INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Endotoxemia contributes to the development of multiple organ failure in sepsis and is associated with the development of the adult respiratory distress syndrome (ARDS) (1). Endotoxemia induced by administration of lipopolysaccharide (LPS), a major constituent of the cell walls of gram-negative bacteria, can initiate production of biologically active molecules from a variety of cells (4, 5), including cytokines, bioactive amines, eicosancoids, and a variety of reactive oxygen species. These mediators alone or through their interaction recruit neutrophils to accumulate in the lung (6, 7). Despite a considerable body of data, there still is some reluctance to proclaim the neutrophil as the causative agent in endotoxin-induced lung injury (8). The intrapulmonary accumulation of neutrophils in ARDS does not necessarily prove that they are responsible for cellular lung injury (9, 10). The pulmonary consequence of neutrophil accumulation within the alveolus following endotoxemia is not completely elucidated.

Tachykinins released from sensory C-fiber endings induce an inflammatory response known as neurogenic inflammation (11), characterized by vasodilatation (12), increased postcapillary venule permeability, and neutrophil adherence to blood vessels (13). Several mediators released in endotoxemia, such as bradykinin, histamine, and prostaglandins, have been shown to enhance the chemosensitive sensory nerve endings (14). Our recent report showed that LPS enhanced substance P (SP)-mediated vascular permeability in guinea pig airways and neutrophil accumulation in the lungs via neurokinin 1 (NK-1) receptors (17). These observations suggest that tachykinin-mediated inflammatory responses may be enhanced in endotoxemia and implicated in endotoxin-induced lung injury. Tachykinins, especially SP through the NK-1 receptor, have been shown to induce a series of leukocyte responses to trigger and amplify the inflammatory processes, including up-regulation of intercellular adhesion molecule-1 (ICAM-1) expression on vascular endothelial cells and enhancement of neutrophil transendothelial migration (18), mediating leukocyte adhesion to the endothelial or epithelial cells in the airways (19, 20). SP and the NK-1 receptor were recently demonstrated to contribute to the intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability in pancreatitis-associated lung injury (21). Thus, it is possible that SP and the NK-1 receptor may be implicated in the leukocyte accumulation in endotoxin-induced lung injury. SP is also shown to stimulate human monocytes to release interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNF-) (22), which increase in sepsis and endotoxin administration (23). In this study, we investigated the synergistic effect of SP and LPS on neutrophil adherence to alveolar epithelial cells and release of proinflammatory cytokines, including IL-1 and TNF-, which in turn may amplify neutrophil migration and sequestration in the lungs.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Epithelial Cell Cultures

A549 type II-like lung epithelial cells were purchased from the American Type Culture Collection, Rockville, MD. A549 cells are a cell line derived from a patient with alveolar cell carcinoma of the lung. These cells retain features of type II alveolar epithelial cells, including cytoplasmic multilamellar inclusion bodies and the synthesis of surfactant (24), as well as endocytic ability and localization of cytochrome P-450 (25). However, A549 cells possess a higher permeability property than type II epithelial cells in vivo (25). A549 cells were grown as monolayers in tissue flasks or dishes incubated in 100% humidity and 5% CO₂ at 37° C. Ham's F12K medium supplemented with fetal calf serum (FCS) (10%) was used as growth media. The cells from the monolayers were harvested with trypsin (0.25%) and ethylenediaminetetraacetic acid (EDTA) (0.1%) in phosphate-buffered saline (PBS), centrifuged at low speed (250 × g, 5 min), and resuspended in fresh medium before growing them in 48-well plates. Confluent monolayers of culture were used for experiments.

