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Am J Physiol Lung Cell Mol Physiol 288: L317-L325, 2005. First published October 1, 2004; doi:10.1152/ajplung.00125.2004
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Modulatory effects of ozone on THP-1 cells in response to SP-A stimulation

Branislava Janic,1 Todd M. Umstead,2 David S. Phelps,2 and Joanna Floros1,2,3

Departments of 1Cellular and Molecular Physiology, 2Pediatrics, and 3Obstetrics and Gynecology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania

Submitted 5 April 2004 ; accepted in final form 28 September 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Ozone (O3), a major component of air pollution and a strong oxidizing agent, can lead to lung injury associated with edema, inflammation, and epithelial cell damage. The effects of O3 on pulmonary immune cells have been studied in various in vivo and in vitro systems. We have shown previously that O3 exposure of surfactant protein (SP)-A decreases its ability to modulate proinflammatory cytokine production by cells of monocyte/macrophage lineage (THP-1 cells). In this report, we exposed THP-1 cells and/or native SP-A obtained from bronchoalveolar lavage of patients with alveolar proteinosis to O3 and studied cytokine production and NF-{kappa}B signaling. The results showed 1) exposure of THP-1 cells to O3 significantly decreased their ability to express TNF-{alpha} in response to SP-A; TNF-{alpha} production, under these conditions, was still significantly higher than basal (unstimulated) levels in filtered air-exposed THP-1 cells; 2) exposure of both THP-1 cells and SP-A to O3 did not result in any significant differences in TNF-{alpha} expression compared with basal levels; 3) O3 exposure of SP-A resulted in a decreased ability of SP-A to activate the NF-{kappa}B pathway, as assessed by the lack of significant increase and decrease of the nuclear p65 subunit of NF-{kappa}B and cytoplasmic I{kappa}B{alpha}, respectively; and 4) O3 exposure of THP-1 cells resulted in a decrease in SP-A-mediated THP-1 cell responsiveness, which did not seem to be mediated via the classic NF-{kappa}B pathway. These findings indicate that O3 exposure may mediate its effect on macrophage function both directly and indirectly (via SP-A oxidation) and by involving different mechanisms.

inflammation; tumor necrosis factor-{alpha}; nuclear factor-{kappa}B; I{kappa}B{alpha}; surfactant protein A

OZONE (O3) IS A MAJOR COMPONENT of photochemical air pollution. Approximately 113 million people live in areas with O3 levels above the National Ambient Air Quality (NAAQ) standards for the daily exposure limit, noted as a maximum of 0.12 ppm for 1 h or 0.08 ppm cumulative for 8 consecutive hours (1). O3 is a strong oxidizing agent that can be rapidly converted into a number of reactive oxygen species (ROS). O3 exposure has been associated with impaired lung function (53) and with the majority of pathological changes localized in the lower airways. Due to its very low water solubility, O3 cannot effectively penetrate through the thick epithelial lining fluid in the upper airways. However, the thinner lining in the lower airways allows O3 to interact with various elements of the fluid and the underlying cells.

O3 and other oxidants exhibit their toxicity by reacting with cell proteins and lipids. This interaction often results in edema, inflammation, and epithelial cell damage causing lung injury and surfactant derangement (66, 67). Furthermore, both morphological (6, 7) and biochemical (26, 27) changes in surfactant are observed following O3 exposure. An extensive body of work has been generated by various groups to support the hypothesis that O3-induced changes lead to a compromised immune response in the lung. However, it is not clear how exactly these changes modulate lung immune function or what elements of the lung immune system are affected the most. In a rat model of surfactant deficiency, instillation of oxidized bovine lipid extract surfactant (BLES) resulted in an inferior physiological response and in an increase in the levels of inflammatory cytokines compared with instillation of unoxidized BLES. Supplementation with surfactant protein A (SP-A) did not ameliorate these responses (5), although in in vitro studies SP-A has been shown to reverse the effects of surfactant oxidation on the biophysical properties of surfactant. Disulfide bonds and the COOH-terminal domain of SP-A protein are important for this SP-A function (68). Moreover, high variability in response to O3 exposure within the general population has been observed (33, 45), suggesting the contribution of genetic factors to the observed individual variability in susceptibility to lung injury.

