Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15260
Received May 10, 2004; accepted June 22, 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: residual oil fly ash; mycoplasma; interleukin-6; particulate matter; MALP-2; human lung fibroblasts; nickel; transition metals; innate immunity; cytokines.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mycoplasmas (class Mollicutes) are a class of cell wall-free bacteria that represent the simplest self-replicating microorganisms known (Baseman and Tully, 1997). Due to their extremely small genome (0.582.20 Mb) and their limited metabolic options for replication and survival, these fastidious microorganisms have adopted a strict parasitic lifestyle in intimate relationship with a variety of animal and human hosts. With the exception of M. pneumoniae (Chanock et al., 1962
), these microorganisms are not usually considered severely pathogenic; however, they may modulate host defense mechanisms (Rottem, 2002
; Ruuth and Praz, 1989
). Mycoplasma spp. are commonly found on various mucosal surfaces of healthy individuals, however, systemic dissemination and opportunistic growth of these microorganisms has been detected in patients with a variety of chronic inflammatory conditions including asthma (Cassell, 1998
; Kraft et al., 1998
). M. fermentans serves as a good example of these "stealth" pathogens (Ainsworth et al., 2000
; Johnson et al., 2000
; Saillard et al., 1990
; Shibata et al., 1999
; Vojdani et al., 1998
).
We have previously observed that several early passage fibroblast cell lines derived from human lung were infected with M. fermentans (Fabisiak et al., 1993). Infection induced production of immune-modulating cytokines such as interleukin-6 (IL-6) and strongly potentiated the ability of known inducers such as TNF-ß. Since exposure to PM, in general, and ROFA, in particular, can serve as potent stimuli for cytokine-dependent inflammatory responses in vitro and in vivo, we sought to test the hypothesis that M. fermentans exacerbates release of immune-modulatory factors induced by these chemical agents. We used our model of human lung fibroblasts (HLF) deliberately infected with M. fermentans to examine the ability of various components of PM to induce the induction of a prototypic immunomodulatory marker cytokine, IL-6. M. fermentans and ROFA synergistically interacted to increase the production of IL-6. These effects were mimicked with concurrent exposure to the Toll-like receptor-2 (TLR-2) agonist, M. fermentans-derived macrophage-activating lipopeptide-2 (MALP-2), and nickel. Thus, exposure to microbial-derived agents can strongly determine the cellular response to specific chemical stresses and warrants that the microbial ecology be taken into consideration in the risks and mechanisms of toxicity posed by atmospheric pollutants such as particulate-derived metals.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Preparations of PM were provided by Dr. Andrew Ghio, U.S. EPA. ROFA was collected by Southern Research Institute (Birmingham, AL) downstream from a cyclone scrubber at a power plant in FL burning a low sulfur #6 oil (Hatch et al., 1985). Other PM types included urban dust collected from Dusseldorf Germany (Dussel), volcanic ash from Mt. St. Helens (MSH), and an aqueous extract of PM collected from Provo Valley near Salt Lake City, Utah (SLC). The chemical and physical characteristics of these particles have been described in detail. (Becker et al., 1996
; Frampton et al., 1999
; Hatch et al., 1985
; Prahalad et al., 1999
). The ROFA particles were devoid of endotoxin activity as measured by limulus amoebocyte assay.
Cell culture. Human lung fibroblasts (HLF) were isolated as outgrowths from explanted surplus transbronchial biopsy tissues obtained during routine follow-up bronchoscopy of lung transplant recipients as previously described (Fabisiak et al., 1993) in accordance with a protocol approved by University of Pittsburgh Institutional Review Board. The individual cell lines used here were recovered from frozen stocks prepared at passage three and used for experiments over no more than eight additional subcultures. Greater than 95% purity of fibroblasts was determined by positive immunohistochemical staining for vimentin and negative staining for cytokeratin A3 or Factor VIII. Cells were maintained in Minimal Essential Medium (MEM) supplemented with FBS (10%, final concentration), glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 µg/ml), and Fungizone (1.25 µg/ml) in a humidified incubator at 37°C with 5% CO2/95% air. All cultures were negative for mycoplasma as determined by fluorescent microscopy using Hoechst 33258 dye (Chen, 1977
) prior to the deliberate introduction of M. fermentans.
