In Vitro Pro-inflammatory Regulatory role of Substance P in Alveolar Macrophages and Type II Pneumocytes after JP-8 Exposure

Abstract

The effects of JP-8 on pro-inflammatory cytokine interleukin (IL)-1α,β and nitric oxide (NO) secretion as well as the role of substance P (SP) in these processes were examined in cultured alveolar macrophages (AM), type II epithelial cells (AIIE), and AM/AIIE co-cultures. Exposure of AM to JP-8 for 24 hr exhibited release of IL-1α,β, whereas exposure to AIIE showed a concentration-dependent NO overproduction. Data indicate that there are cell-dependent inflammatory mechanisms responsible for the actual level of JP-8 exposure in alveoli. However, treatment with substance P significantly attenuated JP-8 induced the IL-1α,β secretion. This finding was confirmed by using [Sar⁹ Met (O₂)¹¹] SP (10^{− 10} M), an agonist of substance P, suggesting that substance P may have signal pathway(s) to AM in the inflammatory response mediated by IL-1. Moreover, AM/AIIE co-culture obviously reduced NO overproduction observed in AIIE alone, suggesting that there may be cell interactions or communications between AM and AIIE in response to the JP-8 exposure.

Keywords :

• JP-8
• cytokines
• nitric oxide
• co-cultures
• type II pneumocytes
• macrophages

INTRODUCTION

Jet Propulsion Fuel-8 (JP-8, also known as MIL-T-83133D, AVTUR, or, F-34) is a kerosene-based hydrocarbon distillate fuel containing approximately 228 long-/short-chain aliphatic and aromatic hydrocarbons (C₆–C₁₄). The low volatility characteristics of JP-8 that enable it to be distilled at between 170 and 325°C still allow it to be a potentially toxic irritant on the respiratory system. JP-8 has been reported to increase the occupational health risk amongst personnel working on aircraft flight lines, mechanics, fuel handlers, and pilots. A substantial effort was made to examine JP-8 pulmotoxicity, revealing that the adverse effects of JP-8 in the lungs clearly manifest by mild inflammation and functional impairment, as demonstrated by a series of in vitro (Wang et al., 2002; Hays et al., 2003) and in vivo studies (Mattie et al., 1991; Robledo et al., 1999a; 2000; Witzmann et al., 1999; Wang et al, 2001; Wong et al., 2004). However, fundamental questions remain unclear for the pathophysiological mechanisms, especially the pro-inflammatory signal pathways that may be activated.

Initial morphological analyses found that obvious alterations in affected alveolar macrophages (AM) and type II epithelial cell (AIIE), which may affect the increased respiratory permeability, leukocytic infiltration, and morphological alterations in the distal lung (Robledo and Witten 1998; Robledo et al., 1999b; 2000; Wong et al., 2004). The alterations were accompanied by an activated neurogenic response, which was characterized by substance P (SP) decrease (Pfaff et al., 1995) and its regulatory enzyme neutral endopeptidase activation (Pfaff et al., 1996). In a following study, the animals administered [Sar⁹,Met(O₂)¹¹] SP, SP's agonist, after each JP-8 exposure exhibited the appearance of normal pulmonary values and tissue morphology. In contrast, endogenous SP's receptor antagonism by CP-96345 administration exacerbated JP-8 induced respiratory permeability, AM and epithelial injury (Robledo and Witten, 1999). These studies suggest that SP may have a regulatory role in the development of JP-8 induced pulmotoxicity, possibly through the modulation of targeted alveolar cells.

In addition, our recent pilot study has showed a significant change in lung nitric oxide (NO) production in rats after JP-8 exposure. It is well known that NO functions as a signaling molecule in the initiation of the inflammatory response in other studies. Therefore, we believe that NO production in the lung after JP-8 exposure may involve the initiation of the inflammatory cascade. The concept has been supported by a study that changes in NO and inducible nitric oxide synthase (iNOS) expression was observed in the skin after JP-8 exposure (Kabbur et al., 2001).

Collectively, it is hypothesized that regulation of AM and AIIE by SP is important in release of pro-inflammatory cytokines or NO production in the alveolar space against JP-8 exposure. It has been observed that both cells have the ability to secrete cytokines (Crestani and Aubier, 1998; Wang et al., 2002) and NO (Hahon and Castranova, 1989; Janssen et al., 1998). They are located predominantly at the alveolar septal junction, in close proximity to each other, which is directly targeted by JP-8 exposure (Parra et al., 1986). Consequently, we have examined the role of substance P in regulation of JP-8 induced IL-1α,β and NO release in AM, AIIE, and AM/AIIE cultures. Data show that there is a cell-dependent inflammatory mechanism responsible for the actual level of JP-8 exposure and this process may possibly be regulated by substance P.

