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Stress Hormone Epinephrine Increases IgA Transport across Respiratory Epithelial Surfaces
Stress Hormone Epinephrine Increases IgA Transport across Respiratory Epithelial Surfaces Abubaker Ali,
MD,
Lawrence Diebel,
MD, FACS,
David Liberati,
MS
Secretory immunoglobulin A (sIgA) is the principle antibody produced at the respiratory surface. Respiratory sIgA levels are increased early after injury in both human and laboratory animals; the mechanisms are uncertain. Stress hormones, including epinephrine (Epi) and norepinephrine (NE), increase early after injury. In addition, respiratory epithelial cells are known to be responsive to b2-agonists. We therefore studied the effect of Epi, NE, and albuterol on IgA transport in vitro. STUDY DESIGN: Calu-3 respiratory epithelial cell monolayers grown in a 2-chamber cell culture system were treated for 24 hours with Epi, NE, or albuterol (106M). Dimeric IgA was added to the basal chamber of Calu-3 cells and IgA transcellular transport was indexed by recovery of SIgA in the apical chamber by enzyme-linked immunosorbent assay. In separate experiments, Klebsiella pneumoniae (105 colony-forming units/mL) was added to the apical chamber of treated Calu-3 cell monolayers and bacterial passage across Calu-3 cells was determined by bacterial recovery from basal chamber media. Calu-3 cells not treated with Epi, NE, or albuterol served as control. Cell monolayer integrity was confirmed by transepithelial electrical resistance. RESULTS: Calu-3 cells treated with Epi led to a significant increase in sIgA transport, this was associated with an increase in polyimmunoglobulin receptor expression. Calu-3 cells treated with NE or albuterol showed no statistical difference compared with control. Only cells treated with Epi led to a significant increase in pro-inflammatory cytokine expression and decrease in bacterial passage. CONCLUSIONS: Epinephrine is likely an early upstream signal in the enhanced IgA response at respiratory surfaces after injury. (J Am Coll Surg 2014;218:450e458. 2014 by the American College of Surgeons)
BACKGROUND:
are intubated and who are represented by the ventilator care bundles.4,5 Despite all of these efforts, ventilatorassociated pneumonia after trauma remains a challenge, so investigators are now aiming toward prevention, and to do that it is key to understand the pathophysiology behind lung protective mechanisms. The lungs are protected by a mucosal barrier usually present in the upper airway, and are also protected by a more complex immune system represented by the innate and adaptive immune system. Immunoglobulin (Ig) A is the major immunoglobulin at mucosal surfaces, including the respiratory airway. Immunoglobulin A is mainly concentrated in the upper airway, and IgG is found mainly in the lower airway, however, the latter can cross to the luminal surface by means of diffusion. Previous clinical investigators have studied the relationship between IgA and pneumonia, correlating reduced IgA with higher incidence of pneumonia.6 Immunoglobulin A is produced at the basolateral surface of the respiratory epithelial cells by plasma cells and is then transported to the epithelial cells by means of a special receptor expressed in the basolateral surface, known as polyimmunoglobulin receptor (pIgR). Once
Pneumonia is a common cause of morbidity and mortality after trauma and hemorrhagic shock. The CDC defines ventilator-associated pneumonia as pneumonia in patients who had a device to assist or control respiration continuously through a tracheostomy or by endotracheal intubation within the 48-hour period before the onset of infection, inclusive of the weaning period. Ventilatorassociated pneumonia is associated with a 25% to 50% mortality rate.1,2 The economic impact of ventilatorassociated pneumonia is substantial, with costs ranging from $19,633 to $28,538.00 per patient with ventilatorassociated pneumonia.3 Therefore, a lot of effort has been made to reduce the incidence of pneumonia by applying standards of care in critically ill patients who Disclosure Information: Nothing to disclose. Received First Place, Basic Laboratory Research, the American College of Surgeons Committee on Trauma, 2013. Received September 25, 2013; Revised November 12, 2013; Accepted November 12, 2013. From the Department of Surgery, Wayne State University, Detroit, MI. Correspondence address: Abubaker Ali, MD, General Surgery, Wayne State University, 6C University Health Center, 4201 St Antoine, Detroit, MI 48201. email: [email protected]
ª 2014 by the American College of Surgeons Published by Elsevier Inc.
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ISSN 1072-7515/13/$36.00 http://dx.doi.org/10.1016/j.jamcollsurg.2013.11.011
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Abbreviations and Acronyms
dIgA DMEM ELISA Epi Ig IL NE pIgR sIgA TEER TNF
¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
dimeric immunoglobulin A Dulbecco’s Modified Eagle medium enzyme-linked immunosorbent assay epinephrine immunoglobulin interleukin norepinephrine polyimmunoglobulin receptor secretory immunoglobulin A transepithelial electrical resistance tumor necrosis factor
the complex is internalized and reaches the luminal surface, pIgR becomes unstable and is cleaved, releasing a small protein known as the secretory component. Dimeric IgA along with secretory component forms secretory IgA (sIgA), which protects the airway through various mechanisms, including bacterial exclusion, which prevents bacterial attachment to the mucosal surface. As a result, bacteria cannot attach to the respiratory surface and will not cause infection.7,8 Several known factors affect the concentration of IgA at the mucosal surface. We were able to demonstrate in our laboratory, using an in vitro model, that IgA transport was significantly increased by stimulation of toll like receptor4 or estradiol.9,10 Recent clinical and laboratory studies demonstrated that early in trauma, there is an increase in IgA transport, which was accompanied by an increase in proinflammatory cytokine recovery from bronchoalveolar
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lavage samples after trauma. In a different series of experiments, the pro-inflammatory cytokine receptors were blocked, which led to abrogation of the airway IgA response to injury. They concluded that early after trauma, there is an increase in pro-inflammatory cytokines that leads to an increase in IgA transport.11-13 On the other hand, studies also found that early after injury and hemorrhagic shock, there is an increase in stress hormones and neurotransmitters that can alter cytokine release in the early phase after trauma.14 We hypothesized that early after trauma, there is an increase in stress hormones that alter pro-inflammatory cytokine release, which eventually leads to an increase in IgA transport. In addition, respiratory epithelial cells are known to be responsive to b2 agonists. We therefore conducted an in vitro study with the following objectives: first, we studied the effect of epinephrine (Epi), norepinephrine (NE), and albuterol on IgA transport across cultured human respiratory epithelial cells in vitro (Calu-3 cells); our second objective was to examine if Epi, NE, or albuterol had any effect on pro-inflammatory cytokines; and, lastly, we examined whether induced modulation of IgA transport effects bacterial passage across Calu-3 cells.
METHODS Respiratory epithelial cells Calu-3 cells were obtained from American Type Culture Collection and routinely cultured with Dulbecco’s Modified Eagle medium (DMEM) containing 10% fetal bovine serum, 4.5 g/L glucose, and 1% antibiotic
Figure 1. Diagram of experimental design. A schematic representation of the 2-chamber cell culture system used in the design of all experiments. CFU, colony-forming unit; dIgA, dimeric immunoglobulin A; Epi, epinephrine; pIgR, polyimmunoglobulin receptor; Norepi, norepinephrine.