Isolation and culture of M. fermentans. M. fermentans was isolated from previously described de novo infected HLF cell lines (Fabisiak et al., 1993) by inoculating 50 ml of SP-4 media (Tully et al., 1977
) with 10 ml of spent tissue culture medium obtained from MP-infected HLF. Cultures were incubated in airtight flasks at 37°C until a red to yellow color change was observed indicative of microbial growth. Aliquots of MP cultures were cryopreserved by addition of 0.8 ml of culture to 0.2 ml glycerol and freezing at 80°C. The total amount of MP was quantified by fluorometric determination of DNA content using a modification of Hoechst 33258 assay (Cesarone et al., 1979
). The amount of viable organisms recovered from the frozen stocks was determined for each infection by determining the number of color changing units (ccu) measured with a limiting dilution assay (Rodwell and Whitcomb, 1983
). The isolated strain of MP showed classical "fried-egg" morphology typical for the genus when grown on solid media and PCR positivity using M. fermentans sequence-specific primers (data not shown). In addition, the M. fermentans species was verified using a species-specific monoclonal antibody (personal communication, Dr. Shyh-Ching Lo, Armed Forces Institute of Pathology.). All experiments presented here utilized a single strain of M. fermentans derived from one infected cell line.
In vitro infection with M. fermentans. Uninfected HLF were seeded into T-75 flasks (6 x 105 cells/flask) and one P60 dish (2 x 105 cells /dish) and incubated for 24 h. At the time of infection, MP were rapidly thawed, centrifuged 12,000 x g for 15 min and washed twice with 0.25 M NaCl. Final pellet was resuspended in complete tissue culture, and each T-75 flask and P60 dish received 450 ng or 150 ng of mycoplasma DNA, respectively. Uninfected control cells were set up under identical conditions but did not receive mycoplasma and were maintained in a separate incubator. At 4 days post-infection cells were trypsinized, counted, and seeded into appropriate plates for experiments. Mycoplasma infection was verified by staining the P60 dish with Hoechst 33258 dye. Based on organism load expressed as ccu/ml and DNA content these conditions represent the introduction of 100 viable organisms and
300 total organisms per cell.
Cell viability. Cell viability was measured by quantifying the reduction of the fluorogenic dye, Alamar Blue (Biosource, Camarillo, CA). Briefly, MP-infected or uninfected cells were seeded into 24-well plates (4 x 104/ml) and allowed to attach for 24 h. Cells were then exposed to various concentrations of ROFA for 24 h. Media was then removed, cells washed once with PBS, and media replaced with 0.9 ml serum-free MEM. Alamar Blue (0.1 ml of a 10% solution made in serum-free MEM) was then added to each well, and cells returned to the incubator for 3 h. Fluorescence in each well was measured using a Cytofluor 2300 fluorescence plate reader using excitation 530 ± 25 nm and emission 590 ± 25 nm.
In vitro exposures and IL-6 measurement. M. fermentans-infected cells and uninfected cells were seeded into either 6-well plates (34.5 x 105 cells/3 ml/well) or 24-well plates (0.61 x 105 cells/1 ml/well) and cultured for 48 h. Media was removed and replaced with the same volume of serum-free media containing 0.1% BSA with or without various stimuli. Conditioned media was collected at indicated time points and stored at 80°C until further analysis. IL-6 content of conditioned media was measured using a specific enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. For experiments in 6-well plates IL-6 content was normalized to the producing cell number measured by nuclei counts obtained after lysis in 0.1% citric acid/0.1% crystal violet (Fabisiak et al., 1993). For experiments in 24-well plates IL-6 was normalized to the DNA content of the monolayer measured using Hoechst 33258 fluorescence (Cesarone et al., 1979
).
Measurement of cytokine mRNA. Total cellular RNA was isolated using TRIzolTM reagent (Invitrogen, Gaithersburg, MD) according to the manufacturer's instructions. cDNAs were generated from 0.5 µg total RNA by reverse transcription in a 30 µl reaction mixture containing Ambion 103 first strand buffer, 10 U RNase inhibitor, 0.33 mM each dNTP, 1.7 mM oligo dT, and 100 U M-MLV reverse transcriptase. Reactions were incubated at 44°C for 60 min in a MJ Research PTC-100 thermocycler. cDNA was stored at 20°C until further analysis. Specific primer pairs for IL-6 (forward 5'-GCCCAGCTATGAACTCCTTCTC; reverse 5'-GACTTGTCATGTCCTGCAGCC), IL-8 (forward 5'-ATGACTTCCAAGCTGGCCGTGGCT; reverse 5'-TCTCAGCCCTCTTCAAAAACTTCTC) and ß-actin (forward 5'-GGGACCTGACCGACTACCTC; reverse 5'-GGGCGATGATCTTGATCTTC) were used to amplify the specific cDNAs. Specific cDNAs were amplified using 5 µl aliquots cDNA mixed with 1.25 µl of IL-6, IL-8, or ß-actin forward and reverse primers, 2.5 U of Taq DNA polymerase, and 0.125 mM dNTP in Ambion complete PCR buffer in a final volume of 50 µl. PCR reactions were carried out for 20 s at 95°C, 30 s at 55°C, and 40 s at 72°C for 19 cycles for ß-actin or 24 cycles IL-6 and IL-8. The number of cycles was demonstrated to be within the linear amplification range for each product. PCR products were either detected on 2% agarose gels stained with ethidium bromide or quantified in real-time fashion during the PCR amplification using the double-strand DNA fluorescent dye PicoGreen at 430 nm emission and 525 nm excitation. IL-8 and IL-6 mRNA expression were normalized to the housekeeping gene ß-actin by determining the ratio of the IL-8 or IL-6 fluorescent signals to that for ß-actin.