MATERIALS AND METHODS

Cell Culture

A rat AIIE cell line RLE-6TN (American Tissue Cell Company, Manassas, VA) and/or a rat AM NR8383 (ATCC, Rockville, MD) were cultured in DMEM/F12 1:1 media supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic solution in 6-well plates at a density of 5 × 10⁵ cells/ ml at 37°C and 5% CO₂. AMs were co-cultured with RLE-6TN cells at a ratio of 4:1, which is the approximate ratio of AIIE cells (14%) to AMs (3%) in the normal rat lung (Crapo et al., 1980; Haies et al., 1981). Briefly, AMs were allowed to settle on top of the AIIE monolayer 1 hr before JP-8 and SP exposure. In all the conditions used in the tests, cell viability, as determinedby trypan blue exclusion, was > 95%.

JP-8 Exposure and Substance P treatment

JP-8 (Lot #3509, Fuels Laboratory, Wright-Patterson AFB, OH) was used in this study and its physical and chemical properties were well characterized (DOD, 1992; Henz, 1998) and summarized in previous publications (ATSDR, 1998; NRC, 2003). Briefly, JP-8 is a complex mixture containing > 200 aliphatic and aromatic hydrocarbon compounds with 9–17 (or perhaps more) carbon atoms, including thousands of isomeric forms that distill at 170–325°C. The major hydrocarbon classes in JP-8 are n-alkanes, naphthenes, aromatics, and olefins. In addition, there are 3–5 nonhydrocarbon performance additives, such as diethylene glycol monomethyl ether, stadis 450, and DCL-4A. The experimental concentrations of JP-8 were based on measurement of body burden of JP-8 (1 to 124 ng/ml in blood; Gibson et al., 2001). Considering the absorption and distribution of JP-8 in pulmonary and systematic circulation, the concentration (124 ng/ml) X 1, 5, 10, and 20 was calculated for the range of exposure and a 0.2 and 1.6 μ g/ml JP-8 were chosen in this study in order to reflect the low and high JP-8 exposure levels in alveoli.

JP-8 was first dissolved at 2% in 100% ethanol (20 μ l in 980 μ l EtOH) as stock solution, which was then dissolved in media to obtain the 0.2 and 1.6 μ g/ml concentrations. In addition, substance P or [Sar⁹ Met (O₂)¹¹] substance P was dissolved in culture media to obtain 10^{− 10} M concentrations or a series of combined concentrations of JP-8 and substance P or [Sar⁹ Met (O₂)¹¹] substance P mentioned here. Control AIIE and AM cultures had cell culture media alone or cell culture media with ETOH vehicle. After media was removed from cell culture wells, cells were replaced with either control media or JP-8- or substance P/[Sar⁹ Met (O₂)¹¹] substance P-supplemented media or combinations of both. Cells were then incubated for 24 hr at which time the media were removed and frozen at −70°C until cytokines and nitric oxide analyses.

Cell Viability Assay

The MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Thiazolyl blue, Cell Proliferation Assay, Sigma Co., St. Louis, MO) was performed according to manufacturer's instructions. Briefly, a 10 μ l of MTT dye (0.5 mg/ml) was added directly to the 96-well plates containing growing cells with 90 μ l medium after JP-8 and/or SP treatment. The tested cells were incubated for 2 hr at 37°C and the converted dye was solubilized with acidic isopropanol (0.04–0.10 N HCl in absolute isopropanol). Absorbance of converted dye was measured at a wavelength of 570 nm with background subtraction at 630–690 nm.

Cytokine Analyses

IL-1α and IL-1β in supernatants from AM, AIIE, and AM/AIIE co-cultures were quantified by using commercially available ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's detailed protocol. Spectrophotometer analyses utilized a Bio-Tek EL_x808_IU microplate reader (Bio-Tek Instruments, Winooski, VT). Samples were quantified in triplicate and averaged to obtain the final value. The minimum detectable dose (MDD) of this assay was 1 pg/ml of IL-1α and 1 pg/ml of IL-1β .

NO_x Estimation

The sum of the NO metabolites nitrite (NO₂^–) and nitrate(NO₃^–) is widely used as an index of NO generation andexpressed as NO_x levels per ml, which corresponds to 10⁶ cells in this study. NO_x levels in supernatants from cultured AM, AIIE, and AM/AIIE co-cultures were calculated by measuring conversion of NO₃^– to NO₂^– by the enzyme nitrate reductase using an ELISA assay (R&D Systems, Minneapolis, MN) based on the Griess reaction that absorbs visible light at 540 nm. The minimum detectable dose (MDD) of this assay was 0.8 pg/ml. All samples were tested in duplicate and values were corrected by running samples with culture media without cells to assess background NO_x levels.

