Organic dust-induced lung injury and repair: Bi-directional regulation by TNFα and IL-10.

Exposure to organic dust increases chronic airway inflammatory disorders. Effective treatment strategies are lacking. It has been reported that hog barn dust extracts (HDE) induce TNFα through protein kinase C (PKC) activation and that lung inflammation is enhanced in scavenger receptor A (SRA/CD204) knockout (KO) mice following HDE. Because interleukin (IL)-10 production can limit excessive inflammation, it was hypothesized here that HDE-induced IL-10 would require CD204 to effect inflammatory responses. C57BL/6 wild-type (WT), SRA KO, and IL-10 KO mice were intranasally challenged daily for 8 days with HDE and subsequently rested for 3 days with/without recombinant IL-10 (rIL-10) treatment. Primary peritoneal macrophages (PM) and murine alveolar macrophages (MH-S cells) were treated in vitro with HDE, SRA ligand (fucoidan), rIL-10, and/or PKC isoform inhibitors. HDE induced in vivo lung IL-10 in WT, but not SRA KO mice, and similar trends were demonstrated in isolated PM from same treated mice. Lung lymphocyte aggregates and neutrophils were elevated in in vivo HDE-treated SRA and IL-10 KO mice after a 3-d recovery, and treatment during recovery with rIL-10 abrogated these responses. In vitro rIL-10 treatment reduced HDE-stimulated TNFα release in MH-S and WT PM. In SRA KO macrophages, there was reduced IL-10 and PKC zeta (ζ) activity and increased TNFα following in vitro HDE stimulation. Similarly, blocking SRA (24 hr fucoidan pre-treatment) resulted in enhanced HDE-stimulated macrophage TNFα and decreased IL-10 and PKCζ activation. PKCζ inhibitors blocked HDE-stimulated IL-10, but not TNFα. Collectively, HDE stimulates IL-10 by an SRA- and PKCζ-dependent mechanism to regulate TNFα. Enhancing resolution of dust-mediated lung inflammation through targeting IL-10 and/or SRA may represent new approaches to therapeutic interventions.

Epistatic effect of TLR-1, -6 and -10 polymorphisms on organic dust-mediated cytokine response.

Exposure to organic dust from agricultural environments is associated with inflammatory respiratory conditions. The putative causal agents in organic dust include viral, microbial and fungal components, which are recognized by the family of Toll-like receptors (TLRs) and drive host innate and adaptive responses. Our aim in this study was to determine whether responsiveness to organic dust among agricultural workers was dependent on polymorphisms in the TLR10-TLR1-TLR6 gene cluster. We stimulated whole blood from 509 agricultural workers with organic dust, triacyl lipopeptide N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4 (Pam3CSK4) and the diacyl-lipopeptide peptidoglycan. Several of the tagging polymorphisms and haplotypes conferred hyper-responsiveness to organic dust with an increase in interleukin-6 (IL-6; P<0.005), but not tumor necrosis factor-α (TNF-α), secretion. We conclude that genetic variation in the TLR10-TLR1-TLR6 gene cluster mediates responsiveness to organic dust, but indicates different signaling pathways for IL-6 and TNF-α. These studies provide new insight into the role of the TLR10-TLR1-TLR6 gene cluster and the innate immune response to organic dust.

Effect of elevated carbon dioxide on bronchial epithelial innate immune receptor response to organic dust from swine confinement barns.

Hypercapnia is known to have immunoregulatory effects within the lung. Cell culture systems demonstrate this in both macrophages and alveolar cell lines, suggesting that the alveoli are affected by changes in CO2 levels. We hypothesized that hypercapnia would also modulate human bronchial epithelial cell immune responses. Innate immune responses to Pam3CSK4 (TLR2 ligand), LPS (TLR4 ligand) and a complex innate immune stimulus, an extract from the organic dust of swine confinement barns (barn dust extract or BDE), were tested in a human bronchial epithelial cell line, BEAS-2B. Both TLR ligands showed a decrease in IL-6 and IL-8 production, and an increase in MCP-1 in response to elevated CO2 indicating an enhancement in cytokine production to hypercapnia. This change was not reflected in expression levels of TLR receptor RNA which remained unchanged in response to elevated CO2. Interestingly, barn dust showed an increase in IL-6, IL-8 and MCP-1 response at 9% CO2, suggesting that elevated CO2 exerts different effects on different stimuli. Our results show that airway epithelial cell immune responses to barn dust respond differently to hypercapnic conditions than individual TLR ligands.

