Ann N Y Acad Sci 1996 Oct 31;796:235-244
Department of Pathology, McMaster University, Hamilton, ON, Canada.
In summary, we have been able to demonstrate that adenovirus vectors are valuable tools in examining the roles played by individual cytokines in lung responses and inflammation. These viral vectors have marked trophism for the epithelial cells of the lung and are highly efficient in transferring the genes into these lining cells. This results in significant expression of cytokines both within the lumen and the parenchyma of the lung. As a result of an individual cytokine being overexpressed, there are cytokine-specific changes seen-IL-6 resulting in lymphocytosis, MIP2 resulting in neutrophil accumulation, RANTES resulting in monocyte accumulation, TGF beta resulting in monocytosis but no fibrosis, and GM-CSF most surprisingly resulting in tissue eosinophilia, granuloma formation, and subsequently the onset of fibrosis. These vectors have helped pinpoint the role of a number of the cytokines in inducing chronic inflammatory changes to the lung and imply that a single cytokine may not be the only trigger resulting in chronic changes within the lung parenchyma.
Publication Types:
PMID: 8906231, UI: 97062492
MeSH Terms:
Substances:
Other Formats: Links:
Am J Respir Crit Care Med 1996 Oct;154(4 Pt 1):1092-1098
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
Secretory leukocyte proteinase inhibitor (SLPI) and alpha1-proteinase inhibitor (alpha1(PI)) cannot fully explain the total neutrophil elastase (NE) inhibitory capacity detected in bronchoalveolar lavage (BAL) fluid, suggesting the existence of other NE inhibitor(s). In the present study, we measured the concentrations of elafin, a newly described, low-molecular-weight serine proteinase inhibitor, SLPI, and alpha1(PI) in BAL fluids from eight healthy subjects, 13 asymptomatic farmers, seven farmers with active farmer's lung (FL), and seven farmers with previous (Ex) FL. In addition to SLPI and alpha1(PI), elafin was present in BAL fluids from control subjects and asymptomatic farmers, 13 (7-31) and 12 (7-67) mmol/mol of albumin (median and range) respectively. Elafin concentration increased significantly to 105 (38-207) mmol/mol of albumin in farmers with active FL and was also elevated in farmers with Ex FL. Elafin levels were highly correlated with lung inflammatory cell numbers, especially lymphocytes, and the decrease in single-breath diffusion capacity (DLCO). Elafin and SLPI were linked to yet uncharacterized proteins in BAL fluids. In conclusion, elafin is a constituent of BAL fluid from normal subjects and is found in enhanced concentrations in FL and in farmers with lymphocytic alveolitis. This suggests that elafin may play a role in lung homeostasis and inflammation.
PMID: 8887613, UI: 97042413
Am J Pathol 1996 Oct;149(4):1395-1403
Replication-defective adenoviral vectors are capable of localized transfer and expression of incorporated gene product in lung tissue. We have constructed an adenoviral vector that expresses rat macrophage inflammatory protein (MIP)-2, a C-X-C chemokine specifically chemotactic for neutrophils, Supernatants from 293 cells, infected with the adenoviral MIP-2 (ADMIP-2) construct, showed potent chemotactic activity and the ability of the ADMIP-2 vector to transcribe and make functional protein was confirmed. In vivo analysis of bronchoalveolar lavage fluid from rats after intratracheal instillation of ADMIP-2 (10(9) plaque-forming units) showed a 10-fold increase in the absolute number of neutrophils in bronchoalveolar lavage fluid as opposed to rats treated with an equal titer of an E1-disabled control virus expressing firefly luciferase (ADCA-18). Neutrophils constituted 65% of total BAL cells with alveolar macrophages being the other major cell type recovered. Rat MIP-2 protein was increased (nanograms per milliliter) in bronchoalveolar lavage fluid over a period of 7 days in ADMIP-2-treated animals. MIP-2 mRNA was demonstrated by Northern blot analysis in lung tissue, and histological analysis confirmed the presence of massive localized tissue neutrophilia. Evidence of chronic tissue injury and repair (ie, fibrosis) was not detected up to 2 weeks after the neutrophil infiltrate had resolved, subsequent to decreased chemokine presence. Adenoviral gene transfer proved an effective tool for the assessment of lung tissue expression of this chemokine in vivo and is useful in developing rodent models of tissue neutrophilia.
PMID: 8863686, UI: 97017073
Protein Expr Purif 1996 May;7(3):269-274
Department of Pathology, Molecular Virology and Immunology Program, McMaster University, Hamilton, Ontario, Canada.
Rat interleukin-6 (IL-6) cDNA, coding for an important inflammation- and immune-regulatory polypeptide cytokine, was cloned into the novel expression vector pH6EX3 which directs the synthesis of inserted genes as a fusion protein with histidine hexapeptide (HH). The resultant vector (pRIL6.992) was shown to produce significant amounts of recombinant rat IL-6 fusion protein with HH at its N-terminus in various strains of Escherichia coli. The expression of the HH-IL-6 fusion protein was demonstrated to be under the control of the tac promoter and could be induced by IPTG. This protein was isolated from bacterial inclusion bodies and purified to homogeneity in a one-step procedure by affinity chromatography using a nickel-chelating column. The HH-IL-6 fusion protein isolated in this manner was biologically active as determined by hepatocyte stimulation and B9 hybridoma growth assays. Further, this activity was neutralized with a polyclonal antiserum raised against rat IL-6 protein generated in a novel fashion from rabbits infected with a recombinant human type-5 adenovirus vector expressing rat IL-6 protein (Ad5E3rIL6). The recombinant HH-IL-6 protein was then used to boost Ad5E3rIL6-immunized rabbits. This resulting antiserum was shown to neutralize recombinant and natural rat and murine IL-6 bioactivity in vitro and was useful in Western blot analysis and immunohistochemistry of rat IL-6.