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antimycotic (Invitrogen) in an atmosphere of 5% CO2 at 37 C. Medium was changed twice a week and cells were passaged every 10 to 14 days. Cells (5 105) were seeded on the apical surface of a polycarbonate membrane (3.0-mm pore size) (Transwell; Corning Costar Core) in a 2-chamber cell culture system and allowed to form polarized monolayers. Monolayer integrity was monitored by serial measurement of the transepithelial electrical resistance (TEER) with a Millicell electrical resistance meter (Millipore Corp.). When grown in this system, Calu-3 cells form polarized monolayers that are confluent after 10 to 14 days in culture. Experimental design Calu-3 cells were grown to confluence in complete DMEM. Epinephrine (106M), NE (106M), or albuterol (106M) was added to the apical chambers and cocultured with Calu-3 cells for 24 or 72 hours at 37 C (Fig. 1). After coculture, media was replaced with DMEM þ 0.06% bovine serum albumin (Sigma) and 20 mM HEPES (pH 7.3) for transcytosis experiments. In separate experiments, surface expression of pIgR and monolayer integrity (TEER) was analyzed in Calu-3 cells after coculture with Epi (106M), NE (106M), or albuterol (106M). In still other experiments, basal supernatants, post treatment, were collected for cytokine analysis and Klebsiella pneumoniae was added to assess bacterial translocation after Epi (106M), NE (106M), or albuterol (106M) pretreatment for 24 or 72 hours. Cytokine analysis by enzyme-linked immunosorbent assay Basal supernatants of Calu-3 cells treated with K pneumoniae and Epi (106M), NE (106M), or albuterol (106M) were collected at the end of the experiments and immediately stored at 70 C for subsequent cytokine analysis. Interleukin (IL)-6 and tumor necrosis factor (TNF)ea were quantitated in the previously frozen supernatant samples using a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). These immunoassay kits are commercially available and were used according to manufacturer’s directions (Cytoscreen, Biosource International). The minimal detectable levels of IL-6 and TNF-a with these kits are 2 pg/mL for IL-6 and <1 pg/mL for TNF-a. Bacterial translocation Klebsiella pneumoniae was grown overnight in trypticasesoy broth, centrifuged, and resuspended in phosphatebuffered saline. Bacterial concentration was determined spectraphotometrically and verified by a pour-plate assay. Final concentrations of bacteria used for the experiments were adjusted in fresh DMEM at 105 organisms/mL. In
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one set of experiments, Calu-3 cells were pretreated with Epi (106M), NE (106M), or albuterol (106M) for 24 hours. Then, 100 pg/mL dimeric IgA (dIgA) was added to the basal chamber and allowed to bind at 4 C for 4 hours to promote maximal binding of dIgA to the pIgR. Plates were subsequently placed at 37 C for 12 hours to allow maximal transcytosis and then 105 K pneumoniae was added to the apical chambers (Fig. 1). The cell culture system was reincubated for a total of 240 minutes post inoculum. Basal chamber medium was sampled at baseline and at 60minute intervals thereafter to quantitate bacterial passage across Calu-3 monolayers in the presence of translocated IgA. In another set of experiments, K pneumoniae was added in the absence of transcytosed IgA and basal chamber media was sampled for bacterial translocation, as outlined here. The 0.1-mL aliquots of basal chamber medium sampled were serially diluted and cultured on 10% sheep blood-trypticase soy agar (Becton Dickinson Microbiology System). The magnitude of bacterial translocation was quantitated by counting bacterial colonies after overnight incubation. Transepithelial electrical resistance was measured preinoculum and at 240 minutes post inoculum. Immunoglobulin A transcytosis Calu-3 medium was replaced with DMEM containing 0.6% bovine serum albumin and 20 mmol/L HEPES buffer (N-[2-hydroxyethyl]-piperazine-N-2-ethanesulfonic acid). Dimeric IgA (100 pg/mL) purified from rat myeloma cells was added to the basolateral chamber and allowed to incubate for 4 hours at 4 C to allow maximal binding of the dIgA to the pIgR on the surface of the Calu-3 cells. Cells were removed from the cold and washed 3 times in Hank’s balanced salt solution. Fresh medium at 37 C containing 0.6% bovine serum albumin and 20 mmol/L HEPES was added and cell preparations were allowed to incubate in a 37 C and 5% CO2 environment. Aliquots of apical chamber media were obtained at timed intervals and frozen at 20 C until ready for analysis. A series of experiments was also performed to assess the effect of Epi pretreatment for 24 or 72 hours on IgA transcytosis in HT29 cells. In another series of experiments, Calu-3 cells were pretreated with Epi (106M), NE (106M), or albuterol (106M) for 24 hours, followed by coculture of dIgA, leading to the collection of apical media as outlined here, which was subsequently assayed for free secretory component. Free secretory component was also assayed in apical chamber supernatants not cocultured with dIgA. Determination of immunoglobulin A by enzymelinked immunosorbent assay The amount of transcytosed sIgA in the apical medium samples was determined by a sandwich ELISA. Plates
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were coated overnight by adding 100 mL goat anti-rat IgA primary antibody (ICN Pharmaceuticals) diluted in 0.05 mol/L sodium carbonate coating buffer (pH 9.6) at 4 C. Each well was then washed 3 times and blocked with phosphate-buffered saline e.05% Tween-20 for 2 hours at room temperature. After another 3 washes, 100 mL of the apical media samples were added and the plate was incubated at room temperature for 2 hours. Plates then were washed and 100 mL of the secondary mouse anti-rat IgA antibody conjugated with horseradish peroxidase was added and the mixture was incubated for 2 hours at room temperature. The ELISA plates then were washed and subsequently developed using 1-step Turbo TMB (Pierce Chemical), after a 15-minute incubation at room temperature the color reaction was stopped by adding 1 mol/L H2SO4 and absorbance was measured at 450 nm using an ELISA plate reader. Purified rat myeloma IgA was used to create a standard curve for the determination of sIgA concentrations in the apical samples. In a separate assay, apical media samples collected for the determination of free secretory component were also quantitated. The ELISA plates were prepared as described here and probed with a secondary mouse antiesecretory component (bound and free) antibody conjugated with horseradish peroxidase for 2 hours at room temperature. Development was as described here and absorbance was measured at 450 nm using an ELISA plate reader. Flow cytometry for polyimmunoglobulin receptor expression Expression of the pIgR on the surface of Calu-3 cells was determined using flow cytometry. Calu-3 cells were washed 5 times with phosphate-buffered saline and then trypsinized. Cells then were placed in fresh media and incubated for 2 hours at 37 C to allow re-expression of cell surface proteins. Cells then were washed 3 times with phosphate-buffered saline (10 minutes at 1,200 rpm) and 1 mL goat anti-human secretory component (bound and free) (Sigma) diluted in phosphate-buffered saline was added and the mixture was incubated for 30 minutes at 4 C. Cells then were washed 3 times with ice-cold phosphate-buffered saline and 1 mL rabbit anti-goat IgG conjugated with fluorescein isothiocyanate (Sigma) was added and incubated for 30 minutes at 4 C in the dark. An isotype control was set up in the same manner as described earlier to ensure the secondary antibody was not nonspecifically binding to the surface of the Calu-3 cells. Cells were washed 3 times as described earlier, suspended in phosphate-buffered saline, and pIgR expression on the surface of the cells was determined
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via flow cytometry. Data are expressed as mean fluorescence intensity. Monolayer transepithelial electrical resistance Cell monolayer integrity was assessed by measurement of TEER at the beginning and throughout the various time points of the experiment. TEER measurements were expressed as ohm/cm2. Statistical analysis All samples were compared with an analysis of variance with a post-hoc Tukey test. Statistical significance was inferred at p < 0.001. All data are expressed as mean SD.