CXCR1/CXCR2 antagonist CXCL8(3-74)K11R/G31P blocks lung inflammation in swine barn dust-instilled mice.

Inhalation of agricultural occupational dusts from swine confinement facilities can result in lung inflammation. The innate immune response to organic barn dusts results in production of a number of pro-inflammatory factors in the lungs of barn workers such as cytokines, chemokines, and an influx of neutrophils. Many of these inflammatory factors are influenced by the chemokine CXCL8/IL-8 (KC or MIP-2 in mice). Previously, we have demonstrated that an endotoxin-independent component of swine barn dust extract (SBE) elevates lung chemokines in a protein kinase C (PKC)-dependent manner resulting in the significant formation of lung inflammatory cell infiltrates in a mouse model of SBE injury. In this study we test the ability of a CXCR1/CXCR2 antagonist, CXCL8(3-74)K11R/G31P (G31P) to block many of the features of lung-inflammation in response to challenge with SBE in an established mouse exposure system. Injection of G31P concurrent with SBE nasal instillation over a course of 3 weeks significantly reduced neutrophil accumulation in the lungs of barn dust exposed animals compared to those given SBE alone. There was a similar reduction in pro-inflammatory cytokines and chemokines IL-6, KC, and MIP-2 in SBE plus G31P-treated mice. In addition to excreted products, the receptors ICAM-1, CXCR1, and CXCR2, which all were elevated with SBE exposure, were also decreased with G31P treatment. SBE activation of PKCα and PKCε was reduced as well with G31P treatment. Thus, G31P was found to be highly effective at reducing several features of lung inflammation in mice exposed to barn dust extracts.

Toll-like receptor 2 is upregulated by hog confinement dust in an IL-6-dependent manner in the airway epithelium.

Hog confinement workers are at high risk to develop chronic bronchitis as a result of their exposure to organic dust. Chronic bronchitis is characterized by inflammatory changes of the airway epithelium. A key mediator in inflammation is Toll-like receptor 2 (TLR2). We investigated the role of TLR2 in pulmonary inflammation induced by hog confinement dust. Normal human bronchial epithelial cells (NHBE) were grown in culture and exposed to hog confinement dust extract. Hog confinement dust upregulated airway epithelial cell TLR2 mRNA in a concentration- and time-dependent manner using real-time PCR. There was a similar increase in TLR2 protein at 48 h as shown by Western blot. TLR2 was upregulated on the surface of airway epithelial cells as shown by flow cytometry. A similar upregulation of pulmonary TLR2 mRNA and protein was shown in a murine model of hog confinement dust exposure. Hog confinement dust is known to stimulate epithelial cells to produce IL-6. To determine whether TLR2 expression was being regulated by IL-6, the production of IL-6 was blocked using an IL-6-neutralizing antibody. This resulted in attenuation of the dust-induced upregulation of TLR2. To further demonstrate the importance of IL-6 in the regulation of TLR2, NHBE were directly stimulated with recombinant human IL-6. IL-6 alone was able to upregulate TLR2 in airway epithelial cells. Hog confinement dust upregulates TLR2 in the airway epithelium through an IL-6-dependent mechanism.

Exposure to hog barn dust alters airway epithelial ciliary beating.