PMID: 8860652, UI: 97013816
Cytokine 1996 Apr;8(4):283-287
Recently we examined the expression of the IL-6 receptor (gp80) in three different models of acute inflammation [Geisterfer et al., 1993, Cytokine 5:1]. To continue these studies, we examined the mRNA expression of signal transducing molecule, gp130, and the LIF-R as associated members of a receptor family. Rats were treated with either Freund's complete adjuvant (FA) via intraperitoneal injection, LPS via intravenous injection, or turpentine via subcutaneous injection. The levels of gp130 and LIF-R mRNA expression had a maximum 2-3-fold increase over a 24 h period. However, the time of the maximum increase differed depending on the treatment the rats received. FA treated rats had a maximum induction of gp130 mRNA levels of 2.2-fold (for the 7.5 kb transcript) and 1.3-fold (for the 9.0 kb transcript) at 12 h. LPS treated rats had a maximum increase at 3 h where message levels increased 2.5-fold (7.5 kb) and 1.2-fold (9.0 kb). Turpentine-injected rats showed little difference in gp130 mRNA levels at any time after injection compared to controls. Maximum LIF-R mRNA levels also differed depending on the type of treatment the rats received. FA-injected rats showed a 2.1-fold mRNA increase at 3 h, whereas LPS treated rats show a maximum 2.4-fold increase at 18 h. Turpentine-injected rats showed little increase in mRNA levels compared to controls. Injection of purified recombinant rat IL-6 (rIL-6) had little effect on LIF-R mRNA levels, but had a dramatic inducing effect on gp130 mRNA levels. Rats were also injected (i.p.) with Dexamethasone (Dex) and this had no effect on either gp130 or LIF-R mRNA level expression. These in vivo results indicated that IL-6 has a major role in the regulation of its own receptors, IL-6R (gp80), and gp130, and the onset of acute phase response. We found that the maximum IL-6R (gp80), gp130 and LIF-R mRNA levels peaked at different times depending on the type of acute inflammation induced in the rat. It seems that various combinations of cytokines and hormones are released depending on the type of acute inflammation, and these in turn regulate the expression of diverse receptors on the hepatocyte, resulting in different acute phase kinetics in the various models of inflammation.
PMID: 9162216, UI: 97012545
J Leukoc Biol 1996 Apr;59(4):481-488
Using adenoviral-mediated gene transfer techniques, the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) transgene is efficiently targeted to and highly expressed by the respiratory epithelium of rat lung. This lung tissue-directed expression of GM-CSF induces accumulation of both eosinophils and macrophages at early stages and an irreversible fibrotic reaction at later stages. These tissue responses to GM-CSF appear to be distinct from those induced by other proinflammatory cytokines, interleukin (IL)-5, IL-6, macrophage inflammatory protein-2 (MIP-2), or RANTES overexpressed in the lung. These findings clearly demonstrate that GM-CSF is more than a hematopoietic cytokine in the lung and may play a pivotal role in the multiple pathological processes underlying numerous respiratory illnesses, including asthma. In this overview, the differences in tissue responses induced by GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF and other individual cytokines are highlighted. In addition, the mechanisms by which GM-CSF contributes to the development of eosinophilia, macrophage granuloma, and fibrosis are discussed in conjunction with the recent findings from us and others.
PMID: 8613693, UI: 96201614
Exp Lung Res 1996 Mar;22(2):231-244
Department of Pathology, Chedoke-McMaster Medical Centre, McMaster University, Hamilton, Ontario, Canada.
Fibroblast heterogeneity has been documented in fibrotic tissue from lung and skin. Differences have been demonstrated in proliferative rates in fibroblasts derived from fibrotic lung tissue as compared to normal. Fibroblast lines derived from adult fibrotic lung tissue and neonatal normal lung tissue exhibit colony growth in soft agarose culture, whereas fibroblast cell lines from normal adult lung tissue do not. The characteristic of anchorage-independent growth is consistent with the aggressive nature of the disease and with developmental lung growth. In this study, fibrotic lung fibroblasts were exposed to growth and differentiating factors to determine whether the anchorage-independent phenotype can be modulated. The results indicate that treatment of fibrotic lung fibroblasts with retinoic acid, known to modify matrix gene expression and induce differentiation, inhibits the cells ability to form colonies under soft agarose growth. Treatment with all-trans-retinoic acid yielded the greatest effect inhibiting both IPF and neonatal lung fibroblast anchorage-independent growth approximately 90% at 10(-6) M. Treatment of IPF fibroblasts with all-trans-retinoic acid also inhibited corticosteroid-induced colony growth. Modulation of the "fibrotic" fibroblast phenotype through retinoid therapy may prove beneficial as a potential therapeutic strategy.
PMID: 8706638, UI: 96265461
J Clin Invest 1996 Feb 15;97(4):1102-1110
Department of Pathology, Health Sciences Center, McMaster University, Hamilton, Ontario, L8N3Z5, Canada.
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.
PMID: 8613534, UI: 96193895
Hum Gene Ther 1996 Feb 10;7(3):333-342
Department of Biology, McMaster University, Hamilton, Ontario, Canada.
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.
PMID: 8835220, UI: 96432154