Protocol

A549 epithelial cells grown to confluence were cultured in 5% CO₂ at 37° C in serum-free Dulbecco's modified Eagle medium (DMEM) in the absence or presence of SP (10⁸-10¹² M) or LPS (100 ng/ml) or their combination (LPS 100 ng/ml and SP 10¹⁰ M, LPS + SP) for either 30 min, 2 h, or 6 h. In selected cell cultures, purified neutrophils (3 × 10⁶ cells) were cocultured with the epithelial cell monolayers to evaluate the effect of SP or LPS or their combination on neutrophil- A549 epithelial cell adherence. To examine the specificity of the SP effect, cultured epithelial cells were pretreated with an NK-1 receptor antagonist (N-acetyl-L-tryptophan 3,5-bistrifluoromethyl benzyl ester, L-732,138, 10⁸-10⁵ M) 15 min before LPS + SP concomitant stimulation. The molecular mechanisms underlying the neutrophil-epithelial cell interactions were assessed in some parallel cultured plates in the presence or absence of oversaturated monoclonal antibodies (50 µg/ml) (Becton Dickinson, Mountain View, CA) directed against adhesion molecules on CD11b/CD18 or isotype immunoglobulin G (IgG) or by placing a filter (Seamless Cellulose Tubing, UC No. 20/32-1000; Viskase Sales Corp., Tokyo, Japan) over the epithelial monolayer to prevent neutrophil from adherence. To evaluate whether neutrophil adherence-augmented IL-1 and TNF- release was attributed to the release of serine proteases of neutrophils, the serine protease inhibitors cocktail (₁-antitrypsin [2 mg/ml] + SBTI [100 µg/ml] + eglin C [100 µg/ml]) was added to a group of cultured epithelial cells before neutrophil addition. To determine to what extent IL-1 and TNF- release was attributed to activated neutrophils, neutrophils (at 3 × 10⁶ cells) were left adherent on culture plastic plates in 5% CO₂ at 37° C in serum-free DMEM for 6 h with or without SP (10⁸ M) or LPS (100 ng/ml) or their combination (LPS + SP).

At the completion of incubation, medium was carefully collected, spun down to remove cell pellets, and frozen at 70° C until subsequent analysis for cytokine concentrations. The assay of neutrophil adherence was determined as described below. The epithelial cells were detached with trypsin-EDTA to determine the expression of surface molecules and extract their RNA for reverse transcriptase- polymerase chain reaction (RT-PCR). To determine the cell viability, trypan blue exclusion tests were used to determine the viability of cells after completion of each incubation period.

Neutrophil Adherence Assay

Neutrophils from healthy donors were purified by dextran sedimentation followed by hypotonic lysis and centrifugation on Histopaque. This procedure yielded a population that is 95% to 100% viable determined by trypan blue exclusion test and is 98% pure. The neutrophil adherence assay was performed by incubating the neutrophil suspension at 3 × 10⁶ cells/ml in PBS with 0.2% bovine serum albumin (BSA) with epithelial monolayers for varying periods of time. Then the neutrophil suspension of each well was carefully aspirated, and the wells were gently washed three times with Hanks' balanced salt solution (HBSS, 500 µl). The aspirated cell suspension was pooled, spun down, and resuspended in 500 µl HBSS. Nonadherent neutrophil counts were determined by counting the cell numbers in the cell suspension and the percentage of neutrophils on cytospin cell preparation. Counts of neutrophil adherent to epithelial monolayer were obtained from abstracting nonadherent cell counts from the total cell counts added. The neutrophil adherence was calculated as the ratio of adherent counts to total cell counts added. To validate this assay, the neutrophil adherence assay was also done on epithelial cells grown on coverslips. After incubation, nonadherent cells were aspirated and washed as previously described. The remaining adherent cells were counted under microscopy by peroxidase staining of adherent neutrophils (26), which were visualized as brownish spots. The adherent counts measured by these two methods were highly correlated (rs = 0.93, p < 0.01, n = 8).

Detection of Surface Molecules on Neutrophils and Epithelial Cells

The method used to detect the expression of CD11b/CD18 on neutrophils was described previously (27). Briefly, neutrophils were isolated as previously described and incubated with SP (10¹²-10⁸ M) or LPS (100 ng/ml) or their combination (LPS + SP) for 6 h. After washing three times, the cells were incubated with mouse anti-human CD11b/ CD18 monoclonal antibody (Mac-1, final dilution 1/100; Cedarlane Ltd.) for 15 min at 4° C. After washing, a second antibody was added (FITC-conjugated goat anti-mouse; Cedarlane Ltd.) and the cells were incubated at 4° C for an additional 30 min. After washing to remove unbound second antibody and fixation of cells in formalin, the presence of CD11b/CD18 was detected using flow cytometry (FACScan; Becton Dickinson). Control preparations consisted of neutrophils incubated in medium alone, second antibody alone, and nonspecific mouse IgG. The expression of ICAM-1 on cultured epithelial cells was evaluated in a fashion similar to CD11b/CD18 on neutrophils using freshly isolated A549 cells incubated for 6 h with SP (10¹⁰-10⁸ M) or LPS (100 ng/ml) or LPS + SP for 6 h, and subsequently detached with trypsin-EDTA (GIBCO, Grand Island, NY).