SP-A is one of the molecules in the lung that plays an important role in innate immunity and regulation of inflammatory processes by affecting the expression pattern and thus the phenotype of a variety of cells (31, 62). SP-A belongs to the collectin family of proteins, which has been shown to be involved in many aspects of host defense function (64). Previous studies have demonstrated that SP-A exhibits stimulatory, as well as inhibitory, effects on alveolar macrophages and cells of the monocyte/macrophage lineage. Stimulatory effects of SP-A on these cells include increased oxidative activity (30, 34, 72, 83), increased production of proinflammatory cytokines (43, 44), increased expression of cell surface proteins (42), increased matrix metalloproteinase production (79), enhanced chemotaxis in part via directed actin polymerization (76), and enhanced phagocytosis (21a, 48, 75, 84). Inhibitory effects of SP-A include decreased cytokine expression and nitric oxide production in mouse and rat macrophages pretreated with LPS (3, 11, 51). Moreover, SP-A appears to regulate activities of cells other than those of the monocyte/macrophage lineage (17, 70) as well as to modulate components of adaptive immunity by exhibiting mainly an inhibitory effect. With regard to its role in adaptive immunity, SP-A has been shown to inhibit basal and LPS-mediated expression of the major histocompatibility complex class II and CD86 molecules in murine dendritic cells (DC) as well as DC allostimulation of T cells (15). SP-A also inhibits interleukin (IL)-2 secretion and proliferation of T lymphocytes (12, 13, 62). The latter effect is most likely mediated through the SP-A collagen domain (10). On the other hand, studies on isolated rat splenocytes show that SP-A increased mitogen-induced cell proliferation (43). The described body of work indicates that the overall SP-A effect on lung immunity may be part of a fine tuning mechanism. Such a mechanism may regulate complex multicellular interactions to limit potentially harmful inflammatory responses that could result in lung tissue damage.

Human SP-A is expressed in alveolar type II cells (63) and is encoded by two functional genes, SP-A1 and SP-A2, located on chromosome 10 (16), in opposite transcriptional orientation (16, 32). Both genes are expressed by alveolar type II cells, whereas SP-A2 is predominantly expressed by tracheal and bronchial submucosal gland cells (24, 69). On the basis of sequence differences within the SP-A coding region, more than 30 alleles have been fully or partially characterized (21). However, not all of these are frequently observed in the general population. Amino acid differences among SP-A alleles that are the result of this genetic variability appear to have an impact on the structure, biochemical properties (23, 71, 81), and/or function of SP-A (81, 82) including susceptibility to oxidation by O3 (34, 71, 83). Oxidized residues may in turn influence structural stability and/or the ability of SP-A to regulate immune processes in the lung.

The proinflammatory effect of SP-A on alveolar macrophages is in part mediated through the activation of the NF-{kappa}B cell signaling pathway (41, 73). This pathway has been previously described to be activated in macrophages via LPS activation of Toll-like receptor (Tlr)-4, which transduces the signal through molecules such as myeloid differentiation protein (MyD88), IL-1 receptor-associated kinase, and I{kappa}B kinase (IKK) (14, 29, 85). Stimulation of this pathway results in activation of the IKK complex, which phosphorylates I{kappa}B{alpha} at serine residues (32 and 36). Phosphorylated I{kappa}B{alpha} is then dissociated from NF-{kappa}B enabling it to translocate to the nucleus and facilitate transcription of proinflammatory genes. Although the identity of the macrophage cell surface receptor that mediates SP-A initiation of NF-{kappa}B activation has not been determined yet, previous work from our lab and others indicates interaction of SP-A with Tlr-2 (54, 79) or Tlr-4 (25). Moreover, our published findings have shown that the downstream effect of this activation may involve translocation of cytoplasmic NF-{kappa}B into the nucleus, NF-{kappa}B binding to the specific DNA elements, and subsequent upregulation of TNF-{alpha} expression (41, 73, 79).

We have previously reported an in vitro O3 exposure system (78) that can be used to study the effect of O3 on phagocytic cells (36). Alveolar macrophages are present within the alveolar space, embedded within the very thin lining fluid. As such, depending on their relative position within the fluid and the physiological state of the alveolus, these cells may be affected by O3 directly or by secondary ozonation products. Using the in vitro O3 exposure system, we studied the effects of O3 on lung defense mechanisms by exposing THP-1 cells (a model for alveolar macrophages) to O3. Previous work has shown that the THP-1 cell line is a suitable model for such study (4, 82, 83) and that human SP-A enhances the expression of proinflammatory cytokines and cell surface proteins in THP-1 cells (42, 44). Moreover, the ability of recombinant SP-A variants to stimulate cytokine production by THP-1 cells has been shown to be reduced following O3 exposure of the variants (34, 83).