RESULTS A schematic representation of the 2-chamber cell culture system used in the design of all the experiments is shown in (Fig. 1). Immunoglobulin A transport through Calu-3 cells after 24-hour pretreatment with Epi, NE, or albuterol using the same concentration (106M) is shown in (Fig. 2). Epinephrine significantly increased sIgA recovery from the apical media in a time-dependent fashion, 3 vs 12 hours compared with control (p < 0.001). Norepinephrine and albuterol increased sIgA recovered from the apical media compared with control; this increase did not reach statistical significance (p < 0.001). Figure 3 assesses the role pIgR plays in the increased sIgA transport demonstrated with Epi. Calu-3 cells were challenged with Epi, NE, or albuterol using the same concentration 106M. Epinephrine led to a significant increase in pIgR expressed on the basolateral surface compared with control (p < 0.001), and Calu-3 cells treated with NE or albuterol did not lead to any significant increase in pIgR expression compared with control (p < 0.001). After demonstrating that only the group of Calu-3 cells pretreated with Epi using 106M led to a significant increase in IgA transport and a parallel increase in pIgR expression, we examined if stress levels of Epi would affect IgA transport. Calu-3 cells were pretreated with a higher concentration of Epi (103 M) and IgA recovery was indexed by sampling the apical media at 3 and 12 hours using ELISA. Calu-3 cells pretreated with Epi (103 M) led to a significant increase in IgA transport compared with control (p < 0.001), but this increase did not reach statistically significant levels (p < 0.001) when compared with Calu-3 cells treated with 106M Epi (data not shown).
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Figure 2. The effect of epinephrine (Epi), norepinephrine (Norepi), or albuterol (106M) treatment of Calu-3 cells for 24 hours on immunoglobulin (Ig) A transcytosis. Immunoglobulin A transcytosis was significantly increased at both 3 and 12 hours by Calu-3 cells treated with 106M Epi for 24 hours. Black bar, 3 hours; gray bar, 12 hours.
We then examined if Epi, NE, or albuterol had any effect on cytokine expression by Calu-3 cells. Calu-3 cells were treated with Epi, NE, or albuterol using the same concentration (106M) (Fig. 4). The group of Calu-3 cells pretreated with Epi led to a significant increase in both TNF-a and IL-6 production in the basal media compared with control (p < 0.001). Calu-3 cells treated with NE or albuterol did not lead to any significant difference in cytokine production when compared with control (p < 0.001). In Figure 5, Calu-3 cells were pretreated for 24 hours with Epi, NE, or albuterol followed by coculture with K pneumoniae (105 colony-forming units) in the apical media. Passage of the bacteria from apical to basal media was assessed by sampling the basal media at 60-, 120-, and 240-minute intervals. The group of Calu-3 cells pretreated with Epi led to a significant decrease in bacterial translocation compared with control at all time points (p < 0.001).
In contrast, Calu-3 cells treated with NE or albuterol did not lead to any significant difference in bacterial translocation compared with control (p < 0.001). To ensure Epi, NE, and albuterol coculture on sIgA recovery from the apical media and bacterial recovery from the basal media was not due to a change in the monolayer integrity, TEER was measured at T ¼ 0 (beginning of the experiment) and T ¼ END (24 hours) and the cells integrity remained unchanged between the different groups (figure not shown).
DISCUSSION Pneumonia still remains a common cause of morbidity and mortality after trauma and hemorrhagic shock. Recent clinical studies have demonstrated that individuals who are deficient in IgA are more susceptible to pneumonia.6
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Figure 3. Polyimmunoglobulin receptor (pIgR) expression on the surface of Calu-3 cells after 24-hour treatment with stress hormones. Calu-3 cells were cocultured with epinephrine (Epi), norepinephrine (Norepi), or albuterol (106M) for 24 hours and pIgR expression quantitated. Epi led to a significant increase in pIgR surface expression. Black bar, pIgR (mean fluorescence intensity).
In a recent human study by Kudsk and colleagues,11 injury was found to stimulate an innate respiratory IgA response. In a different laboratory study, Jonker and colleagues13 showed that the airway IgA response seen after injury can be replicated by exogenous administration of 3 pro-inflammatory cytokines (TNF-a, IL-1b and IL-6). In an additional study, the same group demonstrated that if the pro-inflammatory cytokine receptors are blocked, then the IgA transport gets impaired.12 From these studies, it appears that after trauma there is an increase in pro-inflammatory cytokine release that eventually leads to an increase in IgA transport to the respiratory secretions. After trauma, there is also an early increase in stress hormones and sympathetic neurotransmission, both of which play a well-established role in the modulation of the immune system.15 In our current study, Epi led to an increase in proinflammatory cytokine production and an increase in
IgA transport, which eventually led to a decrease in bacterial passage across Calu-3 cells. Briefly, we pretreated Calu-3 cells with Epi, NE, or albuterol (106M), which is within the physiologic concentration range described in previous studies.16-18 Calu-3 cells were treated for 24 hours for maximal cell stimulation. Only the group of cells treated with Epi led to a significant increase in IgA transport. We postulate that treatment with Epi led to increased IgA transport via b1 receptors because albuterol works mainly on b2 receptors and NE works mainly on a1 receptor with minimal action on b1 receptors. To make sure that the cell monolayer integrity was maintained between the different groups and throughout the experiment, TEER was measured at the beginning and end of each experiment and there was no statistical difference between the different groups at both points. Previous studies have shown that stimulation of b2 receptors
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Figure 4. Cytokine expression in Calu-3 cells co cultured with 106M stress hormones for 24 hours. Calu-3 cells were exposed to 106M epinephrine (Epi), norepinephrine (Norepi), or albuterol for 24 hours in the apical chamber of a 2-chamber cell culture system. Basal chamber media was collected at 24 hours and cytokine levels quantified by enzyme-linked immunosorbent assay. Epi treatment led to significant increase in tumor necrosis factor (a) and interleukin (IL)-6 levels compared with Norepi or albuterol treatment. Black bar, IL-6; gray bar, a.
on mononuclear cells and macrophages lead to a diminished capacity to release pro-inflammatory cytokines, such as TNF-a and IL-1b; both of which are important in IgA transport across the respiratory epithelium.13 After demonstrating that physiologic concentrations of Epi (106M) led to a significant increase in IgA transport, we used higher concentrations of Epi to resemble stress conditions. Therefore, a subset of Calu-3 cells was treated with Epi at a concentration of 103 M for 24 hours to mimic stress conditions. There was a significant increase in IgA transport compared with control, but this increase noted at 103 M did not reach statistical significance when compared with the group of Calu-3 cells treated with physiologic Epi concentrations (106M). The latter finding might be related to the fact that, at higher Epi
concentrations, Epi tends to have a higher affinity toward
a1 receptors (result not shown).
