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Fibroblasts are considered major contributors to the process of fibrogenesis and the progression of matrix deposition and tissue distortion in fibrotic diseases such as Pulmonary Fibrosis. Recent discovery of the fibrocyte, a circulating possible precursor cell to the tissue fibroblast in fibrosis, has raised issues regarding the characterization of fibrocytes with respect to their morphology, growth characteristics in vitro, their biological role in vivo and their potential utility as a biomarker and/ or treatment target in various human diseases. Characterization studies of the fibrocyte continue as does emerging conflicting data concerning the relationship to or with the lung fibroblast. The source of signals that direct the traffic of these cells, as well as their response to therapeutic intervention with newly available drugs, bring new insights to the understanding of this cell type. The identification of exosomes from fibrocytes that can affect resident fibroblast activities suggest mechanisms of their influence on pathogenesis. Moreover, interesting comparisons with other pathologies are emerging involving the influence of circulating mesenchymal precursor cells on tissue responses.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by myofibroblast proliferation. Transition of epithelial/mesothelial cells into myofibroblasts [epithelial-to-mesenchymal transition (EMT)] occurs under the influence of transforming growth factor (TGF)-β1, with Snail being a major transcription factor. We study here the role of the heat-shock protein HSP27 in fibrogenesis and EMT. In vitro, we have up- and down-modulated HSP27 expression in mesothelial and epithelial cell lines and studied the expression of different EMT markers induced by TGF-β1. In vivo, we inhibited HSP27 with the antisense oligonucleotide OGX-427 (in phase II clinical trials as anticancer agent) in our rat subpleural/pulmonary fibrosis models. We demonstrate that HSP27 is strongly expressed during the fibrotic process in patients with IPF and in different in vivo models. We showed that HSP27 binds to and stabilizes Snail and consequently induces EMT. Conversely, HSP27 knockdown leads to Snail proteasomal degradation, thus inhibiting TGF-β1-induced EMT. Inhibition of HSP27 with OGX-427 efficiently blocks EMT and fibrosis development. Controls in vivo were an empty adenovirus that did not induce fibrosis and a control antisense oligonucleotide. The present work opens the possibility of a new therapeutic use for HSP27 inhibitors against IPF, for which there is no conclusively effective treatment.

Three patients are described in whom platelet aggregation and/or degranulation occurred in blood collected into EDTA. These changes resulted in spurious thrombocytopenia and morphological changes similar to those observed in some thrombocytopathies. The abnormalities were dependent on the presence of EDTA and did not occur in citrate, oxalate or heparin anticoagulants. In two patients the abnormality was shown to be due to a plasma factor which was not IgG, IgM, fibrinogen or albumin. The most likely explanation is that these patients have an unidentified abnormal plasma component which, on exposure to EDTA, develops 'anti-platelet activity'. Althought relatively uncommon a prospective study of the incidence of these phenomena indicates that they are probably more common than either platelet cold agglutinins or platelet satellitism. They have practical significance with respect to the diagnosis of thrombocytopenia and also to the interpretation of abnormal platelet morphology.

Tumor cells can evolve to evade immune responses by down-modulating surface MHC class I expression and become refractory to T cell–directed immunotherapy. We employed a strategy to bypass this escape mechanism using a recombinant adenovirus vector expressing interleukin-12 (Ad5IL-12) to target natural killer (NK) cell–mediated killing of human prostate tumors in NOD.scid mice. Fluorescence-activated cell sorting analysis revealed that LNCaP tumor cells bear negligible levels of MHC class I molecules; yet, they express MICA/B molecules, ligands for the NKG2D receptors found on NK cells. Transduction of LNCaP cells with the Ad5IL-12 vector prevented tumor formation in NOD.scid mice, indicating that NK cells alone can conduct tumor immunosurveillance and mediate protection. Intratumor injection of the Ad5IL-12 vector to established LNCaP tumors in NOD.scid mice resulted in a significant delay of tumor growth mediated by NK cell killing activity. The dependency of NK cells in this protective response was shown by the complete loss of Ad5IL-12 therapeutic efficacy on LNCaP tumors established in NOD.Cg-Rag1tm1MomPrf1tm1Sdz congenic mice, which are devoid of NK cell activity. More pronounced attenuation of tumor growth and enhanced NK killing activity was observed when pharmacologic adrenalectomy with mitotane was done in combination with Ad5IL-12 vector treatment. The Ad5IL-12 vector treatment also induced killing of MICA/B-negative MHC class I–positive PC3 tumors formed in NOD.scid mice. Together, these results indicate that a targeted NK cell response could provide a generic approach for cancer immunotherapy, and that enhancing the NK cell response via control of cortisol levels may provide an additional therapeutic avenue in cancer. [Cancer Res 2007;67(5):2290–7]

In this study, we have examined the in vivo effects of interleukin-5 (IL-5) and IL-6 over-expression on systemic and mucosal immune responses using recombinant human type 5 adenoviruses capable of expressing these cytokines upon infection. A recombinant adenovirus containing the murine IL-5 gene within the E3 region was constructed and found to express high levels of IL-5 protein both in vitro and in vivo. Intranasal inoculation of mice with this vector or a vector expressing murine IL-6 increased adenovirus-specific immunoglobulin A (IgA) titres in lung lavage fluid threefold compared with those elicited by control virus. The simultaneous expression of both cytokines by co-inoculation altered the kinetics of the mucosal anti-adenovirus IgA response and resulted in a more than additive increase in antibody titres. The co-expression effect on IgA synthesis was not due to an increase in numbers of antigen-specific resident lung tissue lymphocytes. When mucosal IgG responses were examined, IL-6 expression had the largest impact on anti-adenovirus levels, whereas co-expression produced an intermediate response. Systemic immune responses were also affected by IL-6 expression as a twofold increase in serum IgG anti-adenovirus titres was observed after a secondary challenge with wild-type adenovirus. These results demonstrate a relevant role for IL-5 and IL-6 in the development of mucosal immune responses in vivo and suggest that the incorporation of either IL-5 and/or IL-6 into recombinant adenovirus vectors may be a useful tool in the development of mucosal vaccines.

