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The Journal of Immunology, 2002, 169: 4522-4530.
Copyright © 2002 by The American Association of Immunologists

Pseudomonas aeruginosa Activates Human Mast Cells to Induce Neutrophil Transendothelial Migration Via Mast Cell-Derived IL-1{alpha} and {beta}1

Tong-Jun Lin2,*,{dagger}, Rafael Garduno*, Robert T. M. Boudreau* and Andrew C. Issekutz*,{dagger},{ddagger}

Departments of * Microbiology and Immunology, {dagger} Pediatrics, and {ddagger} Pathology, Dalhousie University, Halifax, Nova Scotia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The mechanisms of neutrophil (PMN) recruitment to Pseudomonas aeruginosa infection remain incompletely defined. Mast cells (MC) involvement in this process has not been studied previously. In this study, we demonstrate that human cord blood-derived MC phagocytose P. aeruginosa and release mediators that activate HUVEC monolayers for supporting PMN transmigration. Pretreatment of supernatants from P. aeruginosa-MC cocultures with neutralizing anti-IL-1{alpha} plus anti-IL-1{beta} Abs, or IL-1R antagonist before addition to HUVEC for stimulation completely abrogated MC-induced PMN transmigration, while anti-TNF-{alpha} treatment had no effect. The expression of E-selectin and ICAM-1 on HUVEC, the latter a ligand for PMN CD11/CD18, was significantly up-regulated by P. aeruginosa-induced MC mediators. Pretreatment of human PMN with anti-CD18 mAb or pretreatment of HUVEC with a combination of three mAbs (against ICAM-1, ICAM-2, and E-selectin) inhibited by 85% the MC-dependent PMN transmigration. Moreover, P. aeruginosa-induced production of IL-1{alpha} and IL-1{beta} was down-regulated by IL-10 and dexamethasone. This study demonstrates for the first time that MC may mediate P. aeruginosa-induced PMN recruitment via production of IL-1{alpha} and {beta}. These findings have important implications for diseases involving P. aeruginosa infection and suggest novel targets for modulating P. aeruginosa-induced inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Pseudomonas aeruginosa is the major pathogen in cystic fibrosis (CF)3 patients (1) and a common cause of nosocomial pneumonia (2). P. aeruginosa elicits a massive influx of neutrophils (PMN) at the site of infection (3, 4). Efficient recruitment of PMN is essential for eliminating this bacterium. However, the excessive infiltration of PMN and their release of reactive radicals and degradative enzymes may also cause tissue damage (3, 4). Thus, a better understanding of the mechanisms of P. aeruginosa-induced PMN migration is necessary for the development of strategies for the control of PMN recruitment during P. aeruginosa infection.

Migration of circulating PMN from vessels into tissues involves interaction with endothelium through adhesion molecules (5). Depending upon the nature of inflammation and tissue involved, adhesion molecules such as CD11/CD18 ({beta}2) integrins and ICAMs are involved in PMN recruitment to some bacterial infections, but not others (5, 6, 7). Many cytokines and chemokines influence PMN migration into tissues, including the potent proinflammatory cytokines IL-1{alpha}, IL-1{beta}, and TNF-{alpha} (8). Significant production of proinflammatory cytokines in infected tissue has been observed within hours after P. aeruginosa infection (4, 9), suggesting that local resident cells in the tissues are sources for these cytokines (4). Although macrophages are traditionally recognized as a major source of IL-1 and TNF-{alpha}, their roles in P. aeruginosa-induced pulmonary inflammation are still a matter of active debate, because depletion of the alveolar macrophages in mice did not affect the susceptibility of the animal to P. aeruginosa (10). Thus, the cellular sources of those P. aeruginosa-induced proinflammatory cytokines and the cellular components mediating PMN infiltration remain to be defined.

Mast cells (MC) are a potent source of various cytokines and chemokines and are long-term resident (up to months) tissue cells. They are found in large numbers in the mucosal areas, skin, and perivascular tissues. This strategic location provides an ideal opportunity for MC to interact with foreign pathogens as well as to communicate with blood-borne leukocytes such as PMN. Indeed, several studies have demonstrated that in mice, MC are critical for PMN recruitment in Klebsiella pneumoniae-infected lung, in caecal ligation and puncture-induced infection in the peritoneum, and in the gut and skin after IgE-dependent stimulation (11, 12, 13). However, a role for MC in P. aeruginosa-induced PMN recruitment has not been investigated previously.