In additional experiments, Calu-3 cells were treated with Epi (106M) for 72 hours and IgA transport was indexed by recovery of IgA from the apical media using ELISA. We found that the level of IgA transported at 72 hours returned to control levels. This finding was also present in the previous study by Junker and colleagues,13 where IgA transport failed to show consistent significant levels compared with control at different time points during the study. In a study by Uren and colleagues,19 pIgR knockout mice had high serum IgA levels, but were deficient in sIgA from BAL samples compared with control. With each molecule of IgA transported, there is one molecule of pIgR consumed. To have an increase in IgA transport, there
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Figure 5. The effect of stress hormones on Klebsiella pneumoniae bacterial translocation in Calu-3 cells. Calu-3 cells were co cultured with epinephrine (Epi), norepinephrine (Norepi), or albuterol (106M) for 24 hours. Klebsiella pneumoniae (105 colony-forming units [CFU]/mL) was added to the apical chamber media of a 2-chamber cell culture system and basal media was subsequently collected at 60, 120, and 240 minutes post inoculum. Basal media samples were plated on 5% sheep blood agar plates and incubated overnight at 37 C. Colonies were counted and bacterial translocation was expressed as CFU/mL K pneumoniae. Black bar, 60 minutes; light gray bar, 120 minutes; dark gray bar, 240 minutes.
should be a parallel increase in pIgR expression. In our current study, we found that only the group of Calu-3 cells treated with Epi (106M) for 24 hours led to a significant increase in pIgR expression. Polyimmunoglobulin receptor expression in cells treated with NE or albuterol did not reach statistically significant levels when compared with control. Interestingly, when Calu-3 cells were treated for 72 hours with Epi at 106M, pIgR expression returned to control levels. This latter finding can explain why IgA levels are not constant in our study and previous studies. To have sIgA in respiratory secretions, plasma cells are required to produce dIgA and respiratory epithelial cells
to produce pIgR. Both of these steps are controlled by various factors, including pro-inflammatory cytokines, estradiol, toll like receptor4, etc.9,10,20,21 Their production also goes through transcription and translation, which also requires time.7 These facts might aid in explaining why sIgA does not remain at constant levels after trauma. The mucosal-associated lymphoid tissue includes the gut-associated lymphoid tissue and a similar structure found in the lungs, called the bronchial-associated lymphoid tissue.22,23 Secretory IgA is considered the major immunoglobulin in both gut-associated lymphoid tissue and bronchial-associated lymphoid tissue. In separate
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experiments, HT-29 cells (human colonic cell line) were pretreated in the same fashion as Calu-3 cells, with Epi for 24 hours or 72 hours. Secretory IgA in the apical media was then measured using ELISA. Secretory IgA levels did not reach statistical significance with either group (figure not shown). This might be related to the fact that HT29 cells lack b1 receptors.24 This latter finding supports that b1 receptors are required for Epi to exert its affects. Epinephrine was also found to increase production of TNF-a and IL-1b recovered from the basal media. This latter finding supports our hypothesis that there is an intermediate link between trauma and the increase in the pro-inflammatory cytokines. This link might be the increase in Epi; a finding not previously measured in the study by Jonker and colleagues.13 Modulation of IgA transport noticed with Epi led to a significant reduction in bacterial passage from apical to basal media at all time points compared with control, supporting the importance of IgA in mucosal immunity. If bacteria cannot attach to the mucosal membrane, then it cannot cause infection.
CONCLUSIONS In summary, Epi led to an early significant increase in IgA transport across Calu-3 cells, this was in part due to an increase in pIgR expression. The increase IgA transport led to a significant reduction in bacterial passage across Calu-3 cells. Epinephrine also led to a significant increase in proinflammatory cytokines production. From our current study, we conclude that Epi might be an early upstream signal in the transport of IgA to the respiratory luminal surface. Factors that influence outcomes during trauma are multiple and cannot be attributed solely to the stress hormone Epi. Author Contributions Study conception and design: Diebel Acquisition of data: Ali, Liberati Analysis and interpretation of data: Ali, Diebel, Liberati Drafting of manuscript: Ali Critical revision: Diebel REFERENCES 1. Efrati S, Deutsch I, Antonelli M, et al. Ventilator-associated pneumonia: current status and future recommendations. J Clin Monit Comput 2010;24:161e168. 2. Scott RD II. The direct medical costs of healthcare-associated infections in US hospitals and the benefits of prevention. March 2009. Available from: http://www.cdc.gov/hai/pdfs/ hai/scott_costpaper.pdf. Accessed 2012. 3. Morrow LE, Shorr AF. The seven deadly sins of ventilatorassociated pneumonia. Chest 2008;134:225e226. 4. Kollef MH. Prevention of nosocomial pneumonia in the intensive care unit: beyond the use of bundles. Surg inf 2011;12:211e220.
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5. Rello J, Afonso E, Lisboa T, et al. A care bundle approach for prevention of ventilator-associated pneumonia. Clinical Microbiol Infect 2013;19:363e369. 6. Annane D, Clair B, Mathieu B, et al. Immunoglobulin A levels in bronchial samples during mechanical ventilation and onset of nosocomial pneumonia in critically ill patients. Am J Respir Crit Care Med 1996;153:1585e1590. 7. Woof JM, Kerr MA. The function of immunoglobulin A in immunity. J Pathol 2006;208:270e282. 8. Kaetzel CS. The polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces. Immunol Rev 2005;206:83e99. 9. Diebel LN, Liberati DM. Disparate effects of bacteria and Toll-like receptor-dependant bacterial ligand stimulation on immunoglobulin A transcytosis. J Trauma 2011;70:691e700. 10. Ali AA, Diebel LN, Liberati DM. Estrogen modulation of pneumonia? An immunoglobulin A effect. J Trauma Acute Care Surg 2012;72:908e915. 11. Kudsk KA, Hermsen JL, Genton L, et al. Injury stimulates an innate respiratory immunoglobulin a immune response in humans. J Trauma 2008;64:316e323; discussion 323325. 12. Hermsen JL, Sano Y, Gomez FE, et al. Parenteral nutrition inhibits tumor necrosis factor-alpha-mediated IgA response to injury. Surg Infect 2008;9:33e40. 13. Jonker MA, Sano Y, Hermsen JL, et al. Proinflammatory cytokine surge after injury stimulates an airway immunoglobulin a increase. J Trauma 2010;69:843e848. 14. Batistaki C, Kostopanagiotou G, Myrianthefs P, et al. Effect of exogenous catecholamines on tumor necrosis factor alpha, interleukin-6, interleukin-10 and beta-endorphin levels following severe trauma. Vasc Pharmacol 2008;48:85e91. 15. Webster JI, Tonelli L, Sternberg EM. Neuroendocrine regulation of immunity. Annu Rev Immunol 2002;20:125e163. 16. McAuley DF, Frank JA, Fang X, Matthay MA. Clinically relevant concentrations of beta2-adrenergic agonists stimulate maximal cyclic adenosine monophosphate-dependent airspace fluid clearance and decrease pulmonary edema in experimental acid-induced lung injury. Crit Care Med 2004;32:1470e1476. 17. Du Q, Min S, Chen LY, et al. Major stress hormones suppress the response of macrophages through down-regulation of TLR2 and TLR4. J Surg Res 2012;173:354e361. 18. Straub RH, Mayer M, Kreutz M, et al. Neurotransmitters of the sympathetic nerve terminal are powerful chemoattractants for monocytes. J Leukocyte Biol 2000;67:553e558. 19. Uren TK, Johansen FE, Wijburg OL, et al. Role of the polymeric Ig receptor in mucosal B cell homeostasis. J Immunol 2003;170:2531e2539. 20. Nosocomial meningitis caused by Pseudomonas aeruginosa: overview of 20 cases in 20 years. Neuroendocrinol Lett 2012;33[Suppl 1]. 21. Takenouchi-Ohkubo N, Takahashi T, Tsuchiya M, et al. Role of nuclear factor-kappaB in the expression by tumor necrosis factor-alpha of the human polymeric immunoglobulin receptor (plgR) gene. Immunogenetics 2000;51:289e295. 22. Bienenstock J, Johnston N, Perey DY. Bronchial lymphoid tissue. I. Morphologic characteristics. Lab Invest 1973;28:686e692. 23. Bienenstock J, Johnston N, Perey DY. Bronchial lymphoid tissue. II. Functional characterisitics. Lab Invest 1973;28:693e698. 24. Abel PW, Zeng W, Wildrick DM, et al. Characterization of beta-adrenergic receptors in DiFi and HT-29 cells. Anticancer Res 1992;12:1655e1658.