Statistical analysis. Data presented are expressed as mean ± SEM collected from at least three or four individual experiments for studies employing pooled HLF or cells derived from individual donors, respectively. Comparisons were made using a one-way ANOVA followed by appropriate group comparisons such as Dunnett's multiple comparison to control or Bonferonni's correction for multiple t-tests. Significance of response to a range of concentrations to single agents was determined by a test for linear trend across the applied concentrations. To control for variability between multiple individually derived cell lines repeated measures ANOVA and post hoc tests were applied with pairing of data collected within a specific cell line. When variability between cell lines precluded the assumption of a normal distribution, nonparametric Wilcoxon signed rank tests were used to compare groups. To compare observed dose-response relationships with combined stimuli to a theoretical additive model, concentrations were transformed to log scale, and a predicted additive response was derived as the algebraic sum to similar concentrations of each agent alone. Logdose response curves were subjected to linear regression and the slopes compared by analysis of covariance. Statistical analyses were performed using GraphPad PRISMTM, version 3.0 (GraphPadTM Software, San Diego, CA), and differences were considered significant at the p < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Comparison of ROFA to PM from Other Sources
We next examined whether other types of PM might similarly augment IL-6 release from MP-infected cells. We measured IL-6 release during a 24-hour exposure of MP-infected and control uninfected HLF to equivalent concentrations (20 µg/ml) of urban dust collected from Dusseldorf, Germany (Dussel); aqueous extract of PM obtained from Provo Valley near Salt Lake City, Utah (SLC); Mt. St. Helens volcanic ash (MSH); as well as ROFA. Figure 2A shows that 20 µg/ml ROFA significantly increased IL-6 release about four-fold above that seen in untreated uninfected cells. In contrast, PM derived from other sources had little effect on IL-6 release at this same concentration. The slight increase observed with SLC did not reach statistical significance. When these same exposures were carried out on MP-infected cells, it was again observed that the combination of ROFA nearly tripled the release of IL-6 relative to untreated MP-infected cells alone (Fig. 2B). In keeping with the synergistic response, it is important to note that the amount of IL-6 release seen with the combination of ROFA plus MP is nearly 20-fold and 100-fold greater than that seen in uninfected cells in the presence and absence of ROFA, respectively. Figure 2B also shows that the other PM types at this concentration lacked sufficient ability to induce IL-6 in MP-infected cells similar to uninfected cells. Thus, the observed potency of ROFA to induce IL-6 release and interact with MP appears to arise from some unique property of this PM or relative enrichment of some chemical component compared to other PM.
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dose-response relationships determined in specific pathogen-free animals and sterile cell culture models are often used to set risk assessment guidelines for various environmental agents. This rarely, however, mimics relevant "real-life" exposures, where individuals may be exposed in the presence of microbial infection/colonization. Such infection can range from clinically evident disease (pneumonia, tracheobronchitis), to subclinical latent chronic infection, to colonization with various "harmless" commensals. Moreover, ambient PM itself is a complex mixture that contains particulates from many sources, including those of biological origin. In fact, bacterial endotoxin and other microbial products have been implicated in some of the biological activity of ambient PMs (Becker et al., 2002). Thus, the synergistic interactions between different components of the PM mixture and concurrent presence of microorganisms need to be considered when evaluating their overall toxicity.