Cytokines Assay

Supernatant of culture medium is sampled at various time points and the samples were then resuspended in one-tenth of the original volume of PBS and stored at 80° C for later measurement of IL-1, and TNF- concentrations. The concentrations of cytokines were measured using a specific ELISA kit (R&D Systems, Minneapolis, MN) employing the quantitative immunometric sandwich enzyme immunoassay technique. The limit of detection of IL-1 and TNF- is 3 pg/ml, respectively.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)

Total RNA was extracted using the guanidine thiocyanate/phenol/ chloroform method described previously (28). The cDNA was reverse transcribed from isolated RNA by use of established techniques involving the incubation of 25 ng of DNase-treated RNA in a reaction buffer containing 10 mM dithiothreitol, 1 mM deoxynucleotide triphophate mixture (dNTP), 50 mol of oligo(DT), 10 U RNasin, and 50 U Moloney murine leukemia virus (MMLV) reverse transcriptase in 10 µl volumes. The PCR reactions were run in amplification buffer containing 1.5 mM MgCl₂, 10 pmol each of forward (5') and reverse (3') primers, 2.5 U of KlenTaq polymerase (Clontech, Palo Alto, CA), 1 mM dNTP, and 10 µl of the RT reaction products in a 25-µl volume. After 25 cycles, 20 µl of the PCR products was run on a 2% agarose gel. The gel was stained with ethidium bromide, photographed, and the bands quantitated by densitometer scan. Each gel was run with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene for comparison of expression. All PCR primers were synthesized by GIBCO-BRL (Gaithersburg, MD).

Reagents

Cytokines TNF- and IL-1 ELISA kits were obtained from R&D Systems Inc. N-Acetyl-L-tryptophan 3,5-bistrifluoromethyl benzyl ester, L-732,138, lipopolysaccharide, substance P, PBS, and serine protease inhibitors cocktail [₁-antitrypsin + SBTI + eglin C] were purchased from Sigma Chemical Co. (St. Louis, MO). Ham's F12K medium, Hank's balanced salt solution, trypsin, and EDTA, were obtained from GIBCO. Monoclonal antibodies reacting with human surface molecules, CD11b/CD18, and intercellular adhesion molecule-1 (ICAM-1) were obtained from Cedarlane Ltd. As irrelevant isotype-matched antibodies, rabbit IgG₂ were obtained from Boehringer Mannheim (Mannheim, Germany).

Statistics

Data were expressed as the mean ± SEM. One-way analysis of variance (ANOVA) for mixed design was used to compare values of more than two different experimental groups. If variance among groups was noted, a Bonferroni test was used to determine significant differences between specific points within groups. The data were analyzed by Student's t test for paired or unpaired data. For data with uneven variation, a Mann-Whitney U test or Wilcoxon signed rank test was used for unpaired or paired data, respectively. The correlation between two methods for neutrophil adherence assay was sought by Spearman's test. A p value of less than 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Neutrophil Adherence to Epithelial Cells

Addition of neutrophils to cultured epithelial cells spontaneously caused a time-dependent increase in the magnitude of neutrophil adherence to epithelial cells (Figure 1). Stimulation with SP (10¹⁰ M) or LPS (100 ng/ml) alone did not induce any significant change in the neutrophil adherence ratio (Figure 1). However, concomitant administration of LPS and SP (10¹⁰ M) induced an increase in the neutrophil adherence to epithelial cells significantly at 2 and 6 h of culture compared with the corresponding time controls (Figure 1). This enhancement response was dose dependent (Figure 2) and appeared to be mediated via the NK-1 receptor, as the NK-1 receptor antagonist L-732,138 dose dependently and significantly inhibited the response (Figure 3). Because there was a significant portion of neutrophils adhering to epithelial cells at 6 h of coincubation with LPS and SP (10¹⁰ M) (LPS + SP), the following studies for the effect of neutrophil-epithelial cells interaction were examined at 6 h of culture.