Swine confinement workers are at increased risk of airway diseases, including mucus membrane irritation syndrome, chronic rhinosinusitis and chronic bronchitis. Dust extracts from swine confinement facilities stimulate the production of pro-inflammatory cytokines in bronchial epithelial cells, including interleukin (IL)-8. As IL-8 is capable of blocking beta-agonist-stimulated increases in cilia beating, which impacts on mucociliary clearance, it was hypothesised that hog barn-dust exposure might alter cilia responses to stimulation. To test this hypothesis, ciliated bovine bronchial epithelial cell cultures were exposed to hog barn-dust extract (HDE) and ciliary beat frequency (CBF) was assayed. An elevation in baseline CBF was observed. This effect appeared to be independent of endotoxin but dependent upon nitric oxide. HDE also stimulated nitric oxide production in bronchial epithelial cells; however, stimulation of cilia beating by a beta-agonist did not occur in cells pre-exposed to HDE. These data demonstrate that hog barn dust can alter normal stimulation of cilia, suggesting a mechanism for the abrogation of stimulated increases in mucociliary clearance in response to inhaled dust exposure.

IL-8 inhibits isoproterenol-stimulated ciliary beat frequency in bovine bronchial epithelial cells.

Mucociliary clearance is a critical host defense that protects the lung. The mechanisms by which mucociliary function is altered by inflammation are poorly defined. Chronic exposure to cigarette smoke decreases ciliary beating and interferes with proper airway clearance. Bronchoalveolar lavage (BAL) fluid from smokers and ex-smokers has increased amounts of IL-8, which has played a critical role in airway inflammation. We hypothesized that IL-8 might interfere with stimulated ciliary beating in airway epithelium. To test this hypothesis, we stimulated bovine ciliated bronchial epithelial cells (BBECs) with a known activator of ciliary beat frequency (CBF), isoproterenol (ISO; 100 microM), in the presence or absence of IL-8 (100 pg/mL). We measured CBF digitally using the Sisson-Ammons Video Analysis (SAVA) system. CBF increased in untreated cells exposed to ISO (approximately 3 Hz) over baseline. In contrast, cells pre-incubated with IL-8 failed to respond to ISO. Pretreatment with IL-8 also blocked ISO-stimulated cAMP-dependent kinase (PKA) activation, which is known to control ISO-stimulated CBF. In addition, IL-8 pretreated cells revealed a marked decrease in PKA activity when cells were stimulated with forskolin (FSK; 10 microM). Cells were assayed specifically for cAMP-phosphodiesterase (PDE) activity. ISO-stimulated cells demonstrated an increase in PDE activity as compared to control. Pretreatment with IL-8 had no effect on ISO-stimulated PDE activity. Collectively, these data suggest that IL-8 appears to mediate its effect at the level of adenylyl cyclase. It is also possible that IL-8 may not only act as a chemotactic agent, but also as a potential autocrine/paracrine inhibitor of PKA-mediated stimulation of ciliary motility. In conclusion, IL-8 inhibits beta-agonist dependent ciliostimulation and such inhibition of stimulated ciliary activity may contribute to the impaired mucociliary clearance seen in airway diseases. Furthermore, since IL-8 levels are increased in the airway of cigarette smokers, it is likely they may be more resistant to the cilio and muco-ciliostimulating effects of beta-agonists.

Hog barn dust extract augments lymphocyte adhesion to human airway epithelial cells.

The dust of hog confinement facilities induces airway inflammation. Mechanisms by which this dust modulates inflammation are not completely defined, although it is clear that exposure to dust can modulate both epithelial cell and inflammatory cell function. In this work, we demonstrate that airway epithelial cell (BEAS-2B) treatment with hog barn dust extract (HDE) results in augmentation of peripheral blood lymphocyte adhesion to epithelial cell cultures in vitro. The augmentation of lymphocyte adhesion to epithelial cells is dependent on the concentration of HDE and time of HDE exposure, with twofold increases observed by 3 h and maintained at 24 h. Similar results are seen with primary human bronchial epithelial cells in culture. Lymphocyte adhesion to epithelial cells is inhibited in a concentration-dependent fashion by the treatment of epithelial cells with antibody to intercellular adhesion molecule-1 (ICAM-1). In addition, HDE exposure of epithelial cells results in an approximate twofold increase in ICAM-1 expression as determined by flow cytometry analysis. Pretreatment of epithelial cells with a protein kinase C-alpha (PKC-alpha) inhibitor, Gö-6976, also inhibited subsequent lymphocyte adhesion to HDE-exposed epithelial cells. These data suggest that airway epithelial cell HDE exposure enhances subsequent lymphocyte adhesion to epithelial cells that is mediated in part by HDE modulation of ICAM-1 expression and PKC-alpha.