The role of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the differentiation of dendritic cells (DCs) during pulmonary viral infection was investigated by using a mouse model of GM-CSF transgene expression established with an adenoviral vector (AdGM-CSF). GM-CSF gene transfer resulted in increased levels of GM-CSF in the lung, which peaked at day 4 and remained increased up to day 19. A striking cellular response composed predominantly of macrophage-like cells was observed in the lung receiving AdGM-CSF but not control vector. By FACS analysis, the majority of these cells were identified at an early time point as macrophages and later as mature/activated myeloid DCs characterized by CD11bbright, CD11cbright, MHC class IIbright, and B7.1bright. In contrast, GM-CSF had a weak effect on a small DC population that was found present in normal lung and was characterized by CD11cbright and CD11blow. By immunohistochemistry staining for MHC II, the majority of activated antigen-presenting cells were localized to the airway epithelium and peribronchial/perivascular areas in the lung. A concurrently enhanced Th1 immune response was observed under these conditions. The number of CD4 and CD8 T cells was markedly increased in the lung expressing GM-CSF, accompanied by increased release of interferon (IFN)γ in the lung. Furthermore, lymphocytes isolated from either lung parenchyma or local draining lymph nodes of these mice but not the control mice released large amounts of IFNγ on adenoviral antigen stimulation in vitro. These findings reveal that GM-CSF promotes the differentiation and activation of a myeloid DC population primarily by acting on macrophages during pulmonary immune responses.

The understanding of cytokine networks and the exploitation of these networks for the treatment of immune and inflammatory diseases as well as cancer depend on in vivo delivery of cytokines. Due to instability of recombinant cytokine proteins, investigators have employed cytokine-encoding gene therapy vectors to induce high levels of cytokine expression in vivo. Numerous gene therapy vectors have been developed recently which are suitable for this purpose. Recent advances in the design of adenovirus, adeno-associated virus, poxvirus, retrovirus, lentivirus, and nonviral vectors are described here. Properties of the various vector systems which determine their usefulness for cytokine gene delivery are compared. The implementations of cytokine-encoding gene therapy vectors for analyzing immune responses and for the therapy of inflammatory disorders, immune disease, infections and cancer are reviewed.

Partial nerve injury is a potential cause of distressing chronic pain for which conventional analgesic treatment with opiates or anti-inflammatory agents is not very effective. Constriction nerve injury, widely used to study neuropathic pain, was shown here to induce interleukin-6 (IL-6) mRNA in a subset of rat primary sensory neurons. When we inflicted chronic nerve constriction on mice with null mutation of the IL-6 gene, the hypersensitivity to cutaneous heat and pressure that is induced in wild-type mice was not evident, the loss of substance P in sensory neurons was excessive and the induction of galanin in central sensory projections was reduced. In additional experiments, intrathecal infusion of IL-6 in rats was shown to stimulate synthesis of galanin in approximately one-third of lumbar dorsal root ganglion neurons. The results of these experiments indicate that endogenous IL-6 mediates some of the hypersensitive responses that characterize peripheral neuropathic pain, and influences two neuropeptides that have been implicated in pain transmission.

Inappropriate release of proteases from inflammatory and stromal cells can lead to destruction of the lung parenchyma. Antiproteinases such as alpha-1-proteinase inhibitor (alpha1-Pi), secretory leukocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor (elafin) control excess production of human neutrophil elastase. In the present study, the concentrations of alpha1-Pi, SLPI and elafin found in bronchoalveolar lavage (BAL) fluid from control subjects, patients at risk of developing acute respiratory distress syndrome (ARDS) and patients with established ARDS were determined. Levels of all three inhibitors were raised in patients compared with normal subjects. SLPI was increased in the group of patients who were at risk of ARDS and went on to develop the condition, compared with the "at-risk" group who did not progress to ARDS (p=0.0083). Alpha1-Pi and elafin levels were similar in these two populations. In patients with established ARDS, both alpha1-Pi and SLPI levels were significantly increased, compared to patients at risk of ARDS who did (p=0.0089) or did not (p=0.0003) progress to ARDS. The finding of increased antiproteinases shortly before the development of acute respiratory distress syndrome provide further evidence for enhanced inflammation prior to clinical disease.

We have previously demonstrated that intratumoral injection with Ad vectors expressing IL-2 or IL-12 can induce regression in a murine breast cancer model. These IL-2- or IL-12-induced antitumor responses were mainly mediated by Ag-specific T cells. Lymphotactin is a novel lymphocyte chemokine that can cause local accumulation of CD4+, CD8+, and NK cells. We hypothesized that addition of lymphotactin may enhance the antitumor immune responses induced by locally produced IL-2 and IL-12 as we have previously shown. To this end we constructed two double-recombinant adenoviral vectors expressing lymphotactin along with either IL-2 (Ad5 Lym/IL-2) or IL-12 (Ad5 Lym/IL-12). Subcutaneous injection of polyoma middle T (PyMT) or Neu (8142) transgenically derived breast adenocarcinoma cells, in the hind flank of FVB/n mice, results in the formation of tumor nodules in 14-21 days. We show that these constructs elicit potent antitumor responses when administered intratumorally. The antitumor responses are long lasting as determined by rechallenge experiments and hence demonstrate a protective immunity. These observations indicate that by augmenting the antitumor response with adenoviral vectors expressing lymphotactin in combination with IL-2 or IL-12 is a novel way to enhance immunotherapeutic approaches.

