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Published ahead of print on May 8, 2003, doi:10.1165/rcmb.2002-0107OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 606-612, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0107OC

Lipopolysaccharide Induces Preprotachykinin Gene Expression

Hsueh-Yin Huang and Yih-Loong Lai

Department of Physiology, National Taiwan University College of Medicine, Taipei, Taiwan

Address correspondence to: Yih-Loong Lai, Ph.D., Department of Physiology, College of Medicine, National Taiwan University, No. 1, Sec. 1, Jen-Ai Road, Taipei 100, Taiwan. E-mail: tiger{at}ha.mc.ntu.edu.tw


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was performed to test whether biosynthesis of tachykinins plays a pivotal role in lipopolysaccharide (LPS)-induced airway alteration by analyzing preprotachykinin-I (PPT-I, a precursor of tachykinins) gene expression. Brown-Norway rats (11–12 wk old) were divided into four groups: control; LPS; dimethylthiourea (DMTU, an effective hydroxyl radical scavenger); and DMTU+LPS. Each animal in the control group received saline treatment. Forty-nine animals in the LPS group were further divided into seven subgroups to test effects of doses and length of the LPS treatment. Total RNA extracted from nodose ganglia and lungs was used to assay relative amount of PPT-I mRNA using the real-time quantitative reverse transcriptase–polymerase chain reaction. In addition, LPS-induced alterations in airway responses to bronchial constrictors, neutral endopeptidase (NEP) gene expression, leukocyte counts, and SP and calcitonin gene–related peptide (CGRP) levels were determined. LPS (4 mg/kg, intraperitoneal) raised significantly PPT-I mRNA level after 4 h in nodose ganglia and 12 h in the lung, and this elevation sustained for 5 d. Also, LPS caused significant increases in NEP mRNA, SP and CGRP levels, airway reactivity to capsaicin and SP, and neutrophil counts, but a significant decrease in macrophage count. Our data support that LPS-induced bronchial hyperreactivity to capsaicin is related closely to the upregulation of tachykinin gene expression, but not the upregulation of NEP.

Abbreviations: bronchoalveolar lavage, BAL • calcitonin gene–related peptide, CGRP • dimethylthiourea, DMTU • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • interleukin, IL • lipopolysaccharide, LPS • maximal expiratory flow-volume, MEFV • neutral endopetidase, NEP • neurokinin, NK • preprotachykinin-I, PPT-I • reactive oxygen species, ROS • substance P, SP • tachykinin, TK • total lung capacity, TLC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Endotoxin is a potent proinflammatory lipopolysaccharide (LPS) in the outer membrane of gram-negative bacteria. Systemic endotoxemia has been recognized as one of the leading causes of acute respiratory distress syndrome (1). Evidence has demonstrated that tachykinins (TKs) released from sensory C-fibers play a pivotal role in LPS-induced airway inflammation (2, 3). Substance P (SP), a TK, is translated through three types of mRNA encoding {alpha}-, ß-, and {gamma}-preprotachykinin-I (PPT-I) transcribed by one gene (4). Sensory neurons, which synthesize TKs, supplying the lung are located in jugular and nodose vagal ganglia (5, 6). SP regulates inflammation via plasma extravasation, cell proliferation, and effects on the immune system (7). In vitro studies also show that LPS induces an increase in SP in sympathetic ganglia via ganglionic interleukin-1 (IL-l) production (8), and SP enhances LPS-induced IL-l release from primary cultures of neuroglial cells (9). Thus, SP has been documented as both a neurotransmitter and an immunomodulator substance (10).

Reactive oxygen species (ROS) have been implicated in the pathogenesis of inflammatory lung diseases. LPS increases the release of various enzymes and reactive oxygen intermediates, which are capable of injuring pulmonary endothelial cells (11, 12), involved in the development of TK-mediated neurogenic plasma exudation (13) and airway constriction (14). Evidence has suggested that ROS seem to play an important role in TK-mediated biological functions, but the underlying mechanisms for the interaction between TKs and ROS are not clear.