MD,
Lawrence Diebel,
MD, FACS,
David Liberati,
MS
Secretory immunoglobulin A (sIgA) is the principle antibody produced at the respiratory surface. Respiratory sIgA levels are increased early after injury in both human and laboratory animals; the mechanisms are uncertain. Stress hormones, including epinephrine (Epi) and norepinephrine (NE), increase early after injury. In addition, respiratory epithelial cells are known to be responsive to b2-agonists. We therefore studied the effect of Epi, NE, and albuterol on IgA transport in vitro. STUDY DESIGN: Calu-3 respiratory epithelial cell monolayers grown in a 2-chamber cell culture system were treated for 24 hours with Epi, NE, or albuterol (106M). Dimeric IgA was added to the basal chamber of Calu-3 cells and IgA transcellular transport was indexed by recovery of SIgA in the apical chamber by enzyme-linked immunosorbent assay. In separate experiments, Klebsiella pneumoniae (105 colony-forming units/mL) was added to the apical chamber of treated Calu-3 cell monolayers and bacterial passage across Calu-3 cells was determined by bacterial recovery from basal chamber media. Calu-3 cells not treated with Epi, NE, or albuterol served as control. Cell monolayer integrity was confirmed by transepithelial electrical resistance. RESULTS: Calu-3 cells treated with Epi led to a significant increase in sIgA transport, this was associated with an increase in polyimmunoglobulin receptor expression. Calu-3 cells treated with NE or albuterol showed no statistical difference compared with control. Only cells treated with Epi led to a significant increase in pro-inflammatory cytokine expression and decrease in bacterial passage. CONCLUSIONS: Epinephrine is likely an early upstream signal in the enhanced IgA response at respiratory surfaces after injury. (J Am Coll Surg 2014;218:450e458. 2014 by the American College of Surgeons)
BACKGROUND:
are intubated and who are represented by the ventilator care bundles.4,5 Despite all of these efforts, ventilatorassociated pneumonia after trauma remains a challenge, so investigators are now aiming toward prevention, and to do that it is key to understand the pathophysiology behind lung protective mechanisms. The lungs are protected by a mucosal barrier usually present in the upper airway, and are also protected by a more complex immune system represented by the innate and adaptive immune system. Immunoglobulin (Ig) A is the major immunoglobulin at mucosal surfaces, including the respiratory airway. Immunoglobulin A is mainly concentrated in the upper airway, and IgG is found mainly in the lower airway, however, the latter can cross to the luminal surface by means of diffusion. Previous clinical investigators have studied the relationship between IgA and pneumonia, correlating reduced IgA with higher incidence of pneumonia.6 Immunoglobulin A is produced at the basolateral surface of the respiratory epithelial cells by plasma cells and is then transported to the epithelial cells by means of a special receptor expressed in the basolateral surface, known as polyimmunoglobulin receptor (pIgR). Once
Pneumonia is a common cause of morbidity and mortality after trauma and hemorrhagic shock. The CDC defines ventilator-associated pneumonia as pneumonia in patients who had a device to assist or control respiration continuously through a tracheostomy or by endotracheal intubation within the 48-hour period before the onset of infection, inclusive of the weaning period. Ventilatorassociated pneumonia is associated with a 25% to 50% mortality rate.1,2 The economic impact of ventilatorassociated pneumonia is substantial, with costs ranging from $19,633 to $28,538.00 per patient with ventilatorassociated pneumonia.3 Therefore, a lot of effort has been made to reduce the incidence of pneumonia by applying standards of care in critically ill patients who Disclosure Information: Nothing to disclose. Received First Place, Basic Laboratory Research, the American College of Surgeons Committee on Trauma, 2013. Received September 25, 2013; Revised November 12, 2013; Accepted November 12, 2013. From the Department of Surgery, Wayne State University, Detroit, MI. Correspondence address: Abubaker Ali, MD, General Surgery, Wayne State University, 6C University Health Center, 4201 St Antoine, Detroit, MI 48201. email: [email protected]
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Abbreviations and Acronyms
dIgA DMEM ELISA Epi Ig IL NE pIgR sIgA TEER TNF
¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼
dimeric immunoglobulin A Dulbecco’s Modified Eagle medium enzyme-linked immunosorbent assay epinephrine immunoglobulin interleukin norepinephrine polyimmunoglobulin receptor secretory immunoglobulin A transepithelial electrical resistance tumor necrosis factor
the complex is internalized and reaches the luminal surface, pIgR becomes unstable and is cleaved, releasing a small protein known as the secretory component. Dimeric IgA along with secretory component forms secretory IgA (sIgA), which protects the airway through various mechanisms, including bacterial exclusion, which prevents bacterial attachment to the mucosal surface. As a result, bacteria cannot attach to the respiratory surface and will not cause infection.7,8 Several known factors affect the concentration of IgA at the mucosal surface. We were able to demonstrate in our laboratory, using an in vitro model, that IgA transport was significantly increased by stimulation of toll like receptor4 or estradiol.9,10 Recent clinical and laboratory studies demonstrated that early in trauma, there is an increase in IgA transport, which was accompanied by an increase in proinflammatory cytokine recovery from bronchoalveolar
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lavage samples after trauma. In a different series of experiments, the pro-inflammatory cytokine receptors were blocked, which led to abrogation of the airway IgA response to injury. They concluded that early after trauma, there is an increase in pro-inflammatory cytokines that leads to an increase in IgA transport.11-13 On the other hand, studies also found that early after injury and hemorrhagic shock, there is an increase in stress hormones and neurotransmitters that can alter cytokine release in the early phase after trauma.14 We hypothesized that early after trauma, there is an increase in stress hormones that alter pro-inflammatory cytokine release, which eventually leads to an increase in IgA transport. In addition, respiratory epithelial cells are known to be responsive to b2 agonists. We therefore conducted an in vitro study with the following objectives: first, we studied the effect of epinephrine (Epi), norepinephrine (NE), and albuterol on IgA transport across cultured human respiratory epithelial cells in vitro (Calu-3 cells); our second objective was to examine if Epi, NE, or albuterol had any effect on pro-inflammatory cytokines; and, lastly, we examined whether induced modulation of IgA transport effects bacterial passage across Calu-3 cells.
METHODS Respiratory epithelial cells Calu-3 cells were obtained from American Type Culture Collection and routinely cultured with Dulbecco’s Modified Eagle medium (DMEM) containing 10% fetal bovine serum, 4.5 g/L glucose, and 1% antibiotic
Figure 1. Diagram of experimental design. A schematic representation of the 2-chamber cell culture system used in the design of all experiments. CFU, colony-forming unit; dIgA, dimeric immunoglobulin A; Epi, epinephrine; pIgR, polyimmunoglobulin receptor; Norepi, norepinephrine.