In this report, we investigated a modulatory effect of O3 on the THP-1 cells or SP-A alone and on the interaction of THP-1 cells and SP-A. We studied 1) the responsiveness of THP-1 cells following O3 exposure and mechanisms involved, 2) mechanisms through which the impaired functional state of O3-exposed SP-A is expressed, and 3) the combined impact of O3-exposed cells and O3-exposed SP-A on TNF-{alpha} production.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and ozone exposure conditions. THP-1 cells obtained from American Type Culture Collection (ATCC, Rockville, MD) were maintained in RPMI 1640 (HEPES modified; Sigma, St. Louis, MO) supplemented with 10% heat-inactivated fetal calf serum (FCS; Summit Biotechnology, Ft. Collins, CO) and 0.05 mM 2-mercaptoethanol (Invitrogen Life Technologies, Carlsbad, CA) as recommended by ATCC. After differentiation with 1{alpha},25-dihydroxycholecalciferol (10–8 M) (vitamin D3; Biomol, Plymouth Meeting, PA) for 72 h, cells were washed and resuspended in Hanks' balanced salt solution (Invitrogen Life Technologies) and exposed to either O3 or filtered air (FA), as previously described (36).

Preparation of native SP-A and exposure of SP-A to ozone. The native human SP-A used in the experiments was purified from bronchoalveolar lavage (BAL) fluid obtained from an alveolar proteinosis patient by a butanol extraction method as previously described (82). The experiments described were performed with native human SP-A preparation from a single patient. This specific SP-A preparation, chosen among all available protein stocks, exhibited the lowest level of protein oxidation before exposure to O3 (the pre-O3-induced oxidation reflects the in vivo oxidation status of the protein). The level of oxidation was detected using the OxyBlot oxidized protein detection kit (Intergen, Purchase, NY), as previously described (78). If needed, SP-A was concentrated with Centriprep-10 concentrators (Amicon, Beverly, MA), and the final concentration was determined by the Micro-BCA method (Pierce Biotechnology, Rockford, IL) using RNase A as the standard. The purified SP-A was stored at –80°C in aliquots until use. SP-A was exposed to O3 at 1 ppm for 4 h in 24-well culture plates (100 µl/well), as previously described by Umstead et al. (78). This method describes the optimal conditions for SP-A oxidation that result in the highest level of oxidation, as determined by a subsequent OxyBlot analysis.

SP-A treatment of THP-1 cells, ribonuclease protection assay, and TNF-{alpha} ELISA. O3-exposed and unexposed THP-1 cells were suspended in RPMI supplemented with 0.5% FCS at a concentration of 2 x 106 per ml. Cells were transferred into 24-well culture plates and incubated for 30 min, 45 min, and 2 h (for mRNA), and 4 h (for protein) in the presence of human SP-A (50 µg/ml) or human SP-A exposed to O3 (50 µg/ml). Cell pellets and supernatants were collected, immediately frozen in liquid nitrogen, and stored at –80°C. Experiments that involved incubation of cells for 30 and 45 min in the presence of SP-A included vitamin D3 control cells that were differentiated for 72 h at the same time as were the cells used in the experiments. At 72 h upon vitamin D3 differentiation, the control cells were pelleted, immediately frozen in liquid nitrogen, and stored at –80°C. RNA was extracted from cell pellets using the RNA Extraction kit (Qiagen, Valencia, CA) according to the protocol supplied by the manufacturer. We determined TNF-{alpha} mRNA levels by analyzing 2 µg of total RNA using the Multi-probe ribonuclease protection assay system, according to the manufacturer's instructions (BD Biosciences, San Diego, CA). TNF-{alpha} protein levels in the supernatants were determined using the OptEIA ELISA kit (BD Biosciences). We obtained reference curves by plotting the TNF-{alpha} concentrations of several dilutions of standard protein vs. absorbance.

Nuclear and cytoplasmic protein extraction. Nuclear and cytoplasmic protein extracts were prepared with the nuclear extraction kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. In brief, cell pellets were resuspended in 1x hypotonic buffer (Active Motif) and incubated on ice for 15 min. Detergent was then added, and the suspension was centrifuged for 30 s at 14,000 g in a microcentrifuge precooled to 4°C. The supernatant (i.e., the cytoplasmic fraction) was collected, while the nuclear pellet was resuspended in complete lysis buffer (Active Motif), vortexed for 10 s at the highest setting (Vortex Gente 2; VWR International, Bridgeport, NJ), incubated for 30 min on ice on a rocking platform at 150 rpm, vortexed for 30 s at the highest setting, and centrifuged for 10 min at 14,000 g in a microcentrifuge precooled to 4°C. The nuclear fraction was collected into a prechilled microcentrifuge tube. Protein concentration was determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA), using BSA as a standard.