In the present study, we investigated whether LPS induces an increase in PPT-I mRNA in nodose ganglia and the lung using the real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) method. We tested also whether a hydroxyl radical scavenger dimethylthiourea (DMTU) alters LPS-induced PPT-I gene expression. TKs and calcitonin gene–related peptides (CGRP) are present together in capsaicin-sensitive sensory nerves in the internal organs (15, 16). TKs and CGRP are costored in the same large dense vesicles in sensory neurons (7, 17), and these peptides are coreleased upon activation of sensory nerves by, for example, capsaicin (16, 18). Thus, we attempted to detect LPS-induced changes in bronchial reactivity to capsaicin, methacholine, and SP, as well as alterations in SP and CGRP levels in the bronchoalveolar lavage (BAL).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal Preparations
The study was conducted according to the Guidelines of the American Physiological Society, and was approved by the Animal Care and Use Committee of the National Taiwan University.

Seventy-seven Brown Norway strain rats weighing 250 ± 10 g (11–12 –wk old) were divided into four groups: saline control (n = 14); DMTU (n = 7); LPS (n = 49); and DMTU+LPS (n = 7). In the control group, the animal received the same amount of saline via intravenous (the saline control-1 subgroup, n = 7) or intraperitoneal injection (the saline control-2 subgroup, n = 7). DMTU was intraperitoneally injected for 3 d before the study. The three consecutive daily doses of DMTU were 250 mg/kg (14). Animals in the LPS group were further divided into seven subgrougps with seven animals each: LPS–2 mg–4 h; LPS–4 mg–0 h; LPS–4 mg–2 h; LPS–4 mg–4 h; LPS–4 mg–12 h; LPS–4 mg–1 d; and LPS–4 mg–5 d. LPS (2 or 4 mg/kg) was intraperitoneally administered in the LPS group for a fixed period of time (from 0 h to 5 d, as indicated above) before the study. For the DMTU+LPS group, animals were treated with DMTU as mentioned above. Then, these animals were intraperitoneally administered with LPS (4 mg/kg) 12 h before the study. On the day of the study, each animal was anesthetized with sodium pentobarbital (35–45 mg/kg) and its trachea was cannulated. Subsequently, the lung (50–100 mg) and bilateral nodose ganglia were excised and immersed in 1 ml ice-cold Trizol reagent (Gibco-BRL, Grand Island, NY) for later RNA extraction.

To explore LPS-induced change in airway reactivity to capsaicin, an additional 25 Brown Norway rats were divided into three groups: saline control (n = 10), LPS (n = 8), and DMTU+LPS (n = 7). In the control group, animals were treated with saline (intraperitoneally) in the same manner as that described above. For the LPS group, each animal was treated with LPS (4 mg/kg, intraperitoneally) 24 h before the functional study. DMTU was given according to the method mentioned above. On the third day, LPS administration was added in addition to DMTU. Bronchial constriction was measured before and 1–30 min after an intravenous injection of capsaicin (40 µg/kg). To explore LPS-induced change in airway reactivity to methacholine and SP, an additional 15 Brown Norway rats were divided into two groups: saline control (n = 7) and LPS (n = 8). These two groups of animals were treated with the same methods as those described above before the study. Bronchial constriction was measured before and 1–30 min after an intravenous injection of methacholine (4 mg/kg), and then followed 40 min later by SP (8 x 10-8 mol/kg, intravenous).

To determine the effects of LPS on CGRP level, 26 animals were divided into four groups: saline control (n = 8); LPS–4 mg–4 h (n = 5); LPS–4 mg–12 h (n = 5); and LPS–4 mg–1 d (n = 8). Animals were treated in the same manner as those described above for the determination of PPT-I gene expression.

An additional 16 rats were used to collect BAL samples for SP analysis. Animals were divided into two groups: saline control (n = 8) and LPS (n = 8). Twenty-four hours after LPS (4 mg/kg, intraperitoneally) treatment or equivalent time, BAL samples were collected to analyze SP.