Gamma radiation inhibits fibroblast-mediated collagen gel retraction.

Radiation exposure is known to impair healing in irradiated areas. Fibroblasts play a major role in the production and modification of extracellular matrix in wound repair. Since one important aspect of wound repair is the contraction of the wound, this study investigated the effects of radiation on the ability of fibroblasts to mediate collagen gel contraction in an in vitro model of wound retraction. After irradiation, the cells were detached and suspended in a solution of rat tail tendon collagen. Radiation exposure decreased retraction, and this effect was dose dependent. In order to define the mechanism of reduced gel retraction, we investigated alpha2beta1 cell surface integrin and fibronectin, which are thought to mediate contraction, and prostaglandin E2 (PGE2), which is known to inhibit this process. PGE2 release increased dose responsively following radiation. The cyclooxygenase inhibitor indomethacin could partially restore the contractile activity of irradiated fibroblasts. Fibronectin production in gel culture showed a significant decrease. In contrast, there was no decrease in alpha2beta1 integrin expression in radiated cells. In conclusion, radiation decreases fibroblast-mediated gel contraction. Increased PGE2 production and decreased fibronectin production by irradiated fibroblasts may contribute to this effect and may be in part responsible for poor healing of radiated tissue.

Red blood cells stimulate human lung fibroblasts to secrete interleukin-8.

Following lung injury, red blood cells (RBC) may interact with extracellular matrix (ECM). Fibroblasts, the resident cell in the ECM, have the capacity to produce and secrete a variety of mediators including interleukin-8 (IL-8). In the present study we hypothesized that RBC, or soluble factors released from them, may stimulate IL-8 production by fibroblasts. Fibroblasts were cultured in a three-dimensional collagen gel culture system in the presence or absence of RBC or conditioned medium from RBC (RBC-CM). IL-8 release from fibroblasts was significantly increased when cultured with RBC or RBC-CM and both tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) further stimulated this IL-8 secretion. The enhanced production of IL-8 within fibroblasts was accompanied by increased IL-8 mRNA expression. To evaluate whether RBC-fibroblast interaction may lead to recruitment of neutrophils, a functional migration assay was performed. RBC and RBC-CM, in the presence of IL-1beta and TNF-alpha, increased the transmigration of neutrophils. Our results indicate that RBC, when interacting with ECM, may participate in the recruitment of inflammatory cells by stimulating fibroblasts to secrete IL-8. This might be an important mechanism regulating tissue repair after injury.

Hog barn dust extract stimulates IL-8 and IL-6 release in human bronchial epithelial cells via PKC activation.

Hog barn workers have an increased incidence of respiratory tract symptoms and demonstrate an increase in lung inflammatory mediators, including interleukin (IL)-8 and IL-6. Utilizing direct kinase assays for protein kinase C (PKC) activation, we demonstrated that dust from hog confinement facilities, or hog dust extract (HDE), augments PKC activity of human airway epithelial cells in vitro. A 5% dilution of HDE typically stimulates an approximately twofold increase in human bronchial epithelial cell (HBEC) PKC activity compared with control medium-treated cells. This increase in PKC is observed with 15 min of HDE treatment, and kinase activity reaches peak activity by 1-2 h of HDE treatment before returning to baseline PKC levels between 6 and 24 h. The classic PKC inhibitor, calphostin C, blocks HDE-stimulated PKC activity and associated IL-8 and IL-6 release. Desensitization to HDE stimulation of PKC activation does not appear to occur because subsequent exposures to HDE after an initial exposure result in further augmentation of PKC. Detoxification of HDE with polymyxin B to remove endotoxin did not change PKC activation or IL-8 release, suggesting that endotoxin is not solely responsible for HDE augmentation of PKC. These data support the hypothesis that HDE exposure augments HBEC IL-8 and IL-6 release via a PKC-dependent pathway.