IL-5 is induced locally in the lung and systemically in the circulation during allergic airways eosinophilic inflammation both in humans and experimental animals. However, the precise role of local and systemic IL-5 in the development of allergic airways eosinophilia remains to be elucidated. In our current study, we demonstrate that compared with their IL-5(+/+) counterparts, IL-5(-/-) mice lacked an IL-5 response both in the lung and peripheral blood, yet they released similar amounts of IL-4, eotaxin, and MIP-1alpha in the lung after ovalbumin (OVA) sensitization and challenge. At cellular levels, these mice failed to develop peripheral blood and airways eosinophilia while the responses of lymphocytes, neutrophils, and macrophages remained similar to those in IL-5(+/+) mice. To dissect the relative role of local and systemic IL-5 in this model, we constructed a gene transfer vector expressing murine IL-5. Intramuscular IL-5 gene transfer to OVA-sensitized IL-5(-/-) mice led to raised levels of IL-5 compartmentalized to the circulation and completely reconstituted airways eosinophilia upon OVA challenge, which was associated with reconstitution of eosinophilia in the bone marrow and peripheral blood. Significant airways eosinophilia was observed for at least 7 d in these mice. In contrast, intranasal IL-5 gene transfer, when rendered to give rise to a significant but compartmentalized level of transgene protein IL-5 in the lung, was unable to reconstitute airways eosinophilia in OVA-sensitized IL-5(-/-) mice upon OVA-challenge, which was associated with a lack of eosinophilic responses in bone marrow and peripheral blood. Our findings thus provide unequivocal evidence that circulating but not local lung IL-5 is critically required for the development of allergic airways eosinophilia. These findings also provide the rationale for developing strategies to target circulating IL-5 and/or its receptors in bone marrow to effectively control asthmatic airways eosinophilia.

SUMMARYTo investigate the role of GM-CSF in asthmatic airways inflammation, we have targeted GM-CSF transgene to the airway cells in a mouse model of ovalbumin (OVA)-induced allergic airways inflammation, a model in which there is marked induction of endogenous IL-5 and IL-4 but not GM-CSF. Following intranasal delivery of a replication-deficient adenoviral gene transfer vector (Ad), transgene expression was found localized primarily to the respiratory epithelial cells. Intranasal delivery of 0.03 × 109 plaque-forming units (PFU) of AdGM-CSF into naive BALB/c mice resulted in prolonged and compartmentalized release of GM-CSF transgene protein with a peak concentration of ≈ 80 pg/ml detected in bronchoalveolar lavage fluid (BALF) at day 7, but little in serum. These levels of local GM-CSF expression per se resulted in no eosinophilia and only a minimum of tissue inflammatory responses in the lung of naive mice, similar to those induced by the control vector. However, such GM-CSF expression in the airways of OVA-sensitized mice resulted in a much greater and sustained accumulation of various inflammatory cell types, most noticeably eosinophils, both in BALF and airway tissues for 15–21 days post-OVA aerosol challenge, at which times airways inflammation had largely resolved in control mice. While the levels of IL-5 and IL-4 in BALF and the rate of eosinophil apoptosis were found similar between different treatments, there was an increased number of proliferative leucocytes in the lung receiving GM-CSF gene transfer. Our results thus provide direct experimental evidence that GM-CSF can significantly contribute to the development of allergic airways inflammation through potentiating and prolonging inflammatory infiltration induced by cytokines such as IL-5 and IL-4.

Bronchiolitis obliterans, a form of chronic allograft rejection characterized by progressive fibrous obliteration of the airways, is the major obstacle limiting prolonged survival of lung transplant recipients. To date, no effective therapy against this fatal complication exists. Interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine, inhibits various T cell and antigen-presenting cell functions. We examined the effect of IL-10 in an animal model for bronchiolitis obliterans. A heterotopic tracheal transplant model was used. IL-10 was administered to the recipient either in its recombinant form by osmotic minipump or by adenoviral-mediated IL-10 gene transfer (Ad5E1mIL-10). Successful gene transfection and expression was confirmed by measuring circulating IL-10 protein. Tracheal allografts were assessed histologically based on a scoring system. IL-10 administration (in recombinant form or by gene transfer) inhibited the development of fibrous airway obliteration 3 weeks after transplantation in comparison to untreated controls (p < 0.05). This was demonstrated only if the delivery was initiated 5 days after transplantation and not if it was started at the time of transplantation. A single administration of the gene construct was sufficient to achieve the desired effect. We have shown that IL-10 can prevent the development of airway fibro-obliteration in this model. Gene transfection at a site distant from a graft can be used to produce a desired effect on the graft. IL-10 may be of major importance in the control of post-transplant bronchiolitis obliterans. The timing of its administration is critical and further studies are required to determine the mechanisms underlying the observed effects of IL-10.

A number of complex mechanisms regulate the size and cellularity of an Ag-dependent granulomatous reaction and their accompanying cytokine production profiles. In the present study, Th1 (purified protein derivative (PPD)) and Th2 (schistosome egg Ag)-type granulomas were established to examine the role of IL-4 in lesion development and procollagen type III expression. PPD-sensitized mice were transfected via the airway with an adenovirus construct containing an IL-4 cDNA cassette, and the lungs were embolized with sized Ag-coated Sepharose beads. Granuloma development in response to the PPD bead challenge in control mice primarily consisted of mononuclear cells. In contrast, the overexpression of IL-4 in the IL-4 adenovirus-transfected animals demonstrated a significant increase in cellularity, size, procollagen type III expression, and accumulation of eosinophils. Analysis of mRNA and protein within the lung demonstrated significant expression of IL-4 in only the IL-4 adenovirus-transfected animals. The granuloma lesion size was significantly increased in the IL-4 adenovirus-transfected animals on days 2 and 5, reaching an approximate 50% increase compared with the control groups. Furthermore, procollagen type III mRNA expression was increased in the IL-4 adenovirus-transfected, PPD bead-embolized lungs. In contrast, when IL-4 was neutralized during Th2-type schistosome egg Ag bead granulomas, a decrease in procollagen type III was observed. These data indicate that the progression of certain granulomas may be regulated by the production of IL-4, thus altering the cellularity, size, and matrix composition of the inflammatory response.