To analyze cell counts in BAL samples, 25 animals were divided into four groups: saline control (n = 7); LPS–4 mg–4 h (n = 5); LPS–4 mg–12 h (n = 6); and LPS–4 mg–24 h (n = 7). Animals of these groups were treated in the same fashion as those described above for the determination of PPT gene expression. Then, BAL was collected to analyze cell counts.

RNA Extraction and Analysis
Total RNA was extracted from nodose ganglia and lung tissue using Trizol reagent as previously described (19). Total RNA pellet was dissolved in RNase-free H2O made with diethyl pyrocarbonate (DEPC; Flauka, Buch, Switzerland). Optical density of the RNA extraction was detected with an ultraviolet spectrophotometer under 260 nm (OD260) and 280 nm (OD280) spectra. In addition, the bands of 18 s and 28 s appeared under ultraviolet light on 1.2% agarose gels containing 1 µl of 10 mg/ml ethidium bromide (BioRad, Glattbnrugg, Switzerland), indicating the integrity of nucleic acids in RNA extraction (20).

Real-Time Quantitative RT-PCR
Real-time quantitative RT-PCR was performed mainly according to our previous method (21). The theoretical basis of the ABI PRISM 7,700 Sequence Detection System (TaqMan) real-time quantitative RT-PCR (Perkin-Elmer Applied Biosystems, Foster City, CA) has been described in detail elsewhere (22). Fluorescent signal from each PCR reaction was collected as peak-normalized values plotted versus the cycle number. Reactions were characterized by comparing threshold cycle (Ct) values. The Ct was a unitless value defined as the fractional cycle number at which the sample fluorescence signal passes a fixed threshold above baseline. Sample with high starting copy number showed an increase in fluorescence early in the PCR process, resulting in a low Ct number, whereas lower starting copy number resulted in higher Ct number.

Oligonucleotide Primers and TaqMan Probe Design
The PPT-I mRNA sequence was evaluated using the Primer Express software (Perkin-Elmer) to design the primer set from base pair 211 to base pair 277 (reverse primer from base pair 211 to base pair 232; forward primer from base pair 250 to base pair 277), as well as a corresponding probe (from base pair 233 to base pair 257 of PPT-I gene) with FAM (6-carboxylfluorescein, as the reporter dye) and TAMRA (6-carboxyl-tetramethyl-rhodamine, as the quencher dye). The primer set and probe were purified with HPLC following synthesis. The forward and reverse primers were designed to lie in adjacent exon to prevent amplification of genomic DNA that may be contained in samples. We also quantitated transcripts of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene using the sequence from base pair 379 to base pair 441 (reverse primer from base pair 379 to base pair 397; forward primer from base pair 422 to base pair 411, as well as a corresponding probe [from base pair 399 to base pair 420] with FAM and TAMRA) as the internal control, with each unknown sample normalized to GAPDH content.

RT-PCR Thermal Cycle Condition
The reaction was performed in 50 µl with 5x TaqMan buffer, 3.0 mM Mn(OAc)2, dNTPs (0.3 mM each), 0.5 U AmpErase uracil N-glycosylase (UNG) (1 U/µl), and 5.0 U rTth DNA polymerase (2.5 U/µl) from the TaqMan EZ RT-PCR kit (Perkin-Elmer). Final PPT-I and GAPDH forward and reverse concentrations were 4.1 µM, and probe concentration was 2.05 µM. To reduce variability between replicates, PCR premixes, which contained all reagents except total RNA (200 ng), were prepared and aliquoted into 0.2-ml optical tube (MicroAmp; Perkin-Elmer). The calibrator sample containing the same reagents as described above except the RNA sample was substituted by water containing DEPC. Thermal cycling conditions were 2 min at 50°C (for UNG activation), 30 min at 60°C (reverse transcription step) following 5 min at 95°C (for UNG deactivation); PCR reaction was then performed in 40 cycles of 15 s at 94°C and 1 min at 60°C. The products (PPT cDNA and GAPDH cDNA) were identified as bright bands under ultraviolet light in 2% agarose gel containing ethidium bromide with size of 63 base pairs and 66 base pairs, respectively.