Cigarette smoke inhibits human bronchial epithelial cell repair processes.

By interfering with the ability of airway epithelial cells to support repair processes, cigarette smoke could contribute to alterations of airway structures and functions that characterize chronic obstructive pulmonary disease (COPD). The current study assessed the ability of cigarette smoke extract (CSE) to alter human airway epithelial cell chemotaxis, proliferation, and contraction of three-dimensional collagen gels, a model of extracellular matrix remodeling. The volatile components contained in cigarette smoke, acetaldehyde and acrolein, were able to inhibit all three processes. Nonvolatile components contained within lyophilized CSE also inhibited chemotaxis but displayed no activity in the other two bioassays. CSE also inhibited the ability of airway epithelial cells to release transforming growth factor (TGF)-beta and fibronectin. Exogenous fibronectin was unable to restore epithelial cell contraction of collagen gels. Exogenous TGF-beta partially restored the ability of airway epithelial cells to contract collagen gels and to produce fibronectin. This supports a role for inhibition of TGF-beta release in mediating the inhibitory effects of cigarette smoke. Taken together, the results of the current study suggest that epithelial cells present in the airways of smokers may be altered in their ability to support repair responses, which may contribute to architectural disruptions present in the airways in COPD associated with cigarette smoking.

Retinoic acid attenuates cytokine-driven fibroblast degradation of extracellular matrix in three-dimensional culture.

Proteolytic degradation of extracellular matrix is thought to play an important role both in emphysema and in tissue development and repair. Retinoic acid has been suggested to modify tissue injury, and in an animal model of emphysema may induce alveolar repair. Since cytokines can induce matrix metalloproteinase (MMP) production in fibroblasts and neutrophil elastase (NE) can activate MMPs, we hypothesized that retinoic acid could attenuate collagen degradation by modifying MMP production and activation. To evaluate this, human lung fibroblasts were cast into native type I collagen gels and floated in medium containing cytomix (TNF-alpha, IL-1beta, and IFN-gamma) alone or in combination with NE in the presence and absence of retinoic acid (1 microM). After 5 d, cytomix with elastase induced significant degradation of the collagen gels assessed by quantifying total hydroxyproline (41.6 +/- 1.6 microg versus 3.3 +/- 1.5 microg, P < 0.01). Retinoic acid significantly inhibited this degradation (23.3 +/- 1.5 microg versus 3.3 +/- 1.5 microg, P < 0.01). Gelatin zymography and Western blot revealed that MMP-1, MMP-3, and MMP-9 were induced by cytomix and that co-exposure to NE resulted in increased production of activated forms of these enzymes. Retinoic acid attenuated the induction and activation of MMP-1 and MMP-3. The current study, therefore, suggests that in addition to stimulating anabolic effects, retinoic acid may modulate proteolytic processes thought to contribute to tissue destruction in emphysema.

Cytokine inhibition of fibroblast-induced gel contraction is mediated by PGE(2) and NO acting through separate parallel pathways.

Contraction of three-dimensional collagen gels is a model of the contraction that characterizes normal healing and remodeling after injury. In the current study, we evaluated the hypothesis that a number of inflammatory factors, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, and interferon (IFN)-gamma, modulate this process by induction of prostaglandin (PG) E(2) and nitric oxide (NO) production and that these secondary mediators function in an autocrine or paracrine manner to modulate contraction. Human fetal lung fibroblasts (HFL) were cultured in type I collagen gels and floated in medium containing TNF-alpha, IL-1 beta, or IFN-gamma alone or in combination (cytomix). All cytokines inhibited the contraction significantly. The potency order was IL-1 beta, TNF-alpha, IFN-gamma. The cytomix was no more potent than was IL-1 beta alone. PGE(2) production was increased by TNF-alpha (5.0 versus 0.16 ng/ml, P < 0.01), IL-1 beta (5.3 versus 0.16 ng/ml, P < 0.01), and cytomix (5.9 versus 0.16 ng/ml, P < 0.01), and was completely inhibited by indomethacin. Indomethacin (P < 0.05) and L-NG-monomethyl arginine citrate (L-NMMA) (P < 0.05) alone both partially attenuated the inhibition of contraction caused by cytokines alone or by cytomix. Indomethacin and L-NMMA together attenuated inhibition more than either alone (P < 0.05). Exogenous PGE(2) and exogenous NO donors (DETA nononate and 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride) inhibited the contraction significantly. The protein kinase A inhibitor KT5270 and the protein kinase G inhibitor Rp-pCPT-cGMPS attenuated the inhibition induced by PGE(2) and NO, respectively. In summary, PGE(2) and NO appear to function in parallel as autocrine/paracrine mediators of cytokine-driven fibroblast inhibition of the contraction of collagen gels and may contribute to remodeling during repair and inflammation in lung disorders.