View larger version (29K): [in this window] [in a new window]   Figure 1.   Time course for the effect of lipopolysaccharide (LPS, 100 ng/ml, hatched bars, n = 5-6), or substance P (SP, 1010 M, dotted bars, n = 5-8), or their combination (LPS + SP, open bars, n = 5-8) on neutrophil adherence to A549 epithelial cells. Data are mean ± SE. *p < 0.05 compared with the corresponding time controls (control, solid bars, n = 5-8).

View larger version (14K): [in this window] [in a new window]   Figure 2.   The enhancement effect of lipopolysaccharide (LPS, 100 ng/ml) on substance P (SP, 1012-108 M) (closed circles, n = 5-6) induced neutrophil adherence to A549 epithelial cells after 6 h of incubation. Data are mean ± SE. *p < 0.01 compared with the corresponding concentration of SP (open circles, n = 5-6) or LPS (n = 5) alone.

View larger version (13K): [in this window] [in a new window]   Figure 3.   The dose-dependent inhibitory effect of an NK-1 receptor antagonist, L-732,138 (closed circles, 108-105 M, n = 5-6) on the combination of lipopolysaccharide (LPS, 100 ng/ml) and substance P (SP, 1010 M) (closed square, LPS + SP, n = 6)-induced neutrophil adherence to A549 epithelial cells after 6 h of incubation. Data are mean ± SE. *p < 0.05 compared with the LPS + SP group. # p < 0.01 compared with the LPS (n = 6) or SP alone group (n = 6).

Expression of Adhesion Molecules

Stimulation of neutrophils with LPS (100 ng/ml) or SP (10¹²- 10⁸ M) alone did not significantly increased the expression of CD11b/CD18 on neutrophils (Figure 4). However, LPS + SP synergistically up-regulated CD11b/CD18 expression (Figure 4). In contrast, neither SP nor LPS nor LPS + SP induced any significant change in the expression of ICAM-1 on epithelial cells (234.5 + 13.5 mean fluorescence intensity [MFI]; 245.7 ± 23.1 MFI; or 242.6 ± 31.4 MFI, respectively, n = 5) when compared with time control (232.9 ± 18.4 MFI, n = 5). Pretreatment with 2-integrin CD11b/CD18 blocking antibody inhibited the LPS-enhanced SP-induced neutrophil adherence to epithelial cells (54.7 ± 4.5%, n = 5, p < 0.01) compared with that with isotype IgG pretreatment (72.5 ± 5.3%, n = 5), indicating up-regulation of 2-integrin on neutrophils was responsible, at least in part, for the increase in adherence to epithelial cells.

View larger version (14K): [in this window] [in a new window]   Figure 4.   Effect of lipopolysaccharide (LPS, 100 ng/ml, open square), or substance P (open circles, SP, 1012-108 M, n = 5-6), or their combination (closed squares, LPS + SP, n = 5-6) on CD11b/CD18 expression (expressed by mean fluorescence intensity, MFI) on neutrophils. Data are mean ± SE. *p < 0.04 compared with the corresponding SP group or LPS alone. There was no significant difference between control (con, n = 6) and SP or LPS alone groups.

IL-1 and TNF- Release from Epithelial Cells

There was no detectable spontaneous release of IL-1 and TNF- from epithelial cells (Figure 5A). Stimulation of epithelial cells with SP (10¹²-10⁸ M) or LPS (100 ng/ml) alone failed to induce detectable levels of IL-1 and TNF-. LPS + SP induced a small amount of IL-1 and TNF- release (Figure 5A). This was a synergistic effect of LPS on the NK-1 receptor, as the NK-1 receptor antagonist L-732,138 significantly inhibited this response (Figure 6).

View larger version (9K): [in this window] [in a new window]   View larger version (12K): [in this window] [in a new window]   Figure 5.   The effect of lipopolysaccharide (LPS, 100 ng/ml) or substance P (SP, 1012-108 M, n = 5- 7) or their combination (LPS + SP, n = 6) on IL-1 or TNF- release with (B) or without (A) neutrophil coculture for 6 h. Data are mean ± SE. *p < 0.01 compared with the corresponding groups without neutrophil coculture (n = 5-7); *p < 0.01 compared with corresponding concentration of SP or LPS alone.