M. fermentans, serves as an example of a mycoplasma with potential to establish chronic subclinical infection. Originally isolated from the genitourinary (Ruiter and Wentholt, 1950), M. fermentans has also been documented in joint fluid of patients with rheumatoid arthritis but not those with other arthrides (Horowitz et al., 2000
; Johnson et al., 2000
), leukemic bone marrow (Murphy et al., 1970
), and a disproportionately high percentage of blood samples from those with chronic fatigue syndrome (Vojdani et al., 1998
). Recently, Mycoplasma spp. were detected in the airways of humans in absence of symptoms of acute infection, and the incidence was greater in asthmatics (Kraft et al., 1998
). Using sensitive PCR-based detection methods, high incidences of M. fermentans positivity have been noted in saliva (Shibata et al., 1999
), blood (Ainsworth et al., 2000
), and urine (Kovacic et al., 1996
) from apparently normal healthy subjects. Data regarding the presence of M. fermentans within the human lung or its ability to establish chronic "symptomless" pulmonary infection, however, are severely limited.
It remains unresolved as to what particular components of PM contribute to its adverse health effects. ROFA is frequently used as a surrogate to study the adverse effects of PM, particularly in the context of particulate-derived metal (Ghio et al., 2002); however, it represents a very small component of total air PM and does not accurately reflect the complex composition of ambient particulate mixtures. Several studies have pointed to the importance of the "fine" (<2.5 µm) and "ultrafine" (<0.1 µm) fractions in mediating ambient PM toxicity (Laden et al., 2000
; Peters et al., 1997
). Although these size fractions represent only <50% and <10%, respectively, of the total mass fraction of PM10, these fractions are enriched in their metal content. Multiple sources of PM-derived metals exist in addition to ROFA. Whether ambient PMs are capable of interacting with microbial stimuli in a manner analogous to ROFA will undoubtedly depend on their sources, chemical composition, size distribution, and physical properties.
The ability of ROFA to produce cell and tissue injury and stimulate inflammatory cytokines has been linked to its high metal content (Carter et al., 1997; Dreher et al., 1995
; Dye et al., 1999
; Pritchard et al., 1996
; Samet et al., 1997
). In much the same way, our data support a role for particulate-derived metal in mediating the synergistic effects with MP. Vanadium and iron have been implicated as the primary transition metals in ROFA capable of inducing oxidative stress and activating inflammatory responses such as cytokine release. In contrast, our data point to an important role for Ni in mediating the effects observed here. The preparation of ROFA used here contains 37.5 mg Ni/gm, which if completely soluble would produce from 6 to 25 µM Ni with the ROFA concentrations used in Figure 1 (1040 µg/ml). It should be pointed out, however, that the threshold for the effect of NiSO4 was between 20 and 50 µM; therefore, it is likely that other metals or alternate forms of Ni also participate in the overall effects of ROFA. Ni is most often considered as an occupational hazard in exposed workers (Morgan and Usher, 1994
), although considerable exposure does occur in the general population, often in the form of ultrafine metal-rich combustion-derived PM. Nearly 1 million and 200 million people living in the vicinity of Ni-emitting sources are exposed to median concentrations of 200 and 50 ng Ni/m3 respectively (Leikauf, 2002
). Assuming an ambient level of 100 ng/m3, 50% deposition, negligible elimination, and normal respiration, we estimate a daily dose of 864 ng Ni that if uniformly distributed within the noncellular volume of the lung (22 ml) (Weibel, 1985
) would produce
1 µM Ni. While this value is below that necessary to produce synergy, a small change in breathing parameters, potential for uneven distribution, and possible accumulation over time could produce Ni levels closer to those used in our experiments. Using data from the Six Cities studies, Laden et al. (2000)
demonstrated that Ni was positively associated with daily deaths. In animal studies, Ni synergistically interacts with V to initiate untoward cardio-vascular effects following inhalational exposure (Campen et al., 2001
).
Our data do not speak directly to the cellular and molecular mechanisms that account for the synergistic interactions between ROFA and MP. Since ROFA contains a variety of transition metals (Fe, V, Ni, among others) it is possible that enhanced formation of reactive oxygen species (ROS) during metal and microbial exposure can subsequently modulate signal transduction pathways culminating in IL-6 release. The SLC PM, however, was inhibitory towards MALP-2-induced IL-6 despite the fact that this PM is particularly rich in redox-active Cu (Kennedy et al., 1998). Although Ni leads to formation of ROS (Andrew et al., 2001
; Huang et al., 1993
; Misra et al., 1990
) it is not considered as redox-active as other transition metals. It is possible, however, that Ni coordinated to specific ligands enhances its redox activity (Misra et al., 1993
; Shi et al., 1992
) and/or imparts an intracellular regio-specificity that is critical for these effects.