Bradykinin augments fibroblast-mediated contraction of released collagen gels.

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10(-9) and 10(-8) M bradykinin. A B2 receptor antagonist attenuated the effect of bradykinin, whereas a B1 receptor antagonist had no effect, suggesting that the effect is mediated by the B2 receptor. An inhibitor of intracellular Ca2+ mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.

Persistence of TGF-beta1 induction of increased fibroblast contractility.

Fibroblast contraction of collagen gels is regarded as a model of wound contraction. Transforming growth factor (TGF)-beta added to such gels can augment contraction consistent with its suggested role as a mediator of fibrotic repair. Since fibroblasts isolated from fibrotic tissues have been suggested to express a "fibrotic phenotype," we hypothesized that TGF-beta exposure may lead to a persistent increase in fibroblasts' contractility. To evaluate this question, confluent human fetal lung fibroblasts were treated with serum-free Dulbecco modified Eagle medium (DMEM), with or without 100 pM [corrected] TGF-beta1, TGF-beta2, or TGF-beta3 for 48 h. Fibroblasts were then trypsinized and cast into gels composed of native type I collagen isolated from rat tail tendons. After 20 min for gelation, the gels were released and maintained in serum-free DMEM. TGF-beta-pretreated fibroblasts caused significantly more rapid gel contraction (52.5+/-0.6, 50.9+/-0.2, and 50.3+/-0.5% by TGF-beta1, -beta2, and -beta3 pretreated fibroblasts, respectively) than control fibroblasts (74.0+/-0.3%, P < 0.01). This effect is concentration dependent (50-200 nM), and all three isoforms had equal activity. The effect of TGF-beta1, however, persisted for only a short period of time following the removal of TGF-beta, and was lost with sequential passage. These observations suggest that the persistent increase in collagen-gel contractility, mediated by fibroblasts from fibrotic tissues, would not appear to be solely due to previous exposure of these cells to TGF-beta.

Ultrastructural heterogeneity of the alveolar macrophages from tobacco smokers with chronic bronchitis.

Alveolar macrophages recovered by bronchoalveolar lavage from 14 heavy smokers with chronic bronchitis were assessed. Ultrastructural examination revealed marked cellular heterogeneity. Three subpopulations of alveolar macrophages were readily identifiable. These have been termed "young," "mature," and "degrading," reflecting their ultrastructural features. In addition, a majority of the cells were found to be positive by TUNEL staining, indicating DNA damage, but a very small percentage tested positive for Caspase-3, suggesting that apoptosis might not account for the DNA damage in at least some of these cells. A small percentage of proliferating cells were noted.

Contraction of fibroblast-containing collagen gels: initial collagen concentration regulates the degree of contraction and cell survival.