Measurement of Neutral Endopeptidase mRNA
The same as the above method (Real-time quantitative RT-PCR to determine PPT mRNA), total RNA extracted from the lung of the saline control (n = 8) or the 24 h LPS–treated group (n = 8) was used to analyze neutral endopeptidase (NEP) mRNA. The NEP mRNA was evaluated using the Primer Express software (Perkin-Elmer) to design the primer set from base pair 1,986 to base pair 2,067 (forward primer from base pair 1,986 to base pair 2,010; reverse primer from base pair 2,045 to base pair 2,067), as well as a corresponding probe (from base pair 2,025 to base pair 2,042 of NEP gene) with FAM and TAMRA. We also determined transcripts of the GAPDH gene using the method described above.

Evaluation of Airway Function
On the day of the study, each animal was anesthetized with sodium pentobarbital (35–45 mg/kg), and its trachea was cannulated. The animal was then paralyzed with gallamine triethiodide (10 mg/kg) and was artificially ventilated. In the whole-body plethysmograph, the respiratory flow of the animal was monitored with a Validyne DP45 differential pressure transducer as the pressure dropped across three layers of 325-mesh wire screen in the wall of the plethysmograph. Lung volume change was acquired via integration of flow with time. The airway opening pressure (Pao) and the arterial pressure were measured by pressure transducers (DPX/Plus; Viggo-Spectramed, Oxnard, CA) connected to a side hole of the tracheal tube and to the arterial catheter, respectively. All of the above signals were recorded on a recorder (TA11; Gould, Valley View, OH). During the baseline period, we first performed the full maximal expiratory flow-volume (MEFV) maneuver to obtain the baseline total lung capacity (TLC, lung volume at Pao = 30 cm H2O). Before the maneuver, the lung was inflated two times to TLC and deflated spontaneously to establish the volume history of each animal. The full MEFV maneuver includes an inflation of the lung to TLC with a positive pressure of 30 cm H2O and a subsequent deflation to residual volume with a negative pressure of 40 cm H2O, which produced maximal expiratory flow (max). Both positive and negative pressures, controlled by a pressure regulator (Dwyer, Michigan City, IN), were provided by pressure reservoirs. Forced expiratory flow in 0.1 s (FEV0.1) was obtained from the recording paper. In addition, the MEFV plot was also stored on a digital storage oscilloscope (VC-6025; Hitachi, New York) and was used to get the max at 50% baseline TLC (max50). Functional residual capacity (FRC) was determined by the neon dilution method (23). Decreases in FEV0.1 and max50 were used as indicators of bronchoconstriction.

BAL (CGRP and SP Levels, as well as Cell Counts)
According to a previous method (14), BAL was collected from each animal to determine CGRP. Collected BAL samples were purified by filtration through a C-18 column. Then, CGRP level was analyzed in BAL samples using a Rat CGRP Enzyme Immunoassay Kit obtained from SPI-BIO (Massy Cedex, France). Collected BAL samples were purified, and then their SP levels were analyzed by solid-phase enzyme immunoassay with a commercial kit (Cayman Chemical, Ann Arbor, MI).

For BAL cell counts, 4 ml warm (39°C) saline was instilled into the lung of the anesthetized rat via trachea. The fluid in the bronchoalveolar space was withdrawn 30 s after the instillation; and the same procedure for collecting BAL was repeated four times. Pooled BAL was centrifuged, and the cell pellet was resuspended. One hundred microliters of the cell suspension was added to the same volume of 0.5% trypan blue solution, and total cells were counted in a hemacytometer. Differential cell counts were determined from cytospin preparations stained by Liu stain.