Numerous transcription factors such as NF-B and AP-1 play well-established roles in the regulation of cytokine gene expression following diverse microbial and chemical stressors. Activation of these pathways by ROFA (Quay et al., 1998
; Samet et al., 2002
) and MALP-2 (Rawadi et al., 1999
) can occur through activation of protein kinase-dependent and oxidant-sensitive mechanisms. Other mechanisms of Ni-dependent regulation gene expression include stabilization of hypoxia-inducible factor (HIF-1
) (Andrew et al., 2001
), as well as other novel, as yet defined, transcription factors (Barchowsky et al., 2002
). Other possibilities for mechanisms of regulation-include metal-dependent inactivation of specific signal-transducing protein tyrosine phosphatases (Samet et al., 1997
, 1998
) and modulation of mRNA stability (Winzen et al., 1999
). Thus, it is possible that synergy arises via amplified activation of one or more of these specific transcription factors or signaling pathways or, alternatively, that each stimulus produces a unique profile of signaling events that converge to govern gene expression in an interactive way.
HLF are useful as in vitro model for these studies since they (1) support live MP infection, (2) represent a relatively normal untransformed human lung cell type without artifacts of prolonged tissue culture, and (3) play an active role in the cytokine and fibrotic response following tissue injury. Other cells such as macrophages and epithelial cells, however, may arguably be more relevant since they are amongst the first to encounter PM and infectious stimuli. Since regulation of cytokine production in diverse cell types likely follows common mechanisms, we anticipate that microbial stimuli and particulate-derived metals will similarly interact to activate other cell types within the lung.
In summary, our studies demonstrate the potential for profound synergistic interactions between microbial products and PM in the ability of HLF to produce immune-modulating/inflammatory cytokines. These effects occur in absence of cytotoxicity and with concentrations of stimuli that produce minimal effects by themselves. Since it appears that mycoplasma are signaling through TLR-dependent mechanisms, the phenomenon can likely be extended to a range of diverse microorganisms. Our studies provide an experimental model to further examine the cellular and molecular mechanisms by which these microbial and chemical stimuli interact to modulate the expression of immune-modulating cytokines and other gene products important in the response to various environmental stresses.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
NOTES |
---|
1 To whom correspondence should be addressed at Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, 3343 Forbes Avenue, Pittsburgh, PA 15237. Fax: (412) 383-2123. E-mail: fabs{at}pitt.edu.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Andrew, A. S., Klei, L. R., and Barchowsky, A. (2001). Nickel requires hypoxia-inducible factor-1, not redox signaling, to induce plasminogen activator inhibitor-1. Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L607L615.
Barchowsky, A., Soucy, N. V., O'Hara, K. A., Hwa, J., Noreault, T. L., and Andrew, A. S. (2002). A novel pathway for nickel-induced interleukin-8 expression. J. Biol. Chem. 2002, 2422524231.[CrossRef]
Baseman, J. B., and Tully, J. G. (1997). Mycoplasmas: Sophisticated, reemerging, and burdened by their notoriety. Emerg. Infect. Dis. 3, 2132.[ISI][Medline]
Bayram, H., Devalia, J. L., Sapsford, R. J., Ohtoshi, T., Miyabara, Y., Sagai, M., and Davis, R. J. (1998). The effect of diesel exhaust particles of cell function and release of inflammatory mediators from human bronchial epithelial cells in vitro. Am. J. Respir. Cell Mol. Biol. 18, 441448.
Becker, S., Fenton, M. J., and Soukup, J. M. (2002). Involvement of microbial components and toll-like receptors 2 and 4 in cytokine responses to air pollution particles. Am. J. Respir. Cell Mol. Biol. 27, 611618.
Becker, S., Soukoup, J. M., Gilmour, I., and Devlin, R. B. (1996). Stimulation of human and rat alveolar macrophages by urban air particulates: Effects on oxidant radical generation and cytokine production. Toxicol. Appl. Pharmacol. 141, 637648.[CrossRef][ISI][Medline]
Campen, M. J., Nolan, J. P., Schladweiler, M. C., Kodavanti, U. P., Evansky, P. A., Costa, D. L., and Watkinson, W. P. (2001). Cardiovascular and thermoregulatory effects of inhaled PM-associated transition metals: A potential interaction between PM-associated transition nickel and vanadium sulfate. Toxicol. Sci. 64, 243252.