MC can vary in phenotype and function not only between different animal species, but even between MC from different body sites in the same host (14). Human MC differ from their counterparts in mice in many features including morphology, mediator content, histochemical characteristics, responsiveness to growth factors, and sensitivity to secretagogues, drugs, etc. (14, 15). This study used human cord blood-derived MC (CBMC) to investigate: 1) the response of human MC to CF-associated P. aeruginosa, 2) the potential role of this response in induction of human PMN transendothelial migration (TEM), and 3) the regulation of such MC responses to P. aeruginosa.

We have demonstrated for the first time that in response to CF-associated P. aeruginosa, human MC secrete IL-1{alpha} and IL-1{beta}, which account for the stimulation of PMN TEM through up-regulation of endothelial ICAM-1 and E-selectin expression. Moreover, the P. aeruginosa-induced IL-1{alpha} and IL-1{beta} production can be regulated by IL-10 and dexamethasone. This study suggests a role for MC in P. aeruginosa-induced inflammation through the production of IL-1. Thus, strategies specifically targeting MC and MC-derived IL-1 may provide an additional therapeutic approach in the treatment of P. aeruginosa-induced inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mast cells

Highly purified CBMC were obtained by long-term culture of cord blood progenitor cells, as previously described (16, 17). After >8 wk in culture, mature MC (>95% purity) were identified by their morphological features and the presence of metachromatic granules (toluidine blue staining). These cells were >98% positive for c-kit when stained by anti-c-kit Ab (Exalpha Biologicals, Boston, MA) and analyzed by flow cytometry.

The human MC line HMC-1 5C6 was grown in Iscove’s medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS (Life Technologies). After confluent growth, the adherent cells were harvested by subtle pipetting (16, 17).

Bacterial treatment

P. aeruginosa strain 8821 (a gift from A. Chakrabarty, University of Illinois, Chicago, IL) is a mucoid strain isolated from a CF patient (18). P. aeruginosa was cultured in Luria-Bertani broth and harvested when the culture reached OD640 of 2 U (early stationary phase). Bacteria were washed in PBS, and density was adjusted to 1 OD unit before being killed with gentamicin (100 µg/ml for 2 h). MC were treated with P. aeruginosa, for the indicated times, at a MC-bacteria ratio of 1:50.

Electron microscopy and MC phagocytosis assay

CBMC or HMC-1 cells (5 x 105 cells/ml) were incubated with 2.5 x 107 P. aeruginosa in 1 ml vol for 18 h. Cells were then fixed in 2% glutaraldehyde and dehydrated in graded ethanol. Then they were embedded in polyBed A12 resin, sectioned, stained with uranyl acetate and lead citrate, and viewed with a Philips 201 electron microscope (19).

MC were incubated at 5 x 105 cells/ml with 2.5 x 106 P. aeruginosa (CF-associated strain 8821, laboratory strain PAO.1) or Legionella pneumophila for 0–3 h. The cells were then washed and treated with gentamicin to kill extracellular bacteria. After washing, the cells were lysed with 100 µl 0.1% Triton X-100. A total of 5 or 10 µl Triton solutions was plated to MacConkey agar or charcoal-yeast extract agar to determine the number of viable intracellular bacteria (19).

Human PMN purification and endothelial cell cultures

Human PMN were purified and labeled with Na251CrO4 (Amersham, Oakville, Ontario, Canada) (20). PMN of >=95% purity with essentially no red cell contamination and >=98% cell viability were used.

HUVEC were isolated and cultured in gelatin-coated flasks and grown on filters, as previously described (16, 20, 21). The HUVEC formed a tight permeability barrier in 5–6 days and were evaluated for barrier function by 125I-labeled human serum albumin (HSA) diffusion, as previously described (20, 21). Less than 1.5% labeled HSA diffused across the HUVEC/filter unit in 45 min with 1 mm positive hydrostatic pressure, while bare filters showed >=30% diffusion of 125I-labeled HSA in this test.