Data Analysis
The comparative Ct ({Delta}{Delta}Ct) method was used to quantify PPT-I and NEP mRNA levels (21, 24). The advantage of this method is that it eliminates the need for standard curves. The {Delta}{Delta}Ct method uses the DEPC water, termed the calibrator sample or no template control, for comparison of every unknown sample's gene expression level. The calibrator sample is analyzed on every assay plate with unknown samples of interest. For PPT-I gene, the calculation of {Delta}{Delta}Ct was: {Delta}{Delta}Ct = [Ct PPT-I (unknown sample) - Ct GAPDH (unknown sample)] - [Ct PPT-I (calibrator sample) - Ct GAPDH (calibrator sample)] (21, 25). Similarly, for NEP mRNA, the calculation of {Delta}{Delta}Ct was: {Delta}{Delta}Ct = [Ct NEP gene (unknown sample) - Ct GAPDH (unknown sample)] - [Ct NEP gene (calibrator sample) - Ct GAPDH (calibrator sample)]. The formula for obtaining the relative amount of mRNA is: 2-({Delta}{Delta}Ct).

All data are reported as means ± SEM. ANOVA was used to establish differences among groups. If significant difference existed among groups, Duncan's multiple range test was used to differentiate differences between any two of them. Difference was considered significant when P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of LPS on PPT-I and NEP mRNA Expression
After 4 h of LPS, 4 mg/kg (but not 2 mg/kg) treatment induced a significant increase in PPT-I mRNA expression in the nodose ganglia and the lung (Figure 1).



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  		Figure 1. PPT-I mRNA expression of the nodose ganglia and the lung at 4 h after saline (control; open bars), 2 mg/kg of LPS (striped bars) or 4 mg/kg of LPS (hatched bars) treatment. The saline control value was the average of saline-1 and saline-2 subgroups. Significant difference compared with the saline control group: *P < 0.05 and **P < 0.01.

 
Temporal changes in LPS-induced increase in PPT-I mRNA expression of the nodose ganglia and the lung are shown in Figure 2. LPS (4 mg/kg) induced an earlier increase in PPT-I mRNA expression in the nodose ganglia than that in the lung. In the nodose ganglia, the increase reached a significant level at 4 h and climbed to a maximal value at 12 h. On the other hand, the maximal value of PPT-I mRNA expression in the lung occurred at 24 h after the LPS treatment.



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  		Figure 2. PPT-I mRNA expression before (time = 0) and up to 5 d after LPS (4 mg/kg) treatment in the nodose ganglia (open circles) and the lung (filled circles). **Significant difference (P < 0.01) compared with the baseline (time = 0) value.

 
Relative lung NEP mRNA amount in the saline control and LPS groups were 0.083 ± 0.037 and 0.280 ± 0.067, respectively. There was a significant increase in lung NEP mRNA level 24 h after LPS treatment.

Effects of DMTU on PPT-I mRNA Expression
In the nodose ganglia, LPS (4 mg/kg)-induced increase in PPT-I mRNA expression at 12 h was attenuated significantly by the DMTU pretreatment (Figure 3A). Similarly, at 12 h after LPS (4 mg/kg) treatment, LPS-induced increase in PPT-I mRNA expression of the lung was ameliorated significantly by DMTU (Figure 3B).



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  		Figure 3. PPT-I mRNA expression in the nodose ganglia (A) and in the lung (B) at 12 h after intravenous saline (saline-1), intraperitoneal saline (saline-2), DMTU, LPS (4 mg/kg), or DMTU+LPS (D+L) treatment. *Significant difference (P < 0.05) compared with the saline-2 subgroup. #Significant difference (P < 0.05) compared with the LPS group.

 
Effects of LPS and DMTU on Bronchial Function
At baseline, values for FEV0.1 and max50 ranged from 5.46 ± 0.38 to 6.08 ± 0.18 ml/s and 53.7 ± 5.2 to 64.9 ± 4.3 ml/s, respectively, with no significant difference in either FEV0.1 or max50 value among groups. At 10 min after capsaicin challenge, both FEV0.1 (Figure 4), and max50 (Figure 5) decreased markedly, indicating capsaicin-induced airway constriction in the control group. Twenty-four hours after LPS (4 mg/kg) treatment, these capsaicin-induced decreases were augmented significantly. However, this LPS-augmented, capsaicin-induced airway constriction was significantly attenuated in the DMTU+LPS group.