Carter, J. D., Ghio, A. J., Samet, J. M., and Devlin, R. B. (1997). Cytokine production by human airway epithelial cells after exposure to an air pollution particle is metal-dependent. Toxicol. Appl. Pharmacol. 146, 180188.[CrossRef][ISI][Medline]
Cassell, G. H. (1998). Infectious causes of chronic inflammatory diseases and cancer. Emerg. Infect. Dis. 4, 475487.[ISI][Medline]
Cesarone, C. F., Bolognesi, L., and Santi, L. (1979). Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Anal. Biochem. 100, 188197.[ISI][Medline]
Chanock, R. M., Hayflick, L., and Barile, M. F. (1962). Growth on artificial medium of an agent associated with atypical pneumonia and its identification as a PPLO. Proc. Natl. Acad. Sci. U.S.A. 48, 4149.[ISI][Medline]
Chen, T. R. (1977). In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain. Exp. Cell Res. 140, 255262.
Dockery, D. W., Pope, C. A., III, Xu, X., Spengler, J. D., Ware, J. H., Fay, M. E., Ferris, B. G., Jr., and Speizer, P. E. (1993). An association between air pollution and mortality in six US cities. New Engl. J. Med. 329, 17531759.
Dreher, K. L., Jaskot, R. H., Lehmann, J. R., Richards, J. H., McGee, J. K., Ghio, A. J., and Costa, D. L. (1995). Soluble transition metals mediate oil fly ash induced acute lung injury. J. Toxicol. Environ. Health 50, 285305.
Dye, J. A., Adler, K. B., Richards, J. H., and Dreher, K. L. (1999). Role of soluble metals in oil fly ash-induced airway epithelial injury and cytokine gene expression. Am. J. Physiol. Lung Cell. Mol. Physiol. 277, L498L510.
Fabisiak, J. P., Weiss, R. D., Powell, G. A., and Dauber, J. H. (1993). Enhanced secretion of immune-modulating cytokines by human lung fibroblasts during in vitro infection with Mycoplasma fermentans. Am. J. Respir. Cell Mol. Biol. 8, 358364.[ISI][Medline]
Frampton, M. W., Ghio, A. J., Samet, J. M., Carson, J. L., Carter, J. D., and Devlin, R. B. (1999). Effects of aqueous extracts of PM10 filters from Utah Valley on human airway epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 277, L960L967.
Ghio, A. J., Silbajoris, R., Carson, J. L., and Samet, J. M. (2002). Biologic effects of oil fly ash. Environ. Heath Prespect. 11(Suppl. 1), 8994.
Hatch, G. E., Boykin, E., Graham, J. E., Lewtas, J., Pott, F., Loud, K., and Mumford, J. L. (1985). Inhalable particles and pulmonary host defense: In vivo and in vitro effects of ambient and combustion particles. Environ. Res. 36, 6780.[ISI][Medline]
Hiura, T. S., Kaszubowski, M. P., Li, N., and Nel, A. E. (1999). Chemicals in diesel exhaust particles generate reactive oxygen radicals and induce apoptosis in macrophages. J. Immunol. 163, 55825591.
Horowitz, S., Evinson, B., Borer, A., and Horowitz, J. (2000). Mycoplasma fermentans in rheumatoid arthritis and other inflammatory arthritides. J. Rheumatol. 27, 27472753.[ISI][Medline]
Huang, X., Frenkel, K., Klein, C. B., and Costa, M. (1993). Nickel induces increased oxidants in intact cultured mammalian cells as detected by dichlorofluorescein fluorescence. Toxicol. Appl. Pharmacol. 120, 2936.[CrossRef][ISI][Medline]
Johnson, S., Sidebottom, D., Bruckner, F., and Collins, D. (2000). Identification of Mycoplasma fermentans in synovial fluid samples from arthritis patients with inflammatory disease. J. Clin. Microbiol. 38, 9093.
Kennedy, T., Ghio, A. J., Reed, W., Samet, J., Zagorski, J., Quay, J., Carter, J., Dailey, L., Hoidal, J. R., and Devlin, R. B. (1998). Copper-dependent inflammation and Nuclear Factor-B activation by particulate air pollution. Am. J. Respir. Cell Mol. Biol. 19, 366378.
Kovacic, R., Launay, V., Tuppin, P., Lafeuillade, A., Feuillie, V., Montagnier, L., and Grau, O. (1996). Search for the presence of six Mycoplasma species in peripheral blood mononuclear cells of subjects seropositive and seronegative for human immunodeficiency virus. J. Clin. Microbiol. 34, 18081810.[Abstract]
Kraft, M., Casell, G. H., Hensen, J. E., Watson, H., Williamson, J., Marimion, B. P., Gaydos, C. A., and Martin, R. J. (1998). Detection of Mycoplasma pneumoniae in the airways of adults with chronic asthma. Am. J. Respir. Crit. Care Med. 158, 9981001.