PMN TEM assay

PMN migration assays were performed as described previously (20, 21). MC supernatant dilutions or IL-1{alpha} (0.5 ng/ml) or TNF-{alpha} (20 U/ml) were added to the lower compartment (well) for 4 h at 37°C. In some experiments, human rIL-1R antagonist (rIL-1RA, 50 ng/ml; gift from Synergen, Boulder, CO) was added to the HUVEC 30 min before supernatant. Some CBMC supernatants were pretreated for 60 min (on ice) with neutralizing mAb to human IL-1{alpha}, IL-1{beta}, and/or TNF-{alpha} (R&D Systems, Minneapolis, MN; 20 µg/ml) before addition to the HUVEC. After 4-h stimulation, HUVEC/filter units were washed and transferred to a new well. 51Cr-labeled PMN (1 x 105) were added above the HUVEC. In some experiments, the PMN were pretreated (20 min, 22°C) with blocking mAb to CD18 (IB4 30 µg/ml) or control mAb (543 anti-CR1), or the HUVEC was pretreated (20 min, 37°C) with mAbs as F(ab')2 fragments (10 µg/ml) to ICAM-1 (R6.5), ICAM-2 (CBR IC2/2), or E-selectin (BB11) before PMN addition. The mAbs were present throughout the assay. After 90 min to allow PMN transmigration, PMN in the lower compartment were quantified by gamma counting (21). The results are expressed as the percentage of the total 51Cr-labeled PMN added above the HUVEC, which migrated through the HUVEC filter unit.

Quantitation of adhesion molecule expression on HUVEC by ELISA

The expression of ICAM-1, VCAM-1, and E-selectin on HUVEC was determined with whole cell ELISA, as described previously, with minor modifications (20). Briefly, HUVEC monolayers were incubated with MC supernatants or IL-1 for 4 h. After washing, HUVEC were further incubated for 60 min with RPMI 1640 medium containing mAbs to ICAM-1 (R6.5), E-selectin (BB11), or control mAb (3H11 B9 anti-pertussis toxin), and followed by incubation with peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich, St. Louis, MO). o-Phenylendiamine was used as a substrate. The absorbance at 490 nm was measured. Results are expressed as absorbance units minus value for control mAb.

IL-1{alpha}, IL-1{beta}, and TNF-{alpha} assays

Human IL-1{alpha}, IL-1{beta}, and TNF-{alpha} levels in supernatants were measured using Abs from R&D Systems (anti-IL-1{alpha} and anti-TNF-{alpha} Abs) or from Endogen (Woburn, MA) (anti-IL-1{beta} Abs) by ELISA, as described previously (16). The detection limit was 3 pg/ml. TNF-{alpha} bioactivity was determined by a standard L929 fibroblast cytotoxicity (24 h) assay in the presence of actinomycin D (0.5 µg/ml) (22).

Immunocytochemistry

For cytospin preparation, CBMC after treatment with P. aeruginosa strain 8821 (MC-bacteria = 1:50) for 24 h (1 x 105 cells) were cytocentrifuged onto poly(L-lysine) (Sigma-Aldrich)-coated slides. Cells were then fixed in cold (-20°C) acetone for 2 min and stored at -20°C until use. To examine MC purity, slides were fixed with Carnoy solution and used for toluidine blue staining. For IL-1{alpha} and IL-1{beta} staining, slides were fixed with 4% paraformaldehyde for 10 min and washed three times with 0.1% saponin in phosphate buffer. Cells were then permeabilized by 0.1% Triton X-100 (10 min), and the nonspecific binding sites were blocked by 5% goat serum (30 min). Titered primary Abs to IL-1{alpha} (mouse IgG2a, 1:200), IL-1{beta} (mouse IgG1, 1:40) (R&D Systems), or isotype controls were added to the slides for incubation overnight at room temperature, followed by secondary biotinylated goat anti-mouse IgG. The Ab was localized using streptavidin-HRP (Signet Kit, Dedham, MA; IDlabs, London, Ontario, Canada) and 3-amino-9-ethyl-carbozole (Sigma-Aldrich) as a chromogen.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Internalization of P. aeruginosa by human MC

To determine whether human MC recognize and internalize P. aeruginosa, CBMC and HMC-1 cells were incubated with gentamicin-killed P. aeruginosa 8821 for 24 h and examined by transmission electron microscopy. Fig. 1Go, a and b, shows various stages of bacterial internalization by MC. Bacteria were clearly seen being embraced by filapod-like structures. A significant amount of bacteria was phagocytosed by MC. Given that gentamicin-killed P. aeruginosa were used in this assay, the observed phagocytic processes indicate an active role of MC during their interaction with P. aeruginosa. It is noteworthy that more bacteria were phagocytosed by CBMC than by HMC-1 (data not shown).