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antimycotic (Invitrogen) in an atmosphere of 5% CO2 at 37 C. Medium was changed twice a week and cells were passaged every 10 to 14 days. Cells (5 105) were seeded on the apical surface of a polycarbonate membrane (3.0-mm pore size) (Transwell; Corning Costar Core) in a 2-chamber cell culture system and allowed to form polarized monolayers. Monolayer integrity was monitored by serial measurement of the transepithelial electrical resistance (TEER) with a Millicell electrical resistance meter (Millipore Corp.). When grown in this system, Calu-3 cells form polarized monolayers that are confluent after 10 to 14 days in culture. Experimental design Calu-3 cells were grown to confluence in complete DMEM. Epinephrine (106M), NE (106M), or albuterol (106M) was added to the apical chambers and cocultured with Calu-3 cells for 24 or 72 hours at 37 C (Fig. 1). After coculture, media was replaced with DMEM þ 0.06% bovine serum albumin (Sigma) and 20 mM HEPES (pH 7.3) for transcytosis experiments. In separate experiments, surface expression of pIgR and monolayer integrity (TEER) was analyzed in Calu-3 cells after coculture with Epi (106M), NE (106M), or albuterol (106M). In still other experiments, basal supernatants, post treatment, were collected for cytokine analysis and Klebsiella pneumoniae was added to assess bacterial translocation after Epi (106M), NE (106M), or albuterol (106M) pretreatment for 24 or 72 hours. Cytokine analysis by enzyme-linked immunosorbent assay Basal supernatants of Calu-3 cells treated with K pneumoniae and Epi (106M), NE (106M), or albuterol (106M) were collected at the end of the experiments and immediately stored at 70 C for subsequent cytokine analysis. Interleukin (IL)-6 and tumor necrosis factor (TNF)ea were quantitated in the previously frozen supernatant samples using a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). These immunoassay kits are commercially available and were used according to manufacturer’s directions (Cytoscreen, Biosource International). The minimal detectable levels of IL-6 and TNF-a with these kits are 2 pg/mL for IL-6 and <1 pg/mL for TNF-a. Bacterial translocation Klebsiella pneumoniae was grown overnight in trypticasesoy broth, centrifuged, and resuspended in phosphatebuffered saline. Bacterial concentration was determined spectraphotometrically and verified by a pour-plate assay. Final concentrations of bacteria used for the experiments were adjusted in fresh DMEM at 105 organisms/mL. In
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one set of experiments, Calu-3 cells were pretreated with Epi (106M), NE (106M), or albuterol (106M) for 24 hours. Then, 100 pg/mL dimeric IgA (dIgA) was added to the basal chamber and allowed to bind at 4 C for 4 hours to promote maximal binding of dIgA to the pIgR. Plates were subsequently placed at 37 C for 12 hours to allow maximal transcytosis and then 105 K pneumoniae was added to the apical chambers (Fig. 1). The cell culture system was reincubated for a total of 240 minutes post inoculum. Basal chamber medium was sampled at baseline and at 60minute intervals thereafter to quantitate bacterial passage across Calu-3 monolayers in the presence of translocated IgA. In another set of experiments, K pneumoniae was added in the absence of transcytosed IgA and basal chamber media was sampled for bacterial translocation, as outlined here. The 0.1-mL aliquots of basal chamber medium sampled were serially diluted and cultured on 10% sheep blood-trypticase soy agar (Becton Dickinson Microbiology System). The magnitude of bacterial translocation was quantitated by counting bacterial colonies after overnight incubation. Transepithelial electrical resistance was measured preinoculum and at 240 minutes post inoculum. Immunoglobulin A transcytosis Calu-3 medium was replaced with DMEM containing 0.6% bovine serum albumin and 20 mmol/L HEPES buffer (N-[2-hydroxyethyl]-piperazine-N-2-ethanesulfonic acid). Dimeric IgA (100 pg/mL) purified from rat myeloma cells was added to the basolateral chamber and allowed to incubate for 4 hours at 4 C to allow maximal binding of the dIgA to the pIgR on the surface of the Calu-3 cells. Cells were removed from the cold and washed 3 times in Hank’s balanced salt solution. Fresh medium at 37 C containing 0.6% bovine serum albumin and 20 mmol/L HEPES was added and cell preparations were allowed to incubate in a 37 C and 5% CO2 environment. Aliquots of apical chamber media were obtained at timed intervals and frozen at 20 C until ready for analysis. A series of experiments was also performed to assess the effect of Epi pretreatment for 24 or 72 hours on IgA transcytosis in HT29 cells. In another series of experiments, Calu-3 cells were pretreated with Epi (106M), NE (106M), or albuterol (106M) for 24 hours, followed by coculture of dIgA, leading to the collection of apical media as outlined here, which was subsequently assayed for free secretory component. Free secretory component was also assayed in apical chamber supernatants not cocultured with dIgA. Determination of immunoglobulin A by enzymelinked immunosorbent assay The amount of transcytosed sIgA in the apical medium samples was determined by a sandwich ELISA. Plates
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were coated overnight by adding 100 mL goat anti-rat IgA primary antibody (ICN Pharmaceuticals) diluted in 0.05 mol/L sodium carbonate coating buffer (pH 9.6) at 4 C. Each well was then washed 3 times and blocked with phosphate-buffered saline e.05% Tween-20 for 2 hours at room temperature. After another 3 washes, 100 mL of the apical media samples were added and the plate was incubated at room temperature for 2 hours. Plates then were washed and 100 mL of the secondary mouse anti-rat IgA antibody conjugated with horseradish peroxidase was added and the mixture was incubated for 2 hours at room temperature. The ELISA plates then were washed and subsequently developed using 1-step Turbo TMB (Pierce Chemical), after a 15-minute incubation at room temperature the color reaction was stopped by adding 1 mol/L H2SO4 and absorbance was measured at 450 nm using an ELISA plate reader. Purified rat myeloma IgA was used to create a standard curve for the determination of sIgA concentrations in the apical samples. In a separate assay, apical media samples collected for the determination of free secretory component were also quantitated. The ELISA plates were prepared as described here and probed with a secondary mouse antiesecretory component (bound and free) antibody conjugated with horseradish peroxidase for 2 hours at room temperature. Development was as described here and absorbance was measured at 450 nm using an ELISA plate reader. Flow cytometry for polyimmunoglobulin receptor expression Expression of the pIgR on the surface of Calu-3 cells was determined using flow cytometry. Calu-3 cells were washed 5 times with phosphate-buffered saline and then trypsinized. Cells then were placed in fresh media and incubated for 2 hours at 37 C to allow re-expression of cell surface proteins. Cells then were washed 3 times with phosphate-buffered saline (10 minutes at 1,200 rpm) and 1 mL goat anti-human secretory component (bound and free) (Sigma) diluted in phosphate-buffered saline was added and the mixture was incubated for 30 minutes at 4 C. Cells then were washed 3 times with ice-cold phosphate-buffered saline and 1 mL rabbit anti-goat IgG conjugated with fluorescein isothiocyanate (Sigma) was added and incubated for 30 minutes at 4 C in the dark. An isotype control was set up in the same manner as described earlier to ensure the secondary antibody was not nonspecifically binding to the surface of the Calu-3 cells. Cells were washed 3 times as described earlier, suspended in phosphate-buffered saline, and pIgR expression on the surface of the cells was determined
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via flow cytometry. Data are expressed as mean fluorescence intensity. Monolayer transepithelial electrical resistance Cell monolayer integrity was assessed by measurement of TEER at the beginning and throughout the various time points of the experiment. TEER measurements were expressed as ohm/cm2. Statistical analysis All samples were compared with an analysis of variance with a post-hoc Tukey test. Statistical significance was inferred at p < 0.001. All data are expressed as mean SD.