RANTES (regulated on activation, normal T cell expressed and secreted) is a member of the C-C superfamily of chemokines and is reported to function as a potent chemoattractant for monocytes, eosinophils, and a subpopulation of CD4+ T cells. Using a recombinant human type 5 adenovirus containing the murine RANTES cDNA (Ad5E3 mRANTES), which is capable of expressing biologically active cytokine upon infection, we initiated a study to characterize the biologic functions of RANTES cytokine in vivo. Intratracheal administration of Ad5E3 mRANTES targeted transient RANTES expression to the bronchial epithelium of the lung in Sprague-Dawley rats. Bronchoalveolar lavage fluids (BAL) collected at 24 h had increased chemotactic activity vs controls as measured in a murine CD4+ T cell Boyden chamber microchemotaxis assay. There was a dramatic increase in the number of cells (macrophage, monocytes, and neutrophils) recovered from BAL samples taken from Ad5E3 mRANTES-treated animals at 24 h, with a >50-fold increase in monocytes, indicating a proinflammatory effect for this cytokine in vivo. This effect on monocytes was transient, decreasing by 7 days, with evidence of increased eosinophils and lymphocytes at this time. Histologic examination of lung sections at 24 h revealed greatly increased numbers of mononuclear cells, primarily monocytes, within the lungs of Ad5E3 mRANTES-treated animals, with increased extravasation of monocytes around blood vessels, indicating an ongoing process of peripheral blood monocyte recruitment. This study provides further evidence for RANTES to be a monocyte chemoattractant in vivo.

Mice bearing breast tumors were treated with a single dose of an adenovirus expressing interleukin-12 (AdmIL-12.1) injected intratumorally, which produced regressions in greater than 75% of the treated tumors; approximately one-third of the animals remained tumor free. Complete regression was associated with immunity to secondary challenge with fresh tumor cells. Analysis of local cytokine expression demonstrated maximum expression of IL-12 within the tumor between 24 and 72 hr post-injection, reaching 600-800 ng per tumor, with elevated local levels of IL-12 detectable for at least 9 days. This expression was highly localized as serum IL-12 peaked at 40-60 ng/ml at 24 hr and was less than 10 ng/ml from day 3 onward. Interferon-gamma (IFN-gamma) concentrations were markedly increased within the tumor following AdmIL-12.1 administration, demonstrating that IL-12 was acting locally. Tumor-draining lymph node cells spontaneously produced IFN-gamma following AdmIL-12.1 treatment, suggesting these cells were activated by IL-12. These data demonstrate that AdmIL-12.1 can be used to deliver very high levels of localized cytokine production. Moreover, we have confirmed that the IL-12 produced from our vector actually affects the local cytokine environment of the tumor and activates responder cells present within the tumor.

IL-12 is a proinflammatory cytokine that has recently been shown to have beneficial effects in the setting of acquired host immunity. To determine the role of IL-12 in innate immunity against Gram-negative bacterial organisms, CBA/J mice were challenged with 10(2) CFU of Klebsiella pneumoniae intratracheally (i.t.), resulting in the time-dependent expression of IL-12 mRNA (p35 and p40) and protein within the lung. Passive immunization of animals with anti-IL-12 serum i.p. at the time of K. pneumoniae inoculation resulted in a 12-fold increase in K. pneumoniae CFU in lung homogenates at 48 h, as compared with animals receiving control serum. In addition, treatment of Klebsiella-infected mice with anti-IL-12 Abs significantly decreased both short and long term survival. To assess the effect of compartmentalized IL-12 overexpression on outcome in Klebsiella pneumonia, animals were treated i.t. with 5 x 10(8) PFU of a nonreplicating adenoviral vector containing a human cytomegalovirus promoter and cDNAs coding for the p35 and p40 subunits of IL-12 inserted into the E1 and E3 domains (Ad5mIL-12), respectively. In vivo transfection with Ad5mIL-12 resulted in 45% long term survival in Klebsiella pneumonia, whereas no animals with Klebsiella pneumonia receiving control adenovirus survived. Moreover, treatment with anti-IFN-gamma Abs or soluble TNF receptor:Ig construct partially and completely attenuated survival benefits observed in animals receiving Ad5mIL-12, respectively. In conclusion, endogenous IL-12 is a critical component of antibacterial host defense, and the compartmentalized overexpression of IL-12 using recombinant adenoviral gene therapy represents a safe and effective approach to deliver IL-12 to the lung in the setting of murine Klebsiella pneumonia.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine whose expression is increased in numerous respiratory diseases, particularly in asthma. However, the role of GM-CSF in the pathogenesis of these conditions in vivo remains unclear. Here, we report the functional activities of GM-CSF highly expressed in rat lung after intrapulmonary transfer of the gene coding for murine GM-CSF by using an adenoviral vector. This high, transient expression of GM-CSF led to the sustained but self-limiting accumulation of eosinophils and macrophages associated with tissue injury in the lung followed by varying degrees of irreversible fibrotic reactions observed in later stages. These results suggest that GM-CSF plays a previously unrealized role in the development of respiratory conditions characterized by eosinophilia, granuloma and/or fibrosis and provide the rationale for targeting this molecule in these diseases.