Laden, F., Neas, L. M., Dockery, D. W., and Schwartz, J. (2000). Association of fine particulate matter from different sources with daily mortality in six U.S. cities. Environ. Health Perspect. 108, 941947.[ISI][Medline]
Leikauf, G. D. (2002). Hazardous air pollutants and asthma. Environ. Health Prespect. 110, 505526.
Misra, M., Olinski, R., Dizaroglu, M., and Kasprzak, K. S. (1993). Enhancement by L-histidine of nickel(II)-induced DNAprotein cross-linking and oxidative DNA base damage in the rat kidney. Chem. Res. Toxicol. 6, 3337.[ISI][Medline]
Misra, M., Rodriguez, R. E., and Kasprzak, K. S. (1990). Nickel-induced lipid peroxidation in the rat: Correlation with nickel effect on antioxidant defense systems. Toxicology 64, 117.[CrossRef][ISI][Medline]
Morgan, L. G., and Usher, V. (1994). Health problems associated with nickel refining and use. Ann. Occup. Hyg. 38, 189198.[ISI][Medline]
Muhlradt, P. F., Keiss, M., Meyer, H., Sussmuth, R., and Jung, G. (1997). Isolation, structural elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentrations. J. Exp. Med. 185, 19511958.
Murphy, W. H., Bullis, C., Dabich, L., Heyn, R., and Zarafonetis, J. D. (1970). Isolation of mycoplasma from leukemic and nonleukemic patients. J. Natl. Cancer Inst. 45, 243251.[Medline]
Nishiguchi, M., Matsumoto, M., Takao, T., Hoshino, M., Shimonishi, Y., Tsuji, S., Begum, N. A., Takeuchi, O., Akira, S., Toyoshima, K., et al. (2001). Mycoplasma fermentans lipoprotein M161Ag-induced cell activation is mediated by Toll-like receptor 2: Role of N-terminal hydrophobic portion in its multiple functions. J. Immunol. 166, 26102616.
Peters, A., Wichmann, H. E., Tuch, T., Heinrich, J., and Heyder, J. (1997). Rerspiratory effects are associated with the number of ultrafine particles. Am. J. Respir. Crit. Care Med. 155, 13761383.[Abstract]
Pope, C. A., and Kanner, R. E. (1993). Acute effects of PM10 pollution on pulmonary function of smokers with mild to moderate chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 147, 13361340.[ISI][Medline]
Prahalad, A. K., Soukup, J. M., Inmon, J., Willis, R., Ghio, A. J., Becker, S., and Gallagher, J. E. (1999). Ambient air particles: Effects on cellular oxidant radical generation in relation to particulate elemental chemistry. Toxicol. Appl. Pharmacol. 158, 8191.[CrossRef][ISI][Medline]
Pritchard, R., Ghio, A. J., Lehmann, J. R., Winsett, D. W., Tepper, J. S., Park, P., Gilmour, M. I., Dreher, K. L., and Costa, D. L. (1996). Oxidant generation and lung injury after particulate air pollution exposure increase with the concentration of associated metals. Inhal. Toxicol. 8, 457477.[ISI]
Quay, J. L., Reed, W., Samet, J., and Devlin, R. B. (1998). Air pollution particles induce IL-6 gene expression in human epithelial cells via NF-B activation. Am. J. Respir. Cell Mol. Biol. 19, 98106.
Rawadi, G., Garcia, J., Lemercier, B., and Roman-Roman, S. (1999). Signal transduction pathways involved in the activation of NF-B, AP-1, and c-fos by Mycoplasma fermentans membrane lipoproteins in macrophages. J. Immunol. 162, 21932203.
Rawadi, G., and Roman-Roman, S. (1996). Mycoplasma membrane lipoproteins induce proinflammatory cytokines by a mechanism distinct from that of lipopolysaccharide. Infect. Immun. 64, 637643.[Abstract]
Rodwell, A. W., and Whitcomb, R. H. (1983). Methods for direct and indirect measurement of mycoplasma growth. In Methods in Mycoplasmology (J. G. Tully and S. Razin, Eds), pp. 185196. Academic Press, New York.