Western blot analysis. Five micrograms of nuclear protein and 10 µg of cytoplasmic protein were resolved on 10% SDS polyacrylamide gels, transferred to polyvinylidene difluoride membrane (Bio-Rad), and probed with specific primary antibodies: rabbit polyclonal IgG for p65 subunit of NF-{kappa}B (catalog no. sc-372; Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal IgG for I{kappa}B{alpha} (catalog no. sc-371, Santa Cruz Biotechnology), and mouse monoclonal IgM for {beta}-actin (Abcam, Cambridge, MA). Primary antibody binding was detected by incubating the membrane in the presence of horseradish peroxidase-conjugated secondary polyclonal goat antibody (Santa Cruz Biotechnology) with specificity for rabbit IgG or polyclonal goat antibody with specificity for mouse IgM for 1 h. The signal was detected by enhanced chemiluminescence using the ECL detection kit (Amersham Biosciences, Piscataway, NJ)

Statistical analysis. Data were analyzed with SigmaStat statistical software (SPSS, Chicago, IL). Values are presented as means ± SE. Comparison among samples was done by one-way analysis of variance followed by the Student-Newman-Keuls test for pairwise comparison. A value of P < 0.05 was considered to be significantly different.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Ozone-exposed THP-1 cells exhibit a decrease in TNF-{alpha} expression. TNF-{alpha} expression, both at the protein and mRNA levels, was decreased in O3-exposed THP-1 cells in response to SP-A compared with TNF-{alpha} production in FA-exposed (control) THP-1 cells stimulated with SP-A (Fig. 1). This decrease in TNF-{alpha} expression was O3 dose dependent with a statistically significant decrease (P < 0.05) observed when the cells were exposed to 0.2 and 0.5 ppm of O3 (Fig. 1). However, exposure of THP-1 cells for 1 h to different O3 concentrations (0.1, 0.2, and 0.5 ppm) did not abolish their ability to express TNF-{alpha} in response to SP-A. Moreover, TNF-{alpha} levels (mRNA and protein) in the O3-exposed cells were always significantly higher than the TNF-{alpha} basal levels in FA-exposed cells.



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  		Fig. 1. Ozone (O3) dose response of THP-1 cell responsiveness to surfactant protein (SP)-A stimulation. THP-1 cells were exposed to O3 at various concentrations (0.1, 0.2, and 0.5 ppm) for 1 h before stimulation with SP-A and measurement of TNF-{alpha} production. TNF-{alpha} protein levels in supernatants of THP-1 cells (top) and mRNA levels in THP-1 cells (bottom) were determined after a 4- or 2-h stimulation, respectively, with 50 µg/ml of SP-A. Protein levels were assessed by ELISA and mRNA by ribonuclease protection assay (RPA) followed by densitometric band quantification. For each O3 concentration, experiments were repeated 3 times (n = 3) for protein levels and 4 times (n = 4) for mRNA levels. Filled bars, THP-1 cells; open bars, THP-1 cells + O3. Bars are means ± SE. *P < 0.05 compared with THP-1 cells; §P < 0.05 compared with THP-1 cells + SP-A.

 
Ozone-exposed SP-A exhibits a decreased ability to enhance TNF-{alpha} expression in THP-1 cells: involvement of NF-{kappa}B signaling pathway. Exposure of SP-A to 1 ppm of O3 for 4 h reduced the ability of SP-A to enhance TNF-{alpha} expression by THP-1 cells, as shown previously (34, 83). A significant difference (P < 0.05) was observed in TNF-{alpha} protein and mRNA levels (Fig. 2). TNF-{alpha} mRNA and protein production in response to O3-exposed SP-A was still significantly higher (P < 0.05) than basal levels in non-SP-A-stimulated (control) THP-1 cells.



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  		Fig. 2. Ability of THP-1 cells to respond to SP-A stimulation when SP-A is treated with 1 ppm of O3 for 4 h. TNF-{alpha} protein levels were determined in THP-1 cell supernatants after a 4-h stimulation with 50 µg/ml of SP-A or 50 µg/ml of O3-exposed SP-A (top). TNF-{alpha} mRNA levels were determined in THP-1 cells after a 2-h stimulation with 50 µg/ml of SP-A or 50 µg/ml of O3-exposed SP-A (bottom). Protein and mRNA levels were assessed as described in Fig. 1. Experiments were repeated 3 times (n = 3). Filled bars, THP-1 cells; hatched bars, THP-1 cells + O3-exposed SP-A. Bars are means ± SE. *P < 0.05 compared with THP-1 cells; §P < 0.05 compared with THP-1 cells + SP-A.