Statistical Analyses

Analysis of variance (ANOVA) was utilized to test differences between means for greater than two groups. Fisher's protected least-significant difference multiple t-test was utilized to identify post-hoc differences between individual cell culture groups. All statistical analyses utilized StatView 4.5 statistical software (Abacus Concepts, Berkeley, CA) and p < 0.05 was considered significant.

RESULTS

To examine the regulation of inflammatory response by substance P in the alveoli against JP-8 exposure, we conducted experiments of cultured AM, AIIE, and AM/AIIE co-culture with JP-8, SP, and [Sar⁹ Met (O₂)¹¹] substance P, an agonist of substance P. In these experiments, neither JP-8 exposure nor substance P or [Sar⁹ Met (O₂)¹¹] substance P treatment in all culture was found to affect cell viability. The cell viability of the treated groups and controls was > 95%.

Macrophages (AM)

Exposure of AM to 1.6 μ g/ml, not to 0.2 μ g/ml, JP-8 for 24 hr produced an increased release of IL-1α (Figure 1). In cells treated with 10^{− 10} M substance P or [Sar⁹ Met (O₂)¹¹] substance P, no significant change in IL-1α was observed. It suggests that substance P per se at this concentration has no specific effect on AM IL-1α release. However, treatment with substance P (10^{− 10} M) significantly attenuated the JP-8 induced IL-1α release in this culture. This datum indicates that added substance P has an observable inhibitory role of AM IL-1α release induced by JP-8. The effect of substance P was confirmed by using [Sar⁹ Met (O₂)¹¹] substance P, an agonist of substance P.

FIG. 1 IL-1α release from cultured macrophages after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the IL-1α levels in culture supernatant were measured by ELISA kits. Data were presented as mean values ± SEM. * p < 0.05 when compared to the control value. #p < 0.05 when compared to 0.2 μ g/ml JP-8 exposure group. %p < 0.05 when compared to 1.6 μ g/ml JP-8 exposure group. Results are the average of three independent experiments.

In the same setups of cell cultures, we also measured the concentration of IL-1β in cultured media. The change in AM IL-1β release had the similar response pattern as those of IL-1α after exposure to JP-8, substance P, and [Sar⁹ Met (O₂)¹¹] substance P, suggesting that the inhibitory role of substance P on IL-1α,β release may act through the same pathway (Figure 2).

FIG. 2 IL-1β release from cultured macrophages after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the IL-1β levels in culture supernatant were measured by ELISA kits. Data were presented as mean values ± SEM. * p < 0.05 when compared to the control value. #p < 0.05 when compared to 0.2 μ g/ml JP-8 exposure group. %p < 0.05 when compared to 1.6 μ g/ml JP-8 exposure group. Results are the average of three independent experiments.

Exposure of cultured AM to JP-8 or substance P for 24 hr had no significant effect on NO production (Figure 3). However, a significant increase in NO production in AM treated by [Sar⁹ Met (O₂)¹¹] substance P alone was observed. This effect was enhanced by co-treatment with JP-8, suggesting that this agonist of substance P could induce NO production that makes AM more sensitive to JP-8 exposure.

FIG. 3 Nitric oxide (NO) production in cultured macrophages after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the NO production in culture supernatants were measured by Nitric Oxide assay kit. Data were presented as mean values ± standard error of the mean (SEM).* p < 0.05 when compared to the control value. #p < 0.05 when compared to 0.2 μ g/ml JP-8 exposure group. %p < 0.05 when compared to 1.6 μ g/ml JP-8 exposure group. Results are the average of three independent experiments.

Alveolar Type II Epithelium (AIIE)

Exposure of cultured AIIE to JP-8 for 24 hr did not affect release of IL-1α,β (Figures 4 and 5). In AIIE treated with 10^{− 10} M substance P or [Sar⁹ Met (O₂)¹¹] substance P, no significant change in IL-1α,β was observed. These experiments suggest that either JP-8 or substance P treatment at the range of concentrations has no specific effect on IL-1α,β release from AIIE. Furthermore, there is no significant change of IL-1α,β in culture media of AIIE after JP-8 exposure with substance P or [Sar⁹ Met (O₂)¹¹] substance P.