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  		Figure 4. Capsaicin-induced changes (values were obtained 10 min after capsaicin challenge) in forced expiratory volume in 0.1 s (FEV0.1) at 24 h after saline (control), LPS (4 mg/kg), or DMTU+LPS treatment. *Significant difference (P < 0.05) compared with the saline control and the DMTU+LPS groups.

 


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  		Figure 5. Capsaicin-induced changes (values were obtained 10 min after capsaicin challenge) in maximal expiratory flow at 50% total lung capacity (max50) at 24 h after saline (control), LPS (4 mg/kg), or DMTU+LPS treatment. **Significant difference (P < 0.01) compared with the control and the DMTU+LPS groups.

 
Compared with the control group, the same dose of SP, but not methacholine, induced a significantly larger decrease in max50 (more severe airway constriction) in the LPS (24 h) group (Figure 6).



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  		Figure 6. Methacholine (Mch, 4 mg/kg)- and substance P (SP, 8 x 10-8 mol/kg)-induced changes in maximal expiratory flow at 50% total lung capacity (max50) at 24 h after saline (control) or LPS (4 mg/kg) treatment. *Significant difference (P < 0.05) compared with the control group.

 
Effects of LPS on BAL CGRP and SP Levels
LPS (4 mg/kg) caused significant increases in BAL CGRP levels at 4 and 24 h. For the saline control group, BAL CGRP level was 126 ± 24 pg/ml; the value increased significantly (P < 0.05) to 503 ± 58 pg/ml at 4 h, and then to 395 ± 36 pg/ml at 24 h after LPS. Similarly, BAL SP level of the saline control group was 8.5 ± 2.6 pg/ml, and the level increased significantly (P < 0.05) to 18.3 ± 4.2 pg/ml at 24 h after LPS.

Effects of LPS on BAL Cell Counts
LPS (4 mg/kg) significantly increased total BAL cell numbers within 12 h, and this increase continued to 24 h after LPS treatment (Table 1). Percentage of macrophages decreased (Table 1), whereas that of neutrophils increased, after LPS administration. These changes in macrophages and neutrophils reached significant level at 12 h, with more severe alterations at 24 h.


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  		TABLE 1 Lipopolysaccharide-induced changes in cell counts in bronchoalveolar lavage

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrated that LPS treatment increased SP gene expression within 4 h, and this increment reached a maximal level at 12 h in the nodose ganglia. A hydroxyl radical scavenger, DMTU, attenuated this LPS-induced upregulation of SP gene expression. Also, LPS caused significant increases in BAL SP and CGRP levels, lung NEP gene expression, bronchial reactivity to capsaicin and SP, and neutrophil count; but it induced a decrease in the percentage of macrophages. Several features of these results will be discussed below.

LPS caused an increase in airway reactivity in response to capsaicin and SP. Capsaicin- and SP-induced airway constriction is mainly the noncholinergic airway constriction. The increased noncholinergic airway reactivity might be due to increases in TK level, TK receptor number, and/or the interaction of TKs with other mediators. We showed in this study that an increase in BAL SP level appeared 24 h after LPS treatment. This fact was augmented by a simultaneous increase in co-stored and co-released CGRP level. However, it is not clear whether there are increases in TK receptor number and/or the interaction of TKs with other mediators. In guinea pigs, an increase in airway responsiveness to aerosolized histamine occurred 4 h after LPS inhalation (26). Also, an increased airway responsiveness to aerosol histamine was found in awake sheep 5 h after LPS inhalation (27). However, we did not find an increase in airway reactivity to non-TK constrictor (methacholine) 24 h after systemic LPS administration. This difference in non-TK–induced airway hyperreactivity between this and the two previous studies (26, 27) could be due to differences in LPS treatment and/or constrictor agent used. Furthermore, NEP is an important degradation enzyme for TKs. We confirmed the results of a previous study (28) that demonstrated that LPS induced an increase in NEP mRNA, and this increase should enhance the production of NEP and reduce TK level. Thus, LPS-induced elevation in SP should not be contributed by the increased activity of NEP.