 
To determine whether the observed O3-induced change in SP-A function is due to alterations in the NF-{kappa}B signaling pathway, we analyzed the levels of the p65 subunit of NF-{kappa}B in nuclear extracts, as well as the levels of the cytoplasmic I{kappa}B{alpha} protein in THP-1 cells stimulated with unexposed SP-A or O3-exposed SP-A. The time-course experiment (not shown) indicated that the most prominent changes in nuclear NF-{kappa}B levels in THP-1 cells stimulated with 50 µg/ml of SP-A occurred at the 30- and 45-min time points, and thus these time points were used in subsequent experimentation.

The ability of SP-A exposed to O3 to activate the NF-{kappa}B pathway in THP-1 cells is significantly reduced compared with unexposed SP-A. Nuclear levels of the p65 NF-{kappa}B subunit were significantly lower in THP-1 cells incubated with O3-exposed SP-A than in cells incubated with unexposed SP-A (Fig. 3, A and B). In addition, the cytoplasmic I{kappa}B{alpha} levels were significantly increased in O3-exposed SP-A compared with that of unexposed SP-A (Fig. 4, A and B).



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  		Fig. 3. Effects of O3-exposed SP-A on the THP-1 cell nuclear NF-{kappa}B levels. Cells were treated with O3-exposed SP-A and unexposed SP-A for 30 (left) and 45 min (right). Nuclear extracted (NE) proteins (5 µg) were analyzed for the presence of the p65 subunit of the NF-{kappa}B complex. A: densitometric measurements of NF-{kappa}B immunoblots. Images of Western blots of duplicate samples from a single representative experiment are shown in B; open bar, vitamin D3-differentiated cells (negative control); gray bar, THP-1 cells; hatched bar, THP-1 cells + SP-A; black bar, THP-1 cells + O3-exposed SP-A; Bars are means ± SE. *P < 0.05 compared with THP-1 cells; §P < 0.05 compared with THP-1 cells + O3 SP-A.

 


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  		Fig. 4. Effects of O3-exposed SP-A on the THP-1 cell cytoplasmic I{kappa}B{alpha} protein levels. Cells were treated with O3-exposed SP-A and unexposed SP-A for 30 (left) and 45 min (right). Cytoplasmic extracted (CE) proteins (10 µg) were analyzed for the presence of I{kappa}B{alpha} protein. A: densitometric measurements of I{kappa}B{alpha} immunoblots. Western blot images of triplicate samples from a single representative experiment are shown in B. Open bar, vitamin D3 differentiated cells (negative control); gray bar, THP-1 cells; hatched bar, THP-1 cells + SP-A; black bar, THP-1 cells + O3-exposed SP-A. Bars are means ± SE (n = 3). *P < 0.05 compared with THP-1 cells; §P < 0.05 compared with THP-1 cells + O3 SP-A.

 
The combined ozone-induced changes in THP-1 cells and SP-A on TNF-{alpha} expression are additive. Under in vivo conditions, it would be expected that airborne pollutants, in this case O3, would have an effect on all of the components of the lung that are exposed to the inhaled air. In addition, it has been suggested that under in vivo conditions, respiratory derangements are more likely to occur in response to acute, short-term exposures to O3 at concentrations well above the NAAQ standards (1). Therefore, we tried to mimic the in vivo situation by incubating the O3-exposed THP-1 cells with O3-treated SP-A and then measuring TNF-{alpha} production at the highest concentration of O3 (0.5 ppm, 1 h) to which THP-1 cells were exposed in the present study. SP-A exposed to 1 ppm of O3 for 4 h exhibited a decreased ability to stimulate TNF-{alpha} expression in O3-exposed THP-1 cells. TNF-{alpha} mRNA (Fig. 5, A and B) and protein (Fig. 5C) levels were significantly decreased in O3-exposed THP-1 cells when stimulated for 2 and 4 h, respectively, with oxidized (O3-exposed) SP-A compared with the TNF-{alpha} levels in unexposed cells stimulated with unexposed SP-A (positive control). Therefore, exposure of both THP-1 cells and SP-A to O3 resulted in TNF-{alpha} mRNA and protein levels similar to basal levels.