RESULTS A schematic representation of the 2-chamber cell culture system used in the design of all the experiments is shown in (Fig. 1). Immunoglobulin A transport through Calu-3 cells after 24-hour pretreatment with Epi, NE, or albuterol using the same concentration (106M) is shown in (Fig. 2). Epinephrine significantly increased sIgA recovery from the apical media in a time-dependent fashion, 3 vs 12 hours compared with control (p < 0.001). Norepinephrine and albuterol increased sIgA recovered from the apical media compared with control; this increase did not reach statistical significance (p < 0.001). Figure 3 assesses the role pIgR plays in the increased sIgA transport demonstrated with Epi. Calu-3 cells were challenged with Epi, NE, or albuterol using the same concentration 106M. Epinephrine led to a significant increase in pIgR expressed on the basolateral surface compared with control (p < 0.001), and Calu-3 cells treated with NE or albuterol did not lead to any significant increase in pIgR expression compared with control (p < 0.001). After demonstrating that only the group of Calu-3 cells pretreated with Epi using 106M led to a significant increase in IgA transport and a parallel increase in pIgR expression, we examined if stress levels of Epi would affect IgA transport. Calu-3 cells were pretreated with a higher concentration of Epi (103 M) and IgA recovery was indexed by sampling the apical media at 3 and 12 hours using ELISA. Calu-3 cells pretreated with Epi (103 M) led to a significant increase in IgA transport compared with control (p < 0.001), but this increase did not reach statistically significant levels (p < 0.001) when compared with Calu-3 cells treated with 106M Epi (data not shown).
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Figure 2. The effect of epinephrine (Epi), norepinephrine (Norepi), or albuterol (106M) treatment of Calu-3 cells for 24 hours on immunoglobulin (Ig) A transcytosis. Immunoglobulin A transcytosis was significantly increased at both 3 and 12 hours by Calu-3 cells treated with 106M Epi for 24 hours. Black bar, 3 hours; gray bar, 12 hours.
We then examined if Epi, NE, or albuterol had any effect on cytokine expression by Calu-3 cells. Calu-3 cells were treated with Epi, NE, or albuterol using the same concentration (106M) (Fig. 4). The group of Calu-3 cells pretreated with Epi led to a significant increase in both TNF-a and IL-6 production in the basal media compared with control (p < 0.001). Calu-3 cells treated with NE or albuterol did not lead to any significant difference in cytokine production when compared with control (p < 0.001). In Figure 5, Calu-3 cells were pretreated for 24 hours with Epi, NE, or albuterol followed by coculture with K pneumoniae (105 colony-forming units) in the apical media. Passage of the bacteria from apical to basal media was assessed by sampling the basal media at 60-, 120-, and 240-minute intervals. The group of Calu-3 cells pretreated with Epi led to a significant decrease in bacterial translocation compared with control at all time points (p < 0.001).
In contrast, Calu-3 cells treated with NE or albuterol did not lead to any significant difference in bacterial translocation compared with control (p < 0.001). To ensure Epi, NE, and albuterol coculture on sIgA recovery from the apical media and bacterial recovery from the basal media was not due to a change in the monolayer integrity, TEER was measured at T ¼ 0 (beginning of the experiment) and T ¼ END (24 hours) and the cells integrity remained unchanged between the different groups (figure not shown).
DISCUSSION Pneumonia still remains a common cause of morbidity and mortality after trauma and hemorrhagic shock. Recent clinical studies have demonstrated that individuals who are deficient in IgA are more susceptible to pneumonia.6
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Figure 3. Polyimmunoglobulin receptor (pIgR) expression on the surface of Calu-3 cells after 24-hour treatment with stress hormones. Calu-3 cells were cocultured with epinephrine (Epi), norepinephrine (Norepi), or albuterol (106M) for 24 hours and pIgR expression quantitated. Epi led to a significant increase in pIgR surface expression. Black bar, pIgR (mean fluorescence intensity).
In a recent human study by Kudsk and colleagues,11 injury was found to stimulate an innate respiratory IgA response. In a different laboratory study, Jonker and colleagues13 showed that the airway IgA response seen after injury can be replicated by exogenous administration of 3 pro-inflammatory cytokines (TNF-a, IL-1b and IL-6). In an additional study, the same group demonstrated that if the pro-inflammatory cytokine receptors are blocked, then the IgA transport gets impaired.12 From these studies, it appears that after trauma there is an increase in pro-inflammatory cytokine release that eventually leads to an increase in IgA transport to the respiratory secretions. After trauma, there is also an early increase in stress hormones and sympathetic neurotransmission, both of which play a well-established role in the modulation of the immune system.15 In our current study, Epi led to an increase in proinflammatory cytokine production and an increase in
IgA transport, which eventually led to a decrease in bacterial passage across Calu-3 cells. Briefly, we pretreated Calu-3 cells with Epi, NE, or albuterol (106M), which is within the physiologic concentration range described in previous studies.16-18 Calu-3 cells were treated for 24 hours for maximal cell stimulation. Only the group of cells treated with Epi led to a significant increase in IgA transport. We postulate that treatment with Epi led to increased IgA transport via b1 receptors because albuterol works mainly on b2 receptors and NE works mainly on a1 receptor with minimal action on b1 receptors. To make sure that the cell monolayer integrity was maintained between the different groups and throughout the experiment, TEER was measured at the beginning and end of each experiment and there was no statistical difference between the different groups at both points. Previous studies have shown that stimulation of b2 receptors
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Figure 4. Cytokine expression in Calu-3 cells co cultured with 106M stress hormones for 24 hours. Calu-3 cells were exposed to 106M epinephrine (Epi), norepinephrine (Norepi), or albuterol for 24 hours in the apical chamber of a 2-chamber cell culture system. Basal chamber media was collected at 24 hours and cytokine levels quantified by enzyme-linked immunosorbent assay. Epi treatment led to significant increase in tumor necrosis factor (a) and interleukin (IL)-6 levels compared with Norepi or albuterol treatment. Black bar, IL-6; gray bar, a.
on mononuclear cells and macrophages lead to a diminished capacity to release pro-inflammatory cytokines, such as TNF-a and IL-1b; both of which are important in IgA transport across the respiratory epithelium.13 After demonstrating that physiologic concentrations of Epi (106M) led to a significant increase in IgA transport, we used higher concentrations of Epi to resemble stress conditions. Therefore, a subset of Calu-3 cells was treated with Epi at a concentration of 103 M for 24 hours to mimic stress conditions. There was a significant increase in IgA transport compared with control, but this increase noted at 103 M did not reach statistical significance when compared with the group of Calu-3 cells treated with physiologic Epi concentrations (106M). The latter finding might be related to the fact that, at higher Epi
concentrations, Epi tends to have a higher affinity toward
a1 receptors (result not shown).
In additional experiments, Calu-3 cells were treated with Epi (106M) for 72 hours and IgA transport was indexed by recovery of IgA from the apical media using ELISA. We found that the level of IgA transported at 72 hours returned to control levels. This finding was also present in the previous study by Junker and colleagues,13 where IgA transport failed to show consistent significant levels compared with control at different time points during the study. In a study by Uren and colleagues,19 pIgR knockout mice had high serum IgA levels, but were deficient in sIgA from BAL samples compared with control. With each molecule of IgA transported, there is one molecule of pIgR consumed. To have an increase in IgA transport, there
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Figure 5. The effect of stress hormones on Klebsiella pneumoniae bacterial translocation in Calu-3 cells. Calu-3 cells were co cultured with epinephrine (Epi), norepinephrine (Norepi), or albuterol (106M) for 24 hours. Klebsiella pneumoniae (105 colony-forming units [CFU]/mL) was added to the apical chamber media of a 2-chamber cell culture system and basal media was subsequently collected at 60, 120, and 240 minutes post inoculum. Basal media samples were plated on 5% sheep blood agar plates and incubated overnight at 37 C. Colonies were counted and bacterial translocation was expressed as CFU/mL K pneumoniae. Black bar, 60 minutes; light gray bar, 120 minutes; dark gray bar, 240 minutes.