LPS-induced increase in SP may be mainly due to an increase in mRNA coding for the precursor of SP PPT-I. In addition to this mRNA coding process, LPS-associated IL-1 has been shown to enhance the rate of transcription using the nuclear transcription assay (29). Furthermore, LPS caused increases in granulocytes in BAL fluid (Table 1). It is possible that LPS may act through non-neural cells such as macrophages, fibroblasts, and mast cells to release ILs (such as IL-1, IL-6, and tumor necrosis factor-{alpha}) (30), promotes the upregulation of cyclooxygenase-2 (COX-2) expression and then the synthesis of TKs (31). Also, we suggested previously that the increase in TK level in the lungs might be related to increased axonal transport of TKs (32). In this study, we could not rule out the factor of axonal transport of TKs.

In this study, we found that LPS induced increases in PPT-I mRNA gene expressions in both nodose ganglia and the lungs. There are two main sources of TKs for the lungs: neuronal and non-neuronal. The neuronal origin should include vagal C-fiber and sympathetic C-fiber afferents (33). Because nodose ganglia (the origin of vagal C-fiber afferents) and dorsal root ganglia (the origin of sympathetic C-fiber afferents) are the locations for neurons of afferent C-fibers, these two ganglia should be the sites of neuronal PPT-I mRNA production. PPT-I mRNA is a big molecule, and thus should not be transported from the above ganglia to their nerve terminals along the afferent C-fibers. Accordingly, PPT-I mRNA in the lungs may be not originated at all from the above ganglia. Therefore, mRNA in the lungs should be originated from neurons with cell bodies in intrinsic airway ganglia (34, 35) and from non-neuronal origins. The non-neuronal origins, as mentioned above, may include macrophages (10), neutrophils (10), eosinophils (36), and lymphocytes (37). Therefore, LPS-induced temporal increase in PPT-I mRNA should be related closely to the gradual increase in inflammatory cells with time (Table 1).

We showed that LPS-induced upregulation of PPT-I mRNA can be maintained above the baseline level for 5 d. This maintained upregulation might be related to a continuous interaction between TKs and inflammation. Based on data from the literature, the interaction may be generated in several ways. (i) TKs may act on neurokinin (NK) receptors on the surface of inflammatory cells, and lead to the release of IL-1 and COX-2, as mentioned above. (ii) The binding of TKs with NK1 or NK2 receptors may activate a signal transduction in mast cells, which then release histamine and attract leukocytes, with accompanying increase in ILs and activation of afferent C-fibers. (iii) Other inflammatory mediators such as bradykinin (38), leukotrienes (39), prostaglandins (40), and serotonin (7) may also play a role.

LPS-induced upregulation of SP gene expression was attenuated significantly by a hydroxyl radical scavenger DMTU (Figure 3). This may imply that the upregulation of PPT-I mRNA is related closely to ROS, especially hydroxyl radicals. These data are compatible with previous results obtained from our and other laboratories. We found that ROS augmented TK-dependent bronchial reactivity such as hyperpnea- (41), exsanguination- (42), and citric acid–induced (14) noncholinergic airway constriction. Also, other investigators have shown that ROS activate cardiac vagal C-fiber afferents (43) and visceral sympathetic C-fiber afferents (44). In addition to TK levels mentioned above, we made a further step to demonstrate that ROS also play an important role in TK gene expression. It is speculated that ROS may activate vanilloid receptors and lead to an increase in PPT-I mRNA.

In summary, we demonstrated that LPS-induced bronchial hyperreactivity to capsaicin was related closely to an upregulation of TK gene expression, increases in neuropeptide level, and leukocyte (especially neutrophil) counts.


   Acknowledgments
 
The authors thank Dr. Tze-Bin Chou for providing many suggestions, and Miss Y.-C. Tsou for technical assistance in this study. This investigation was supported by the National Health Research Institutes (NHRI-EX90–8833SL) and National Science Council (NSC89-2320-B002-169-M59).

Received in original form July 9, 2002

Received in final form April 1, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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