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  		Fig. 5. Effect of 0.5 ppm of O3 on the ability of THP-1 cells to express TNF-{alpha} in response to unexposed human SP-A and O3-exposed human SP-A. Levels of mRNA (A and B) and protein (C) are shown after 2- and 4-h stimulation, respectively, with 50 µg/ml of SP-A or 50 µg/ml of O3-exposed SP-A. A: densitometric measurements of the RPA. A representative autoradiograph is shown in B. C: protein levels by ELISA. Gray bar, THP-1 cells; black bar, THP-1 cells exposed to 0.5 ppm of O3. Bars are means ± SE (n = 3); §P < 0.05 compared with THP-1 cells + SP-A; *P < 0.05 compared with THP-1 cells.

 
Western blot analysis of the nuclear protein fraction shows that O3-exposed cells or FA-exposed cells upregulated the levels of nuclear p65 NF-{kappa}B protein in response to O3-exposed SP-A or unexposed SP-A (Fig. 6), although no difference was detected between O3-exposed cells and FA-exposed cells. The increase was significantly higher when the cells were stimulated with unexposed SP-A compared with cells stimulated with O3-exposed SP-A (P < 0.05) (Fig. 6, A and B). However, this increase was not followed by anticipated parallel changes in the levels of cytoplasmic I{kappa}B{alpha} protein. I{kappa}B{alpha} levels in O3-exposed cells were significantly and equally reduced in cells stimulated with either unexposed SP-A or O3-exposed SP-A at 30 and 45 min compared with control D3 cells (non-SP-A stimulated) (Fig. 7, A and B). The results obtained by Western blot analysis of the p65 NF-{kappa}B and the I{kappa}B{alpha} levels do not support involvement of the traditional NF-{kappa}B signaling pathway in the observed decrease of TNF-{alpha} mRNA and protein levels by O3-exposed THP-1 cells in response to O3-exposed SP-A.



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  		Fig. 6. Effect of 0.5 ppm of O3 on the THP-1 cell p65 N-{kappa}B nuclear protein levels in response to unexposed human SP-A and O3-exposed human SP-A. A: densitometric measurements of NF-{kappa}B immunoblots. Images of Western blots of duplicate samples from a single representative experiment are shown in B; gray bar, THP-1 cells differentiated with D3 (negative control); open bar, THP-1 cells; black bar, THP-1 cells exposed to O3. Bars are means ± SE (n = 3). *P < 0.05 compared with D3 control; §P < 0.05 compared with O3 SP-A.

 


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  		Fig. 7. Effect of 0.5 ppm of O3 on the THP-1 cell I{kappa}B{alpha} cytoplasmic protein levels in response to native SP-A and O3-exposed native SP-A. A: densitometric measurements of I{kappa}B{alpha} immunoblots. Images of Western blots of duplicate samples from a single representative experiment are shown in B. Gray bar, THP-1 cells differentiated with vitamin D3 (negative control); open bar, THP-1 cells; filled bar, THP-1 cells exposed to O3. Bars are means ± SE (n = 3). *P < 0.05 compared with D3 control; §P < 0.05 compared with O3 SP-A.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we show that THP-1 cells exposed to O3 and followed by treatment with native SP-A or native oxidized SP-A exhibit a decrease in TNF-{alpha} at both mRNA and protein levels. Comparable findings were obtained with IL-8 at both protein and mRNA levels (data not shown). In an attempt to identify underlying mechanisms of the observed O3-induced downregulation of TNF-{alpha} expression in THP-1 cells, we analyzed the effect of O3 on the individual elements of the system. Exposing only one component of our experimental system (either THP-1 cells or SP-A) to O3 significantly reduced TNF-{alpha} expression in THP-1 cells, but the reduced levels were always higher than basal levels (Figs. 1 and 2) (34, 82), whereas when both (cells and SP-A) were exposed to O3, TNF-{alpha} production was similar to basal levels (Fig. 5). Although downregulation of the canonical NF-{kappa}B signaling pathway is involved when SP-A alone is exposed to O3, an additional mechanism appears to be involved when cells are also exposed to O3. The data indicate that, when both components are exposed to O3, the decrease in TNF-{alpha} production appears to be additive and that more than one mechanism may be involved.