should be a parallel increase in pIgR expression. In our current study, we found that only the group of Calu-3 cells treated with Epi (106M) for 24 hours led to a significant increase in pIgR expression. Polyimmunoglobulin receptor expression in cells treated with NE or albuterol did not reach statistically significant levels when compared with control. Interestingly, when Calu-3 cells were treated for 72 hours with Epi at 106M, pIgR expression returned to control levels. This latter finding can explain why IgA levels are not constant in our study and previous studies. To have sIgA in respiratory secretions, plasma cells are required to produce dIgA and respiratory epithelial cells
to produce pIgR. Both of these steps are controlled by various factors, including pro-inflammatory cytokines, estradiol, toll like receptor4, etc.9,10,20,21 Their production also goes through transcription and translation, which also requires time.7 These facts might aid in explaining why sIgA does not remain at constant levels after trauma. The mucosal-associated lymphoid tissue includes the gut-associated lymphoid tissue and a similar structure found in the lungs, called the bronchial-associated lymphoid tissue.22,23 Secretory IgA is considered the major immunoglobulin in both gut-associated lymphoid tissue and bronchial-associated lymphoid tissue. In separate
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experiments, HT-29 cells (human colonic cell line) were pretreated in the same fashion as Calu-3 cells, with Epi for 24 hours or 72 hours. Secretory IgA in the apical media was then measured using ELISA. Secretory IgA levels did not reach statistical significance with either group (figure not shown). This might be related to the fact that HT29 cells lack b1 receptors.24 This latter finding supports that b1 receptors are required for Epi to exert its affects. Epinephrine was also found to increase production of TNF-a and IL-1b recovered from the basal media. This latter finding supports our hypothesis that there is an intermediate link between trauma and the increase in the pro-inflammatory cytokines. This link might be the increase in Epi; a finding not previously measured in the study by Jonker and colleagues.13 Modulation of IgA transport noticed with Epi led to a significant reduction in bacterial passage from apical to basal media at all time points compared with control, supporting the importance of IgA in mucosal immunity. If bacteria cannot attach to the mucosal membrane, then it cannot cause infection.
CONCLUSIONS In summary, Epi led to an early significant increase in IgA transport across Calu-3 cells, this was in part due to an increase in pIgR expression. The increase IgA transport led to a significant reduction in bacterial passage across Calu-3 cells. Epinephrine also led to a significant increase in proinflammatory cytokines production. From our current study, we conclude that Epi might be an early upstream signal in the transport of IgA to the respiratory luminal surface. Factors that influence outcomes during trauma are multiple and cannot be attributed solely to the stress hormone Epi. Author Contributions Study conception and design: Diebel Acquisition of data: Ali, Liberati Analysis and interpretation of data: Ali, Diebel, Liberati Drafting of manuscript: Ali Critical revision: Diebel REFERENCES 1. Efrati S, Deutsch I, Antonelli M, et al. Ventilator-associated pneumonia: current status and future recommendations. J Clin Monit Comput 2010;24:161e168. 2. Scott RD II. The direct medical costs of healthcare-associated infections in US hospitals and the benefits of prevention. March 2009. Available from: http://www.cdc.gov/hai/pdfs/ hai/scott_costpaper.pdf. Accessed 2012. 3. Morrow LE, Shorr AF. The seven deadly sins of ventilatorassociated pneumonia. Chest 2008;134:225e226. 4. Kollef MH. Prevention of nosocomial pneumonia in the intensive care unit: beyond the use of bundles. Surg inf 2011;12:211e220.
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5. Rello J, Afonso E, Lisboa T, et al. A care bundle approach for prevention of ventilator-associated pneumonia. Clinical Microbiol Infect 2013;19:363e369. 6. Annane D, Clair B, Mathieu B, et al. Immunoglobulin A levels in bronchial samples during mechanical ventilation and onset of nosocomial pneumonia in critically ill patients. Am J Respir Crit Care Med 1996;153:1585e1590. 7. Woof JM, Kerr MA. The function of immunoglobulin A in immunity. J Pathol 2006;208:270e282. 8. Kaetzel CS. The polymeric immunoglobulin receptor: bridging innate and adaptive immune responses at mucosal surfaces. Immunol Rev 2005;206:83e99. 9. Diebel LN, Liberati DM. Disparate effects of bacteria and Toll-like receptor-dependant bacterial ligand stimulation on immunoglobulin A transcytosis. J Trauma 2011;70:691e700. 10. Ali AA, Diebel LN, Liberati DM. Estrogen modulation of pneumonia? An immunoglobulin A effect. J Trauma Acute Care Surg 2012;72:908e915. 11. Kudsk KA, Hermsen JL, Genton L, et al. Injury stimulates an innate respiratory immunoglobulin a immune response in humans. J Trauma 2008;64:316e323; discussion 323325. 12. Hermsen JL, Sano Y, Gomez FE, et al. Parenteral nutrition inhibits tumor necrosis factor-alpha-mediated IgA response to injury. Surg Infect 2008;9:33e40. 13. Jonker MA, Sano Y, Hermsen JL, et al. Proinflammatory cytokine surge after injury stimulates an airway immunoglobulin a increase. J Trauma 2010;69:843e848. 14. Batistaki C, Kostopanagiotou G, Myrianthefs P, et al. Effect of exogenous catecholamines on tumor necrosis factor alpha, interleukin-6, interleukin-10 and beta-endorphin levels following severe trauma. Vasc Pharmacol 2008;48:85e91. 15. Webster JI, Tonelli L, Sternberg EM. Neuroendocrine regulation of immunity. Annu Rev Immunol 2002;20:125e163. 16. McAuley DF, Frank JA, Fang X, Matthay MA. Clinically relevant concentrations of beta2-adrenergic agonists stimulate maximal cyclic adenosine monophosphate-dependent airspace fluid clearance and decrease pulmonary edema in experimental acid-induced lung injury. Crit Care Med 2004;32:1470e1476. 17. Du Q, Min S, Chen LY, et al. Major stress hormones suppress the response of macrophages through down-regulation of TLR2 and TLR4. J Surg Res 2012;173:354e361. 18. Straub RH, Mayer M, Kreutz M, et al. Neurotransmitters of the sympathetic nerve terminal are powerful chemoattractants for monocytes. J Leukocyte Biol 2000;67:553e558. 19. Uren TK, Johansen FE, Wijburg OL, et al. Role of the polymeric Ig receptor in mucosal B cell homeostasis. J Immunol 2003;170:2531e2539. 20. Nosocomial meningitis caused by Pseudomonas aeruginosa: overview of 20 cases in 20 years. Neuroendocrinol Lett 2012;33[Suppl 1]. 21. Takenouchi-Ohkubo N, Takahashi T, Tsuchiya M, et al. Role of nuclear factor-kappaB in the expression by tumor necrosis factor-alpha of the human polymeric immunoglobulin receptor (plgR) gene. Immunogenetics 2000;51:289e295. 22. Bienenstock J, Johnston N, Perey DY. Bronchial lymphoid tissue. I. Morphologic characteristics. Lab Invest 1973;28:686e692. 23. Bienenstock J, Johnston N, Perey DY. Bronchial lymphoid tissue. II. Functional characterisitics. Lab Invest 1973;28:693e698. 24. Abel PW, Zeng W, Wildrick DM, et al. Characterization of beta-adrenergic receptors in DiFi and HT-29 cells. Anticancer Res 1992;12:1655e1658.