Interleukin-12 (IL-12) is a heterodimeric cytokine that plays an important role in the development of cellular immunity. Clinical applications for this lymphokine include resolution of infectious disease, cancer immunotherapy, and boosting cellular immunity in AIDS patients. When using IL-12 and other cytokines therapeutically, an approach designed to obtain localized cytokine expression would be beneficial, because this could reduce the problem of systemic toxicity. As a means of developing a suitable delivery vehicle for IL-12, we have produced double-recombinant adenovirus vectors containing the p35 subunit cDNA of murine IL-12 in early region 1 of adenovirus type 5 and the cDNA for p40 in early region 3 (AdmIL-12). Cell lines infected with AdmIL-12 produced up to 42,000 units of IL-12/10(6) cells per 24 hr. Biological activity of the virally expressed product was demonstrated in vitro through its ability to induce proliferation of phytohemagglutinin (PHA)-stimulated lymphoblasts and to stimulate natural killer (NK) activity in naive splenocytes. Mice injected intraperitoneally with these vectors displayed serum IL-12 levels that increased proportionately with the amount of virus administered. IL-12 production in vivo caused a dose-dependent increase in splenic and lung NK cell activity. This work represents the first demonstration of a double-recombinant adenovirus vector expressing a functional heterodimeric protein. The results of these studies support the use of AdmIL-12 as an efficient delivery vehicle for IL-12, and direct studies of its ability to modulate cellular immunity in vivo are currently underway.

The effects of recombinant interleukin-1 (IL-1), given as a continuous infusion for 6 days, on host responses were determined in rats. The development of fever, change in food intake and body weight, and key components of the acute-phase response in energy and protein metabolism were assessed. The effects of IL-1 were compared with those observed in a matched pair-fed group (semistarved), to distinguish the contribution from anorexia, and in a group that received IL-1 for 4 h acutely. IL-1 significantly increased core temperature, plasma levels of IL-6, and acute-phase protein production and decreased food intake and the circulating zinc level. The catabolic effects of IL-1 on nitrogen loss and muscle protein breakdown were independent of, and additive to those from malnutrition. The changes in energy expenditure, cumulative urinary nitrogen, and hydroxyproline excretion in the chronic IL-1 group were increased over semistarved animals. Finally, changes in muscle protein kinetics were only seen with chronic IL-1 infusion, and the changes in acute-phase protein were greater.

We tested the hypothesis that vitronectin (Vn) is regulated as an acute phase reactant in response to inflammatory stimuli. In initial experiments, Vn levels were measured during the surgically induced acute phase response in humans. The plasma concentration of Vn increased approximately twofold following elective orthopedic surgery and remained elevated up to 5 days. To examine the mechanism(s) of increased Vn synthesis, hepatic Vn mRNA expression and serum levels were examined in three rat models of acute inflammation: LPS (i.v.), CFA (i.p.), or turpentine (s.c.) injection. The serum concentration of Vn increased approximately twofold 24 h following treatment with turpentine. The expression of Vn mRNA in the liver increased markedly as early as 3 h after treatment in these models and remained elevated up to 18 h. Northern blot analysis of RNA isolated from fractionated liver cells derived from rats treated with LPS indicated that Vn was mainly expressed in hepatocytes, but not in the endothelial or nonparenchymal cell fractions. To analyze the individual effects of raised corticosterone and IL-6 levels on the expression of hepatic Vn mRNA, rats were injected (i.p.) with either dexamethasone or purified recombinant rat IL-6. Vn mRNA expression was elevated within 1 h after IL-6 injection, whereas dexamethasone-injected rats showed unchanged Vn expression. Vn mRNA also was increased in rats chronically injected with IL-6. These results indicate that the Vn gene is up-regulated in acute and chronic inflammation, and this induction is primarily mediated by IL-6.

The biologic function of cytokines may be best studied in the context of a defined tissue site. To establish a model for studying the function of IL-6 at local tissue sites, we targeted the IL-6 transgene into the bronchial epithelium in the lung of Sprague-Dawley rats by intratracheal administration of a recombinant human type 5 adenovirus with rat IL-6 cDNA incorporated into the E3 region of the viral genome. This approach led to a highly compartmentalized overexpression of the IL-6 transgene and production of bioactive protein within the lung for about 7 days post-infection. Associated with this overexpression of IL-6 was the development of profound local lymphocytic hyperplasia around day 7, characterized by the dramatic expansion of bronchial associated lymphoid aggregates and massive lymphocytic infiltration in the pulmonary parenchyma. Concurrently, there were strikingly increased numbers of lymphocytes in bronchoalveolar lavage fluids. The majority of these lymphocytes were found to be CD3+CD8+ cytotoxic T and CD3+CD4+ helper T cells with the remaining being primarily a small number of CD45R+ B cells. In addition, there was moderate bronchial and alveolar epithelial hyperplasia associated with lymphocytic hyperplasia. However, all of these changes subsided concomitant with the decrease in IL-6 expression and the lung seemed normal at 12 to 14 days post-infection. Thus, our study provides a tissue-specific transient transgene model for investigating cytokine functions in vivo and demonstrates that IL-6 has a profound stimulatory effect on the local lymphoid tissue in the lung.