FIG. 4 IL-1α release from cultured type II epithelial cells after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the IL-1α levels in culture supernatant were measured by ELISA kits. Data were presented as mean values ± SEM. Results are the average of three independent experiments.

FIG. 5 IL-1β release from cultured type II epithelial cells after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the IL-1β levels in culture supernatant were measured by ELISA kits. Data were presented as mean values ± SEM. Results are the average of three independent experiments.

However, exposure of cultured AIIE to JP-8 for 24 hr induced a concentration-dependent increase in NO production, as measured by a [nitrite + nitrate] ELISA kit (Figure 6). Similarly, treatment with [Sar⁹ Met (O₂)¹¹] substance P (10^{− 10} M) also resulted in a significant increase of NO production. This elevation was enhanced when JP-8 exposure with [Sar⁹ Met (O₂)¹¹] substance P, indicating that both JP-8 and [Sar⁹ Met (O₂)¹¹] substance P treatment has induced the release of NO production from AIIE.

FIG. 6 Nitric oxide (NO) production in cultured type II epithelial cells after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the NO production in culture supernatant were measured by Nitric Oxide assay kit. Data were presented as mean values ± SEM. * p < 0.05 when compared to the control value. #p < 0.05 when compared to 0.2 μ g/ml JP-8 exposure group. %p < 0.05 when compared to 1.6 μ g/ml JP-8 exposure group. Results are the average of three independent experiments.

AM/AIIE Co-culture

In an established set-up of AM/AIIE co-cultures, a simulated alveoli model of the in vivo situation, we examined the regulation of JP-8 induced IL-1α,β and NO release by substance P by using the same protocol as that of experiments in AM or AIIE culture. Exposure of AM/AIIE co-cultures to JP-8 for 24 hr induced consistently an increased release of IL-1α in cultured media (Figure 7). However, treatment with substance P or its agonist (10^{− 10} M) significantly attenuated the JP-8 induced IL-1α release, suggesting that substance P or its agonist has an observable inhibitory role in IL-1α release induced by JP-8.

FIG. 7 IL-1α release from co-cultures after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the IL-1α levels in culture supernatant were measured by ELISA kits. Data were presented as mean values ± SEM. * p < 0.05 when compared to the control value. #p < 0.05 when compared to 0.2 μ g/ml JP-8 exposure group. %p < 0.05 when compared to 1.6 μ g/ml JP-8 exposure group. Results are the average of three independent experiments.

As shown in Figure 8, the increase in AM IL-1β release had the similar pattern as those of IL-1β after exposure to JP-8. However, no significant inhibitory effect of substance P or [Sar⁹ Met (O₂)¹¹] substance P on IL-1β release was observed at this endpoint (p > 0.05). In the same setup of co-cultures, there were no significant changes statistically in NO production after treatment of JP-8 with or without substance P including its agonist (Figure 9).

FIG. 8 IL-1β release from co-cultures after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the IL-1β levels in culture supernatant were measured by ELISA kits. Data were presented as mean values ± SEM. * p < 0.05 when compared to the control value. Results are the average of three independent experiments.

FIG. 9 Nitric oxide (NO) production in co-cultures after treatments of JP-8, substance P, [Sar⁹ Met (O₂)¹¹] substance P, and their combinations. Cells were cultured for 24 hr and the NO production in culture supernatant were measured by Nitric Oxide assay kit. Data were presented as mean values ± SEM. Results are the average of three independent experiments.

DISCUSSION

In a simulated JP-8 exposure protocol, we have examined the effects of JP-8, in targeted AM, type II pneumocytes (AIIE), and AM/AIIE cultures, on the production of typical pro-inflammatory cytokines IL-1α,β and NO. Data show that: (1) JP-8 induced IL-1α,β and NO release in alveolar macrophages and type II epithelial cells, respectively, indicating that two cell types exhibit different initial inflammatory mechanisms as cellular results of acute exposure to the low level of JP-8; (2) substance P or its agonist [Sar⁹Met (O₂)¹¹] substance P significantly block JP-8 induced IL-1α,β release. This finding indicates that substance P is a modulator in the inflammatory response mediated by IL-1 production; and, (3) co-culture could prevent JP-8 induced NO overproduction observed in cultured AIIE alone, suggesting that NO production could be regulated possibly by signal communication of AMs, which reside in close proximity to each other.