Remodeling of extracellular matrix involves a number of steps including the recruitment, accumulation, and eventual apoptosis of parenchymal cells as well as the production, organization, and rearrangement of extracellular matrix produced by these cells. The culture of fibroblasts in three-dimensional gels made of type I collagen has been used as a model of tissue contraction which characterizes both wound repair and fibrosis. The current study was designed to determine the effect of initial collagen concentration on the ability of fibroblasts to contract collagen gels and on cell survival. Native type I collagen was extracted from rat tail tendons and used to prepare collagen gels with varying collagen concentrations (0.75-2.0 mg/ml). Human lung fibroblasts (HFL-1) were cast into the gels and cultured in Dulbecco modified Eagle medium with 0.1% fetal calf serum for 2 wk. The gel size, collagen content, and deoxyribonucleic acid (DNA) content were determined. Gels prepared with an initial concentration of 0.75 mg/ml contracted more rapidly and to a smaller final size than gels prepared from 2 mg/ml initial collagen concentration (final size 7.1 versus 36.4% of initial size, P < 0.01). There was no significant degradation of the collagen in the gels under either condition. Hence, the dramatically increased contraction of the lower density gels resulted in a higher final density (P < 0.01). Cell density was estimated from DNA content. In low initial density gels, the final DNA content was significantly less than that in higher initial density gels (0.73 versus 1.88 microg/gel, P < 0.05). This was accompanied by an increased percentage of apoptotic cells at day 14 (43.3 versus 34.1%, P < 0.05). If the gels were maintained in the attached state which largely prevents contraction, apoptosis was significantly reduced, suggesting that contraction rather than matrix composition was a requirement for the increased apoptosis. In summary, these findings indicate that the initial matrix composition can lead to differing outcomes during fibroblast-mediated wound contraction.

Cigarette smoke inhibits osteogenic differentiation and proliferation of human osteoprogenitor cells in monolayer and three-dimensional collagen gel culture.

Cigarette smoke is a risk factor not only for emphysema but also for other disorders characterized by deficient tissue repair, including osteoporosis. We hypothesized, therefore, that smoke might directly impair bone cell repair processes. To evaluate this, bone marrow osteoprogenitor cells were isolated from normal subjects and cultured in monolayer and in three-dimensional type I collagen gel culture. Human osteoprogenitor cells could be induced to differentiate toward osteoblast-like cells in both culture conditions by osteogenic supplements. Under both culture conditions, cigarette smoke extract (CSE) inhibited the proliferation of osteoprogenitor cells in a concentration-dependent manner. CSE also inhibited differentiation of osteoprogenitor cells toward osteoblast-like cells as assayed by alkaline phosphatase activity and calcium incorporation into cell layer. Cells in monolayer culture were more sensitive to the effect of smoke than cells in three-dimensional gel culture. Similar results were obtained with osteoblast-like cells derived from osteosarcomas. This study, therefore, demonstrates that cigarette smoke may affect bone progenitor cells directly and in this manner may contribute to the development of osteoporosis.

Effects of interferons alpha and gamma on cytokine production and phenotypic pattern of human bronchial epithelial cells.

Human bronchial epithelial cells are involved in airway immune mechanisms through secretion of cytokines and through cell-cell contacts with immunocompetent cells. The aim of our study was to assess the ability of interferon (IFN) alpha and gamma alone and in combination to modulate human bronchial epithelial cell (HBECs) release of the inflammatory cytokines IL-8 and IL-6 and fibronectin and to induce the surface expression of HLA-DR and ICAM-1 molecules involved in immune interactions with other cells. HBECs spontaneously secreted a limited amount of IL-8, which was significantly increased by IFN gamma. IFN alpha inhibited IFN gamma stimulated IL-8 secretion in a concentration-dependent manner. Further, IFN gamma induced IL-6 and fibronectin secretion, and this was also inhibited by IFN alpha. The expression of HLA-DR antigens was significantly increased by IFN gamma and partially inhibited by co-stimulation with IFN alpha. In contrast, IFN gamma also induced ICAM-1 expression by HBECs but co-stimulation with IFN alpha had no significant effect on the expression of this surface antigen. IFN alpha modulation of HBEC functions does not seem to be restricted to IFN gamma stimulation since either stimulatory or inhibitory effects of INF alpha on IL-8 production have been found in pilot experiments using IL-1 beta, TNF alpha, and TGF beta as stimuli. In summary, IFN-gamma induces a number of responses in HBECs including increased secretion of IL-6, IL-8 and fibronectin and increased expression of HLA-DR and ICAM-1. IFN alpha can inhibit all these except expression of ICAM-1 which is unaffected. IFN alpha can also interact with other inflammatory cytokines, but whether the effects are inhibitory or augmentive depends on the cytokines.