TNF-alpha is a cytokine with a wide spectrum of proinflammatory activities. Nasal polyps (NP), which occur in association with allergic rhinitis and asthma, are characterized by a marked infiltration of activated eosinophils, epithelial damage, and varying degrees of stromal fibrosis. By using Southern blot analysis after a reverse transcription-PCR, we detected a signal specific for TNF-alpha mRNA in five of seven NP samples, but not in control nasal mucosal samples. With in situ hybridization, we detected cells expressing mRNA specific for TNF-alpha in eight of thirteen NP samples, but not in four control samples. Counterstaining with chromotrope 2R demonstrated that virtually all cells expressing TNF-alpha message were eosinophils. The ratio of eosinophils expressing TNF-alpha to the total number of eosinophils varied greatly among tissues; as an average, we observed 24% TNF-alpha-positive eosinophils. Using a mAb against human TNF-alpha, we demonstrated TNF-alpha localization in 12 NP tissues (48.8 +/- 16.5 positive cells/mm2) but not in three control samples. Morphologically, cells localizing TNF-alpha were both mononucleated and polynucleated with only a small number of eosinophils, as determined by aniline blue counterstaining. Studies of purified blood eosinophils from a patient with hypereosinophilic syndrome and from four healthy donors indicated that TNF-alpha is produced, but rapidly secreted, so that TNF-alpha mRNA-positive cells contain little detectable protein. Furthermore, cells that retain detectable TNF-alpha may not contain sufficient TNF-alpha mRNA to be detected by using the probe developed for our studies. Together, these findings identify a novel mechanism by which eosinophils may contribute to mucosal inflammation and provide an approach to future investigation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF), a 23-kDa polypeptide, was originally identified as a hematopoietic growth factor, but has recently been found to be a multifunctional cytokine with many proinflammatory activities (1,2). GM-CSF can be produced by, and act upon, a broad range of cell types, including both immature and mature granulocyte and monocyte lineage cells, dendritic cells, and tissue structural cells. Abundant in vitro observations have suggested that GM-CSF is able to induce both differentiation and activation of these cells (1).

The majority of biologic functions assigned to cytokines have been characterized by in vitro assay systems which may not necessarily reflect cytokine roles in vivo. Recently, recombinant virus approaches have allowed tissue-specific expression of foreign gene products in experimental animal models. We have constructed recombinant human type 5 adenoviruses, deficient in the E3 region of the genome, with incorporated rodent IL-6 cDNA that express significant levels of biologically active IL-6 on infection both in vitro and in vivo. After i.p. injection, the liver, spleen, and peritoneum appear to be primary sites of expression, whereas the lung and bronchus are the main sites of expression after intratracheal instillation. Injection i.p. of BALB/c mice with the murine rIL-6 virus causes an increase in serum levels of bioactive IL-6 for up to 6 days post-infection, whereas similar changes are not seen in animals infected with control viruses. Coincident with enhanced plasma levels of IL-6, we detect raised serum levels of hepatic-derived acute phase proteins. Associated with the expression of IL-6 in the liver and spleen, at 7 days we note a fourfold splenomegaly with expansion of B and T cell compartments, as well as the presence of lymphoid aggregates in the liver. These morphologic changes had resolved by 16 days. Our findings demonstrate that recombinant human type 5 adenoviruses expressing cDNA for various cytokines could be used as a transient pseudo-transgenic animal model to investigate the biologic function of cytokines in vivo.

Tissue inhibitor of metalloproteinases (TIMP-1) is a potent inhibitor of activated matrix metalloproteinases (MMP) such as collagenase, stromelysin, and gelatinase, and thus helps to control extracellular matrix metabolism and deposition by connective tissue cells. Since various cytokines and growth factors can modify the production of MMP and TIMP-1, we explored the action of oncostatin M (OM), a unique lymphocyte- and monocyte-derived cytokine, on expression of these proteins. We examined the regulation of TIMP-1 expression in cultured human fibroblasts by cytokines including OM, IL-6, leukemia inhibitory factor (LIF), and IL-1 alpha. When used at levels of 5 to 50 ng/ml, OM, IL-6, LIF, and IL-1 alpha elevated the TIMP-1 expression at the RNA level in fibroblasts of lung or synovial origin. Interestingly, OM stimulation resulted in highest levels of TIMP-1 RNA and protein synthesis. However, unlike IL-1 alpha, the cytokines OM, IL-6, and LIF did not induce MMP or PGE2 release. OM also enhanced TIMP-1 mRNA levels in the H2981 lung carcinoma and HepG2 hepatoma cell lines. The results suggest that OM as well as IL-6 and LIF, other cytokines acting through similar receptor pathways, may act to inhibit net MMP activity by specifically up-regulating TIMP-1 expression. The selective induction of TIMP-1 by OM may be influential in altering matrix destruction in chronic inflammation and tumor metastasis.

The contributing role of hepatocytes and IL-6 in the acute phase-like elevation of plasma type 1 plasminogen activator inhibitor (PAI-1) in vivo is not known. We addressed this question by comparing the effects of two inflammatory stimuli known to increase plasma concentrations of IL-6 on the plasma concentrations and site of synthesis of PAI-1 and cysteine proteinase inhibitor (CPI) in rats. Subcutaneous injection of turpentine results in a sustained increase in plasma IL-6 and CPI Ag levels over a 24-h period. In contrast, plasma PAI-1 activity was not altered by turpentine treatment. Northern blot analysis of poly(A)+ mRNA extracted from freshly isolated hepatocytes of saline- or turpentine-treated animals demonstrated induction of CPI mRNA expression but failed to detect basal or induced PAI-1 or IL-6 mRNA expression. However, PAI-1 mRNA was detected in rat hepatocytes in primary culture for 24 h and was induced by dexamethasone. Intravenous infusion of bacterial LPS (4 mg/kg) induced a sustained increase in plasma CPI Ag and transient increases in plasma IL-6 and PAI-1. Northern blot analysis of freshly isolated, fractionated liver cells indicated that LPS treatment (3 h) induced PAI-1 mRNA expression most significantly in the endothelial and Kupffer cell fractions. IL-6 mRNA expression was induced in Kupffer cells and CPI mRNA was induced in hepatocytes. Immunocytochemical analysis revealed LPS-induced accumulation of PAI-1 Ag associated with the vascular endothelium, subendothelial matrix, and sinusoidal lining cells. Our results indicate that PAI-1 mRNA is not significantly expressed by rat hepatocytes in vivo and that plasma PAI-1 levels are not influenced by increased IL-6 expression in Kupffer cells or in plasma.