Previous work has shown that SP-A enhances TNF-{alpha} production via the activation of NF-{kappa}B signaling cascade (41, 73). The present findings indicate that O3 decreases the ability of SP-A to activate the NF-{kappa}B cell signaling pathway by reducing the levels of the free NF-{kappa}B subunit p65 available for translocation to the nucleus and initiation of transcription. This change was accompanied with an expected decrease in cytoplasmic I{kappa}B{alpha} levels. The susceptibility of SP-A to oxidant-induced changes has been documented previously. In vitro experiments have shown that O3 exposure of SP-A reduces its ability to self-associate, aggregate lipid vesicles, and bind to carbohydrates (56). O3 exposure of SP-A also resulted in changes in its absorption spectra (71) and in its gel electrophoretic pattern (71, 81). Moreover, when SP-A was exposed to O3, the SP-A-dependent extracellular surfactant morphology was impaired (56, 61). In addition, the functional abilities of SP-A to regulate phosphatidylcholine secretion by alveolar type II cells (57, 81), stimulate superoxide production by alveolar macrophages, mediate opsonization-dependent Herpes simplex virus phagocytosis (57), and enhance cytokine production by THP-1 cells (34, 83) were also altered. Although the mechanisms for these functional changes are unknown, one possibility is that SP-A oxidation alters its protein conformation and that this in turn leads to a decrease in the affinity of its binding to the putative SP-A cell surface receptor (57). A decrease in its receptor binding affinity may in turn compromise initiation of the signaling cascade events leading to an altered gene expression of cytokines and other molecules. In the present study, we show that this phenomenon is manifested through a decrease in the potency of the oxidized SP-A to activate the NF-{kappa}B pathway, as assessed by a decrease in nuclear NF-{kappa}B and an increase in cytoplasmic I{kappa}B{alpha}.

Although exposure of THP-1 cells to O3 resulted in an O3 dose-dependent decrease in TNF-{alpha} protein and mRNA levels, these levels were still significantly above the basal levels observed in (unexposed) control cells. The mechanisms involved in this relative O3-induced cell unresponsiveness are unknown. Previous flow cytometry studies (36) indicate that this change may be due in part to O3-induced cell death. However, a decrease in cell numbers, as shown previously (36), may account for only a small proportion of the decrease in TNF-{alpha} cytokine levels in the THP-1 cells. Because in vivo studies (18, 40, 77) have shown an overall increase in the BAL levels of proinflammatory mediators following O3 exposure, the decrease in cytokine production by O3-exposed THP-1 cells was rather unexpected. However, in the majority of the in vivo studies, the alveolar macrophages were analyzed for the possible in vivo O3-induced changes at various time points after in vivo exposure of animals to O3 (28, 52). This approach inherently does not account for the preexisting lung microenvironmental factors that may have influenced the physiological status of the cell population under analysis (22, 47, 62). Therefore, it is possible that additional unknown factors account for the differences observed between in vivo and in vitro. For example, Ishii et al. (35) showed that alveolar macrophages isolated from in vivo O3-exposed animals exhibit an increase in IL-1{beta} and TNF-{alpha} cytokines. Conversely, consistent with our findings, alveolar macrophages after in vitro exposure to O3 did not produce increased amounts of proinflammatory cytokines (20), and exposure of human alveolar macrophages to nitric oxide (NO2), a strong oxidant that may share mechanisms of cellular toxicity with O3, resulted in a significant decrease of LPS-stimulated IL-1{beta}, IL-6, TNF-{alpha}, and transforming growth factor-{beta} release (39). Moreover, recent studies where THP-1 cells were used as a model system for cells of monocyte/macrophage lineage showed that short-term exposure to O3 decreased the chemotactic mobility of THP-1 cells in response to N-formyl-methionyl-leucyl-phenylalanine chemoattractant (46). Therefore, it seems that, in general, the in vitro O3 effect on macrophages or macrophage-like cells appears to be a decrease in their functional capabilities. However, it remains to be determined whether the in vivo or in vitro observed O3-induced changes are due to changes in cell membrane lipids and membrane fluidity, resulting in impairment of receptor-ligand interaction (59, 60) and/or to the generation of ROS such as hydrogen peroxide and nitric oxide (NO) (80), causing oxidative loss of functional groups and activities of cellular biomolecules, or other mechanisms. However, we do not exclude the possibility that under in vivo conditions O3's effect on alveolar macrophages is indirect, i.e., due to the generation of secondary ozonation products, such as the reactive molecules generated by lipid oxidation.