View larger version (13K): [in this window] [in a new window]   View larger version (13K): [in this window] [in a new window]   Figure 6.   The dose-dependent inhibitory effect of an NK-1 receptor antagonist, L-732,138 (circles, 108-105 M, n = 5-6) on the combination of lipopolysaccharide (LPS, 100 ng/ml) and substance P (SP, 1010 M) (triangles, SP + LPS, n = 5-6)-induced IL-1 (A) or TNF- (B) release from A549 epithelial cells in the presence (closed symbols) or absence (open symbols) of neutrophil coculture for 6 h. Data are mean ± SE. *p < 0.05 compared with the corresponding LPS + SP group.

Interaction Between Neutrophils and Epithelial Cells

Addition of neutrophils to cultured epithelial cells without specific stimulation induced significant levels of IL-1 and TNF- release in the culture medium (Figure 5B). The release of IL-1, but not TNF-, was significantly elevated by SP (10¹²-10⁸ M) or LPS (100 ng/ml) alone stimulation, and was further augmented by LPS + SP stimulation (Figure 5B). Again, there was a significant inhibitory effect of L-732,138 on the enhanced release of IL-1 and TNF-, indicating this response was mediated via the NK-1 receptor (Figure 6). The augmented NK-1 receptor-mediated neutrophil adherence to epithelial cells was responsible for the enhanced release of IL-1 and TNF-, as pretreatment with CD11b/CD18 blocking antibody to inhibit neutrophil adherence to epithelial cells significantly inhibited the augmented release of IL-1 and TNF- with stimulation of LPS + SP (Figure 7). Placing a filter on cultured epithelial cells to prevent neutrophil adherence also significantly decreased IL-1 and TNF- release from epithelial cells with or without LPS + SP stimulation (Figure 7).

View larger version (38K): [in this window] [in a new window]   View larger version (35K): [in this window] [in a new window]   Figure 7.   Placing a filter (filter, n = 6) to prevent neutrophil adherence or pretreatment with CD11b/CD18 blocking antibody (CD11b, n = 6) or isotype IgG (IgG, n = 5) inhibited LPS + SP-enhanced IL-1 (A) or TNF- (B) release from A549 epithelial cells and neutrophil coculture for 6 h. Data are mean ± SE. *p < 0.01 compared with controls (n = 6); p < 0.01; #p < 0.01 compared with the LPS + SP group.

Neutrophil adherence to epithelial cells may release serine proteases that have been shown to induce IL-8 release from epithelial cells (29, 30). To investigate whether neutrophil-derived serine protease is implicated in neutrophil adherence-augmented IL-1 and TNF- release, the serine protease inhibitors cocktail (₁-antitrypsin [2 mg/ml] + SBTI [100 µg/ml] + eglin C [100 µg/ml]) were added to cultured epithelial cells before addition of neutrophils. The serine protease inhibitors eliminated the neutrophil adherence-augmented IL-1 and TNF- release by LPS + SP stimulation (Figure 8). However, serine protease inhibitors failed to affect the release of IL-1 and TNF- from A549 epithelial cells without neutrophil coculture stimulated by LPS + SP (8.3 ± 1.4 pg/ml, n = 5 and 2.8 ± 0.9 pg/ ml, n = 5, respectively) when compared with those with LPS + SP stimulation alone (7.9 ± 0.3 pg/ml, n = 6 and 3.2 ± 0.7 pg/ml, n = 6, respectively) (Figure 8).

View larger version (23K): [in this window] [in a new window]   View larger version (22K): [in this window] [in a new window]   Figure 8.   The effect of serine protease inhibitor cocktail (cocktail) on release of IL-1 (A) and TNF- (B) from neutrophils, A549 epithelial cells alone, or with neutrophil coculture for 6 h in the presence or absence of stimulation with lipopolysaccharide (LPS, 100 ng/ml, n = 5-6) or substance P (SP, 1010 M, n = 5-7), or their combination (LPS + SP, n = 5-6). Data are mean ± SE. **p < 0.01; *p < 0.05 compared with the corresponding SP or LPS group; #p < 0.01 compared with the corresponding LPS + SP group.