The induction of thrombocytopenia results in elevated levels of thrombopoietin (TPO), which can be detected in the plasma of experimental animals. Acute, severe thrombocytopenia (platelet count less than 5% of control) was produced in mice by the administration of either guinea pig or rabbit antimouse platelet antiserum. Control mice received equal volumes of normal serum. At various times after the induction of thrombocytopenia (0.5, 1, 2, 3, 4, 6, 12, and 24 hours) citrated plasma was collected, and circulating interleukin-6 (IL-6) levels were measured using the IL-6-dependent murine hybridoma cell line B9. At no time points after induction of thrombocytopenia were plasma IL-6 levels significantly different from control animals that received normal serum. However, injection of heterologous serum did result in slightly elevated plasma IL-6 levels (at 2 and 3 hours) compared with basal levels measured in uninjected animals. This brief increase was not related to the production of thrombocytopenia. Protein fractions from the plasma of thrombocytopenic rabbits were also tested for the presence of IL-6. Preparations that contained TPO, as shown by stimulation of megakaryocyte maturation in vitro, did not contain detectable levels of IL-6. The ability of the B9 assay to detect the elevation of IL-6 levels in murine or rabbit plasma was verified after the administration of bacterial endotoxin, which is known to increase circulating IL-6 concentrations. IL-6 levels were highly elevated in rabbit or mouse serum after the administration of 5 mg/kg or 1 mg/kg of endotoxin, respectively. Anti-IL-6 antiserum did not neutralize the in vitro megakaryocyte maturation activity of partially purified TPO from the plasma of thrombocytopenic rabbits. In addition, IgG purified from the same antiserum did not neutralize partially purified TPO, as shown after incubation with TPO and subsequent precipitation with agarose- bound protein A. These results show that, unlike TPO, levels of IL-6 do not increase after the induction of acute, severe thrombocytopenia, and strongly suggest that IL-6 does not mediate the thrombopoietic response to acute thrombocytopenia. Although prolonged administration of IL-6 has been shown to induce thrombocytosis, IL-6 and TPO are apparently different and immunologically distinct molecules.

We have reported an accumulation of T-kininogen mRNA in the liver of aging Sprague-Dawley rats. T-kininogen is a cysteine proteinase inhibitor. Since a disruption of the intracellular protein degradation machinery is known to occur during senescence, we wished to further define the role of this protein in the aging process. As a first step, we have measured T-kininogen levels both in serum and within the liver. We have found that serum protein levels are indeed augmented during senescence, although not as dramatically as the mRNA (2.5-fold versus 8.3-fold). Immunocytochemistry, as well as Western blot analysis suggests that this is due to the presence of T-kininogen within hepatic cells in aged rats. Life-long dietary restriction, a known age-prolonging treatment, decreases the overexpression of the protein in 24-month-old rats. Later, diet-restricted animals still show an increased expression from the gene, the effect being delayed but not abolished by dietary manipulation. Interestingly, a longitudinal study indicated the existence of a positive correlation between the time of increase of serum T-kininogen and the time of death of the animal. Serum T-kininogen was found to increase 2.5-4 months before death.

Acute inflammation is characterized by increased liver output of acute phase proteins (APP). Several cytokines including IL-6, leukemia inhibitory factor, and IL-11 are capable of stimulating APP synthesis by hepatocytes and hepatoma cells. We have tested the activity of a separate and unique cytokine oncostatin M (OM) and have found potent APP-inducing activity of human recombinant OM on hepatocytes. OM acted in a dose-dependent fashion (ED50 5 to 10 ng/ml) in stimulating APP synthesis in human HepG2 cells, rat H35 cells, and primary rat hepatocyte cultures, but not human Hep3B cells. Human OM induced equivalent to or greater responses than IL-6 in HepG2 cells, however, it was less effective than human IL-6 in stimulating rat cells. Northern analysis showed that OM stimulated mRNA levels of haptoglobin and alpha 1-antichymotrypsin in HepG2 cells. OM induced CAT activity in HepG2 cells transfected with CAT constructs containing IL-6-responsive elements, suggesting that OM induces transcription of native proteins through mechanisms involving IL-6-responsive element-like sequences in gene promoters. OM was also shown to act additively with IL-6 or leukemia inhibitory factor and synergistically with glucocorticoid or IL-1 in the induction of specific APP. These results suggest that OM plays a role as a mediator of APP synthesis in inflammatory responses.

Interleukin 6 (IL-6) is a pluripotent cytokine with multiple effects on many different cell types. It is produced by a variety of cells in response to immunological and other stimuli. This unit describes a simple and sensitive assay for human, rat, rabbit, pig, and mouse IL-6, based on IL-6-dependent proliferation of a murine B cell hybridoma cell line, B9. Support protocols discuss maintenance of B9 cells, preparation of IL-6 standards, and production of IL-6-containing supernatant. In addition, IL-6 ELISA kits for the measurement of human or mouse IL-6 are available from a number of companies. These products are reliable, highly sensitive, and specific, and thus should be considered as an excellent (although more expensive) alternative, keeping in mind that bioactivity is not assessed with this approach.

