects that correlated with enhanced tumor growth. However, genetic or pharmacological inhibition of tumor COX-2 expression restored DC function and effective antitumor immune responses. Functional analyses indicated that TSN causes a decrement in DC capacity to (a) process and present antigens, (b) induce alloreactivity, and (c) secrete IL-12. Whereas TSN DCs showed a significant reduction in cell surface expression of CD11c, DEC-205, MHC class I antigen, MHC class II antigen, CD80, and CD86 as well as a reduction in the transporter-associated proteins, transporter associated with antigen processing 1 and 2, the changes in phenotype and function were not evident when DCs were cultured in supernatant from COX-2-inhibited tumors. We conclude that inhibition of tumor COX-2 expression or activity can prevent tumor-induced suppression of DC activities.