Cell-Dependent Effects and Cell Interaction

Alveolar macrophages and type II epithelial cells are the first cells to encounter inhaled JP-8 (Robledo et al., 2000; Boulares et al., 2002; Wang et al., 2002). However, how these types of cells contribute to the inflammatory response and their interaction remain largely unknown. In this regard, we set up an in vitro culture system to understand cellular responses to JP-8 exposure. Our data show that acute exposure of cultured AM to JP-8 induces a significant release of IL-1, both IL-1α and IL-1β, as an important inflammatory source in alveoli, whereas cultured type II epithelial cells generated a significant release of NO production after JP-8 exposure.

It appears that there are alveolar cell-dependent effects through obvious initial response mechanisms. Therefore, excessive IL-1α,β and NO production generated from AM and type II epithelial cells, respectively, could contribute to the development of JP-8 induced lung inflammation that we have observed in our previous in vivo studies (Hays et al., 1995; Robledo and Witten, 1998; Robledo et al., 2000). There is compelling evidence that both endogenous IL-1α,β and NO production are crucial pro-inflammatory mediators in both local and systemic acute inflammatory responses (Kabbur et al., 2001).

However, NO production is significantly attenuated in AM/ AIIE co-culture when compared with those in cultured type II epithelial cells alone, suggesting that there is cell interaction, in the case of NO production, between AM and AIIE in response to the JP-8 exposure. It appears that AMs have the capability to inhibit type II epithelial cells to generate NO through certain mechanisms such as direct cell-cell physical contact or intercellular communication. The change in NO production from this AM/AIIE co-culture system could more closely mimic the in vivo situation of JP-8 exposure in alveoli. As we know, AM play an active role in the control of the immune and inflammatory reactions in alveoli, acting in synergy with alveolar epithelial cells to preserve the lung structure, essential for alveolar homeostasis. Due to complexity of the responses occurring in vivo, therefore, our co-culture system needs to be further characterized to replicate what would exhibit in the intact alveoli during exposure to JP-8.

Role of Substance P Signaling on IL-1 and NO Production

Substance P is a neurotransmitter of the non-adrenergic, non-cholinergic nervous (NANC) system (sensory C-fibers) that serves as an important sensory system in detecting the onset of pathophysiological changes in the lungs. Reflexes generated by the stimulation of C-fibers are both protective/defensive (e.g., apnea, laryngeal narrowing, bronchoconstriction, sneezing, aspiration, and expiration reflexes). These responses mediate the antidromical released neurokinins, particularly substance P (SP), binding to a family of ubiquitous G protein-coupled receptors with at least three distinct members, of which the neurokinin-1 receptor (NK-1R) has the highest affinity for SP. Interestingly, in this study, SP or its agonist [Sar⁹Met (O₂)¹¹] SP significantly block JP-8 induced AM IL-1α and IL-1β .

This observation suggests that substance P may have transduction pathway(s) to signal AM in the inflammatory response mediated by IL-1 production. Our previous in vivo study has shown that mice administered [Sar⁹,Met(O₂)¹¹] substance P after JP-8 exposure had the appearance of normal pulmonary values and tissue morphology (Robledo and Witten, 1999). In contrast, endogenous substance P receptor, neurokinin-1 receptor, antagonism by CP-96345 administration exacerbated JP-8-enhanced permeability and bronchiolar epithelial injury. In another model, SP administration decreased the release of IL-8 normally seen in keratinocytes after JP-8 exposure, a response similar to that reported for SP's effect on JP-8 pulmonary toxicity (Monteiro-Riviere et al., 2004). These data indicate that substance P has a protective role in preventing the development of JP-8-induced injury, possibly through receptor-dependent activation.

In summary, this study showed that alveolar cell-dependent inflammatory mechanisms may be responsible for the inflammatory response after the occupational level of JP-8 exposure. In the same exposure condition, AM exhibited relative elevated release of pro-inflammatory cytokine IL-1α,β, whereas type II epithelial cells generated NO overproduction. Data from lung cell co-cultures indicated that the generation of NO production could be regulated possibly by AM, in close proximity to each other. Obviously, there is a signaling mechanism(s) between the two alveolar-resident cells. Moreover, substance P or its agonist [Sar⁹Met (O₂)¹¹] substance P significantly block JP-8 induced AM IL-1α and IL-1β, thus suggesting the existence of a neuro-immune cell interaction in vivo. This finding illustrates that the activation of the sensory neuropeptide pathway may represent an initial defense mechanism in the respiratory system. However, additional studies are needed to confirm whether AM/AIIE co-culture system is an accurate reflection of in vivo alveolar physiology as well as to examine how substance P is involved in these inflammatory mechanisms.

ACKNOWLEDGMENTS

The research was supported by AFOSR grant F49620-94-1-0297. This study was also supported in part by NIEHS P30 ES006694 and DOD AASERT Program.