Transforming growth factor (TGF)-beta signalling plays important roles in regulating lung development. However, the specific regulatory functions of TGF-beta signalling in developing lung epithelial versus mesenchymal cells are still unknown. By immunostaining, the expression pattern of the TGF-beta type II receptor (TbetaRII) was first determined in the developing mouse lung. The functions of TbetaRII in developing lung were then determined by conditionally knocking out TbetaRII in the lung epithelium of floxed-TbetaRII/surfactant protein C-reverse tetracycline transactivator/TetO-Cre mice versus mesenchyme of floxed-TbetaRII/Dermo1-Cre mice. TbetaRII was expressed only in distal airway epithelium at early gestation (embryonic day (E)11.5), but in both airway epithelium and mesenchyme from mid-gestation (E14.5) to post-natal day 14. Abrogation of TbetaRII in mouse lung epithelium resulted in retardation of post-natal lung alveolarisation, with markedly decreased type I alveolar epithelial cells, while no abnormality in prenatal lung development was observed. In contrast, blockade of TbetaRII in mesoderm-derived tissues, including lung mesenchyme, resulted in mildly abnormal lung branching and reduced cell proliferation after mid-gestation, accompanied by multiple defects in other organs, including diaphragmatic hernia. The primary lung branching defect was verified in embryonic lung explant culture. The novel findings of the present study suggest that transforming growth factor-beta type II receptor-mediated transforming growth factor-beta signalling plays distinct roles in lung epithelium versus mesenchyme to differentially control specific stages of lung development.

Transforming growth factor-β (TGF-β)1, 2 and 3 peptide superfamily signaling is not only essential for both prenatal and postnatal lung morphogenesis, but also plays a key role in the pathobiology of bronchopulmonary dysplasia, pulmonary fibrosis and emphysema. The respective null mutations of TGF-β1 reveals its function to protect against lung inflammation, of TGF-β2 in cardiopulmonary morphogenesis and of TGF-β3 in lung and palatal fusion. TGF-β signal transduction is tightly regulated at all levels from ligand bioavailability in the extracellular space to the nucleus. Protease-antiprotease balance, correct final assembly of lung matrix and hence completion of alveolarization are all important normal functions of the TGF-β signaling pathway. The consequences of excess TGF-β signaling depend on the developmental stage of the lung: alveolar hypoplasia and fibrosis in the growing lung, fibrosis in the adult lung. While inflammation can induce excessive TGF-β signaling, lung fibrosis per se is inflammation independent and mediated by excessive TGF-β and Smad3 signaling. Therapeutic manipulation of TGF-β-Smad3 function is therefore a rational target. However, its application to pulmonary medicine will not be easy because of the narrow therapeutic range and many-layered physiological regulation of this pathway.

We have previously shown that IL-6 is the major monocyte- and fibroblast-derived regulator of acute phase protein gene expression and synthesis in hepatocytes in inflammation. Recently, we and others have shown that rat and human hepatoma cells express IL-6 mRNA, and the question arose as to whether normal hepatocytes express IL-6 and whether any such expression occurs under normal physiologic conditions or is seen in inflammation. Poly A+ mRNA of liver from normal rats and from rats undergoing an acute phase response was not positive when probed with cDNA for rat IL-6 under conditions in which macrophage mRNA was strongly positive. We then compared poly A+ mRNA from purified hepatocytes freshly isolated from normal rats--from rats that were undergoing an acute inflammatory response and from freshly isolated normal hepatocytes that had been cultured for 24 h in the presence or absence of dexamethasone (microM). Only the mRNA from normal hepatocytes cultured for 24 h in the absence of any glucocorticoid was obviously positive for IL-6. The increased expression of gamma-fibrinogen mRNA indicated the presence of inflammation. These results confirm the identification of IL-6 as an exogenous hormone for regulating normal hepatic acute phase protein synthesis in inflammation and rules out an autocrine mechanism being active in the liver in normal homeostasis.

We determined the histochemical characteristics of nonspecific esterase in different populations of rat macrophages. The cells included alveolar and peritoneal macrophages recovered by lavage and a mixed cell population obtained by collagenase digestion of the small intestine. The histochemically localized enzyme activity of alveolar and peritoneal macrophages was cytoplasmic, diffuse, and inhibited by sodium fluoride. Both populations were effectively stained using alpha-naphthyl acetate and alpha-naphthyl butyrate as the esterase substrate. When the intestinal cells were examined for activity, a greater percentage of cells showed positive nonspecific esterase than would be predicted by differential counts for macrophages on the basis of morphological criteria. We confirmed, using cell smears and tissue sections, that rat intestinal epithelial cells, a prominent component of the isolated cell population, possessed esterases that react similarly to macrophage esterases with histochemical procedures.

Emphysema is a major health problem and novel drugs are needed. Animal disease models are pivotal in their development, but the validity and sensitivity of current tools for the evaluation of drug efficacy is limited. The usefulness of micro computed tomography (CT) as an innovative tool to assess emphysema in a mouse model was investigated. Serial CT scans were performed in bi-weekly intervals in Smad3 knockout (KO) mice, which spontaneously develop airspace enlargement. Lung density was quantified in two- and three-dimensional images and correlated to mean linear intercept and lung compliance. CT scans of Smad3 KO lungs revealed a significant decrease in lung density at age 8 weeks and a further progression at age 14 weeks with respect to age-matched wild-type (WT) animals. Emphysema could be reliably assessed with both the two- and three-dimensional approach, but the three-dimensional approach was superior, due to normalisation to lung volumes and less variability. Lung compliance by week 14 was 0.053+/-0.005 and 0.034+/-0.002% of maximum volume.cmH(2)O(-1) for KO and WT mice, respectively, reflecting significant physiologically relevant emphysema. Small animal computed tomography imaging and density quantification in a reconstructed three-dimensional image is a useful tool for quantifying emphysematous changes in an animal disease model. It adds significant information to conventional assessment.