Rottem, S. (2002). Interaction of mycoplasmas with host cells. Physiol. Rev. 83, 417432.[ISI]
Ruiter, M., and Wentholt, H. M. M. (1950). A pleuropneumonia-like organism in primary fusopirochetal gangrene of the penis. J. Invest. Dermatol. 15, 301304.[ISI][Medline]
Ruuth, E., and Praz, F. (1989). Interactions between mycoplasmas and the immune system. Immunol. Rev. 112, 133160.[ISI][Medline]
Saillard, C., Carle, P., Bove, J. M., Bebear, C., Lo, S.-C., Shih, J. W. K., Wang, R. Y.-H., Rose, D. L., and Tully, J. G. (1990). Genetic and serologic relatedness between M. fermentans and a mycoplasma recently identified in tissues of AIDS and non-AIDS patients. Res. Virol. 141, 441448.
Samet, J. M., Dominici, F., Curriero, F. C., Coursac, I., and Zeger, S. L. (2000). Fine particulate air pollution and mortality in 20 U.S. cities. N. Engl. J. Med. 343, 17421749.
Samet, J. M., Graves, L. M., Quay, J., Dailey, L. A., Devlin, R. B., Ghio, A. J., Wu, W., Bromberg, P. A., and Reed, W. (1998). Activation of MAPKs in bronchial epithelial cells exposed to metals. Am. J. Physiol. Lung Cell. Mol. Physiol. 275, L551L558.
Samet, J. M., Silbajoris, R., Huang, T., and Jaspers, I. (2002). Transcription factor activation following exposure of an intact lung preparation to metallic particulate matter. Environ. Health Prespect. 110, 985990.
Samet, J. M., Stonehuerner, J., Reed, W., Devlin, R. B., Dailey, L. A., Kennedy, T. P., Bromberg, P. A., and Ghio, A. J. (1997). Disruption of protein tyrosine phosphate homeostasis in bronchial epithelial cell exposed to oil fly ash. Am. J. Physiol. Lung Cell. Mol. Physiol. 272, L426L432.
Schwartz, J., Slater, D., Larson, T. V., Pierson, W. E., and Koenig, J. Q. (1993). Particulate air pollution and hospital emergency room visits for asthma in Seattle. Am. Rev. Respir. Dis. 147, 826831.[ISI][Medline]
Shi, X., Dalal, N. S., and Kasprzak, K. S. (1992). Generation of free radicals from lipid hydroperoxides by Ni2+ in the presence of oligopeptides. Arch. Biochem. Biophys. 299, 154162.[ISI][Medline]
Shibata, K., Kaga, M., Kudo, M., Dong, L., Hasebe, A., Domon, H., Sato, Y., Oguchi, H., and Watanabe, T. (1999). Detection of Mycoplasma fermentans in saliva sampled from infants, preschool, and school children, adolescents and adults by a polymerase chain reaction-based assay. Microb. Immunol. 43, 521525.[ISI]
Takeuchi, O., Kawai, T., Muhlradt, P. F., Morr, M., Radolf, J. D., Zychlinsky, A., Takeda, K., and Akira, S. (2001). Discrimination of bacterial lipoproteins by toll-like receptor 6. Intl. Immunology 13(7), 933940.[CrossRef]
Tully, J. G., Whitcomb, R. F., Clark, H. F., and Williamson, D. L. (1977). Pathogenic mycoplasmas: Cultivation and vertebrate pathogenicity of a new spiroplasma. Science 195, 892894.[ISI][Medline]
van Eeden, S. F., Tan, W. C., Suwa, T., Mukae, H., Terashima, T., T., F., Qui, D., Vincent, R., and Hogg, J. C. (2001). Cytokines involved in the systemic inflammatory response induced by exposure to particulate matter air pollutants (PM10). Am. J. Respir. Crit. Care Med. 164, 826830.
Vojdani, A., Choppa, P. C., Tagle, C., Andrin, R., Samimi, B., and Lapp, C. W. (1998). Detection of Mycoplasma genus and Mycoplasma fermentans by PCR in patients with chronic fatigue syndrome. FEMS Immunol. Med. Microbiol. 22, 355365.[CrossRef][ISI][Medline]
Weibel, E. R. (1985). Lung cell biology. In Handbook of Physiology: The Respiratory System I. Circulation and nonrespiratory functions (A. P. Fishman, Ed.), pp. 4791. American Physiological Society, Betheseda, MD.
Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, A.-Y. A., Shyum, A.-B., Muller, M., Gaestel, M., Resch, K., and Holtmann, H. (1999). The p38 MAP kinase pathway signals for cytokine-induced mRNA stabilization via MAP-kinase-activated protein kinase 2 and an AU-rich region-targeted mechanism. EMBO J. 18(18), 49694980.