Most of the IL-1 and TNF- appeared to be released from the epithelial cells because there were very low levels of IL-1 and TNF- produced from neutrophils adhering to plastic for 6 h with SP (10¹⁰ M, n = 5, 0.1 ± 0.0 pg/ml and 1.5 ± 0.8 pg/ml, respectively) or LPS (100 ng/ml, n = 5, 0.3 ± 0.1 pg/ml, and 0.3 ± 0.3 pg/ml, respectively) stimulation (Figure 8). There was no potentiation effect of LPS + SP on IL-1 (0.5 ± 0.1 pg/ml, n = 4) or TNF- (1.8 ± 0.6 pg/ml, n = 4) production from activated neutrophils (Figure 8). Neither was there any effect of serine protease inhibitors on IL-1 and TNF- released from adherent neutrophils activated by LPS + SP (Figure 8).

mRNA Expression of IL-1 and TNF-

There was no apparent increase in mRNA expression of IL-1 and TNF- when stimulated with SP or LPS alone or LPS + SP stimulation (Figure 9). Addition of neutrophils enhanced the expression of IL-1 or TNF- with LPS + SP stimulation (Figure 9). Pretreatment with antiprotease cocktails down-regulated these mRNA expressions (Figure 9). The mRNA retrieved from A549 cells with neutrophils adherence is from a mix of two cell types. However, there is no detectable expression of IL- or TNF- mRNA on neutrophils that adhered to a plastic as a control either stimulated with SP or LPS alone or LPS + SP (Figure 9), suggesting neutrophils adherent on A549 cells are not a major cellular source of IL-1 or TNF- mRNA expression.

View larger version (34K): [in this window] [in a new window]   View larger version (26K): [in this window] [in a new window]   Figure 9.   Agarose gels of PCR-amplified mRNA of IL-1 or TNF- on (A) A549 epithelial cells with or without neutrophils coculture (N), after stimulation with vehicle (C), lipopolysaccharide (LPS, 100 ng/ml), or substance P (SP, 1010 M), or their combination (LPS + SP) for 6 h. Lanes 5 and 6 are mRNA for a mix of A549 cells and neutrophils (A549 cells + N). Antiserine protease cocktail (AP) inhibited both IL-1 and TNF- mRNA expression. (B) There is no IL-1 or TNF- mRNA expression on neutrophils either with or without stimulation. GAPDH was used for a housekeeper gene for comparison.

Cytotoxicity

The viability of epithelial cells and neutrophils was more than 95% at 6 h of incubation and epithelial cells persisted viable (> 95% viability) after 24 h of culture. There was no significant difference in viability of either epithelial cells or neutrophils among study subgroups.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our results demonstrate that the neuropeptide SP alone is not a potent inflammatory mediator. However, under LPS stimulation, SP is able to increase neutrophil adherence to lung epithelial cells and induce IL-1 and TNF- release (Figures 3 and 5). These effects appear to be mediated by an enhancement effect of LPS on the tachykinin receptor, NK-1 receptor, as the specific NK-1 receptor antagonist, L-732,138, significantly inhibits these responses (Figure 6). The underlying mechanism for this enhancement response was not further explored in this study. However, LPS has been reported to up-regulate the expression of mRNA encoding for SP receptors in rat macrophages and neuronal cells (31). Thus, it is possible that LPS may increase the number of NK-1 receptors or their affinity to augment SP-mediated response. However, conflicting evidence exists that reject the possibility that an increase in the number or affinity of SP receptors contributes to the LPS enhancement of SP-induced TNF- secretion from neuroglial cells (32). It is also possible that the augmented responses are due to a synergism of LPS and SP activities through their own respective different mechanisms.

Such a synergism response was also previously reported on the stimulatory effect of SP on leukocytes. SP alone can directly stimulate leukocytes to release oxygen radicals and lysosomal enzyme (33, 34) only at extremely high concentrations of the peptide (greater than 10 µM). Under stimulation with chemotactic peptides, such as fMLP and C5a, or LPS, there are potent synergistic effects of nanomolar doses of SP on the migratory and cytotoxic functions of human leukocytes (32, 35). This synergism response of SP reveals a new regulatory activity of SP and suggests that neurogenic stimuli may prepare neutrophils for an exaggerated inflammatory response to other physiological mediators.