Under in vivo conditions, it is expected that both alveolar macrophages and SP-A are exposed to O3, and thus exposure of both THP-1 cells and SP-A to O3 may better mimic the in vivo situation. Under this experimental circumstance, TNF-{alpha} levels were similar to basal levels, indicating that both O3-exposed cells and O3-exposed SP-A contribute to the decrease in TNF-{alpha}. The changes in nuclear p65 NF-{kappa}B and the cytoplasmic I{kappa}B{alpha} observed at the molecular level indicate that the NF-{kappa}B pathway was activated. However, the fact that a decrease (rather than an increase) in TNF-{alpha} (mRNA and protein) expression was observed points to the presence of an additional mechanism through which O3 may counteract the observed activation of NF-{kappa}B. Previous studies have shown that oxidative stress can influence posttranslational modification of intracellular proteins (including NF-{kappa}B). NO alters NF-{kappa}B activation by S-nitrosylation of the p50 subunit, and this modification impairs NF-{kappa}B DNA binding (49, 50). Furthermore, NO induces I{kappa}B{alpha} transcription and translocation to the nucleus, leading to a termination of NF-{kappa}B DNA binding (9, 74). In addition, alveolar macrophages have been shown to generate ROS in response to O3 (80). However, further studies are warranted to characterize the intracellular changes induced by O3 in THP-1 cells to explain the processes involved in O3-induced lung injury and to determine both the impact of O3 on SP-A structure and how O3-exposed SP-A modulates downregulation of TNF-{alpha}.

As noted above, in vivo studies showed an increase in the inflammatory processes in lung in response to O3 exposure. Therefore, an increase in the production of TNF-{alpha}, as one of the major markers of airway inflammation by the residential cells of the pulmonary lining, was the expected outcome after O3 exposure. However, our in vitro findings are in contrast to the anticipated results. In view of our current findings and the fact that the alveolar macrophage is considered as one of the major sources of proinflammatory cytokines in lung (58), we propose that the observed in vivo increase in inflammatory cytokine production following O3 exposure is the consequence of an inadequate inflammatory response. In this scenario, alveolar macrophages exhibit a decrease in their ability to properly respond to an inflammatory challenge. For example, an O3-induced functional impairment may render alveolar macrophages inefficient in their ability to clear pathological agents and/or necrotic cells, which itself may be a sufficient challenge for a prolonged inflammatory response. Therefore, it is plausible that in response to O3 the alveolar macrophage (THP-1 cells) may not be the key element in initiating inflammation by cytokine production but is enabling the prolonged presence of inflammatory challenge due to the decrease in its function.

It is important to emphasize that under in vivo conditions the inflammatory process in the lung may be the result of complex interactions among resident pulmonary cells (such as alveolar macrophages, alveolar epithelial cells, and lung fibroblasts), cells recruited from circulation, cytokines, and other molecules secreted in the course of inflammation. Therefore, it is possible that O3 influences all or some of the elements of the inflammatory cascade, and thus each affected element has a potential to play an important role in O3-induced inflammation. The alveolar epithelial cell, a rich source of proinflammatory cytokines, may be one of the contributors to this O3-induced inflammation (2, 47). Recent work done with human nasal epithelial cells shows that O3 exposure resulted in NF-{kappa}B activation and increased production of TNF-{alpha} by these cells (55). Thus the same may be true for the alveolar epithelial cells (8). In such a situation, the alveolar epithelial cells in response to O3 may initiate an inflammatory cascade by increasing the local levels of inflammatory cytokines, and this may influence the activity of resident alveolar macrophages as well as affect the influx of circulatory inflammatory cells. Another contributing factor to the in vivo inflammatory process could be a derangement in the composition and activity of various surfactant components. Ozone can induce oxidation of surfactant phospholipids (19), creating potent proinflammatory signaling molecules (38, 65). Ozone-induced changes in SP-A and lipids may also influence the overall balance between production and the uptake of surfactant and/or SP-A by alveolar type II cell (57), and this may further disrupt the phospholipid/SP-A balance (22, 47, 62) and contribute to the inflammatory process.

In summary, we have demonstrated that O3 significantly decreases the ability of THP-1 cells to produce TNF-{alpha} in response to SP-A. Exposure of both THP-1 cells and SP-A to O3 exhibited an additive inhibitory effect on TNF-{alpha} production. We further show that the O3-exposed SP-A exhibited a decreased ability to activate the NF-{kappa}B signaling pathway. We speculate that the ozone-induced change in THP-1 cell responsiveness is the result of a mechanism that functionally abrogates the seemingly activated NF-{kappa}B pathway.


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This work was supported by National Institute of Environmental Health Sciences Grant 1ROI ES-09882-01.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Floros, Dept. of Cellular and Molecular Physiology, Pennsylvania State Univ. College of Medicine, P.O. Box 850, Hershey, PA 17033 (E-mail: jfloros{at}psu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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