SP and NK-1 receptors have been shown to mediate leukocyte adhesion to the endothelial cell (19) and bronchial epithelial cell (20) in the airways. The mechanism of the increase in adherence is complex. One important mechanism involves the interaction of CD11b on the neutrophils with ICAM-1 on the epithelial cells (36). The integrin-ICAM-1 adhesive interaction is, at least in part, responsible for the enhanced neutrophil adherence in our study. This is suggested by the observation that anti-CD11b/CD18 antibody decreases adherence. In our results, SP and LPS synergistically up-regulate CD11b/CD18 expression (Figure 4). The expression of ICAM-1 on epithelial cells does not increase with SP or LPS alone or in combination. Although no increase in ICAM-1 immunoreactive epitope expression is reported (39), it is not known whether there is up-regulation of ICAM-1 avidity or adhesivity. Similarly, it is also possible that up-regulation of CD11b/CD18 avidity is contributory to the enhanced neutrophil adherence.

Neutrophil adherence to epithelial cells elicits IL-1 and TNF- release. This response was slightly responsive to the stimulation of SP or LPS alone, but was highly augmented by the concomitant stimulation with LPS and SP (Figure 5). The synergistic response of LPS and SP seems to be mediated by the increased neutrophil adherence to epithelial cells, as pretreatment with anti-CD11b/CD18 antibody or placing a filter to prevent neutrophil adherence significantly inhibits the augmented release of IL-1 and TNF- (Figure 7). Most of the IL-1 and TNF- release is released from the epithelial cells when compared with the levels of IL-1 and TNF- released from neutrophils alone adhering to plastic for 6 h with or without SP or LPS or LPS + SP stimulation (Figure 8). The IL-1 and TNF- mRNA expression is also mostly attributed to epithelial cells, as there is no detectable IL-1 or TNF- mRNA retrieved from activated adherent neutrophils. However, it is also possible that the interaction with epithelial cells through integrin-ICAM-1 adhesivity may potentiate neutrophils to increase IL-1 and TNF- synthesis and release capacity. Further investigation is needed to determine the extent of IL-1 and TNF- release from stimulated neutophils in a plate precoated with immobilized ICAM-1.

Tight adhesion of neutrophils to the endothelial cells through engagement of CD11b/CD18 results in calcium influx and content release of secondary and azurophilic granules (29, 38). Antibody crosslinking of CD18 on neutrophils also induces exocytosis of azurophilic granules (30). Neutrophil-derived serine proteases have been shown to induce proinflammatory cytokines, such as IL-8 release from pulmonary epithelium (39). Although we did not measure the activities of serine proteases in the culture medium, the inhibitory effect of antiprotease cocktail on neutrophil adherence-augmented IL-1 and TNF- release indicates serine proteases from adherent neutrophils may be implicated in this response. The release of serine proteases from neutrophils may increase IL-1 activity through a proteolytic cleavage of an inactive precursor. A monocytic protease has been identified that appears to be involved in the physiological activation of this cytokine (40). However, pretreatment with serine protease inhibitors also down-regulates the expression of IL-1 and TNF- mRNA, suggesting the stimulatory effect of serine proteases on the release of IL-1 and TNF- is mostly at the level of mRNA (Figure 9). The failure of serine protease inhibitor cocktail to influence the release of IL-1 and TNF- from A549 epithelial cells upon LPS + SP stimulation suggests the inhibitory effect of serine protease inhibitor cocktail is not nonspecific (Figure 9).

In conclusion, we have demonstrated LPS enhanced SP-induced neutrophil adherence and associated IL-1 and TNF- release from A549 epithelial monolayer, partly through NK-1 receptors to up-regulate adhesion molecules on neutrophils. Neutrophil adherence to epithelial cells may release serine protease to increase IL-1 and TNF- release and synthesis. Tachykinins released from nerve endings may contribute to endotoxin-related pulmonary inflammatory responses by increasing neutrophil sequestration and cytokines release. However, though A549 cells retain features of type II alveolar epithelial cells (24), it remains to be determined whether SP would synergistically induce neutrophil adhesion to alveolar epithelial cell and induce IL-1 or TNF- release in vivo.