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  		FIGURE 1. Internalization of P. aeruginosa by human MC. a and b, CBMC were incubated with P. aeruginosa strain 8821 and examined by electron microscopy. In both a and b, the bacteria were gripped by MC filapod-like structures (black arrows). Several internalized bacteria can be identified inside MC (white arrow) (b). Original magnification: a, x22,000; b, x7,500. c and d, HMC-1 cells were treated with P. aeruginosa (mucoid strain 8821 and nonmucoid strain PAO.1) or L. pneumophila for 3 h (d) or 1, 2, and 3 h (c). Extracellular bacteria were killed by gentamicin treatment. MC were lysed to release intracellular bacteria, lysate was cultured overnight, and colonies were enumerated.

 
A time course of bacterial internalization by MC was determined. HMC-1 cells after incubation with live P. aeruginosa (mucoid strain 8821 or nonmucoid strain PAO1) or L. pneumophila for 1, 2, or 3 h were treated with gentamicin to kill extracellular bacteria. Intracellular bacteria were released by Triton X-100 and quantitated by colony counting. As shown in Fig. 1Goc, MC internalized P. aeruginosa in a time-dependent manner. CF-associated mucoid strain 8821 was more efficiently internalized than nonmucoid PAO1. Interestingly, MC did not internalize L. pneumophila in these experiments (Fig. 1God), suggesting MC-P. aeruginosa interaction is selective.

P. aeruginosa-induced MC mediators stimulate PMN TEM

Given that PMN recruitment into the local tissue is a predominant feature during P. aeruginosa infection, we determined whether P. aeruginosa-MC interaction could elicit PMN TEM. Cell-free supernatants were collected from CBMC that had been treated with or without P. aeruginosa 8821 for 24 h. Endothelial monolayers (HUVEC) were stimulated for 4 h with various dilutions of MC supernatants and used to evaluate their capacity to support human PMN transmigration. As shown in Fig. 2Go, PMN TEM was dramatically stimulated by P. aeruginosa-induced MC mediators. IL-1{alpha} (0.5 ng/ml) and TNF-{alpha} (20 U/ml) were used as positive controls. Interestingly, 1/15 dilutions of supernatant from P. aeruginosa-activated MC demonstrated similar stimulatory effects, as did IL-1{alpha} or TNF-{alpha} (Fig. 2Go). No effect on PMN TEM was observed when HUVEC monolayers were treated with equivalent 1/15 dilution of MC supernatant from sham-treated CBMC (no bacteria) or P. aeruginosa alone without MC or medium alone (Fig. 2Go).



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  		FIGURE 2. Induction of human PMN TEM by P. aeruginosa-induced MC mediator(s). IL-1{alpha} (0.5 ng/ml) or TNF-{alpha} (20 U/ml) or supernatants from CBMC cultured with or without P. aeruginosa strain 8821 for 24 h (diluted 1/15) were used to treat HUVEC monolayers on filters for 4 h. After washing, 51Cr-labeled human PMN were added above the monolayer/filter unit for 90 min. The percentage of added PMN that migrated is shown. Results are means ± SD of triplicate determinations. *, p < 0.01 compared with HUVEC treated with medium alone or supernatants from nonstimulated MC (MC-untreated).

 
To assess the potency of MC supernatants in mediating PMN TEM, we tested dilutions (1/50, 1/100, or 1/300) of supernatants from P. aeruginosa-stimulated CBMC. HUVEC monolayers were incubated with these supernatants for 4 h. After washing, these HUVEC monolayers induced marked PMN TEM, even after 300-fold dilutions. The TEM (%) of PMN was 35.6 ± 3.6, 23.8 ± 0.5, or 21.8 ± 0.9, following HUVEC stimulation by 1/50, 1/100, or 1/300 dilutions, respectively, of P. aeruginosa-stimulated CBMC supernatants. Baseline PMN transmigration was 2.0 ± 0.5% in this experiment (data are mean ± SD of triplicates).

Roles of IL-1{alpha}, IL-1{beta}, and TNF-{alpha} in MC-dependent PMN TEM

To determine the factors in P. aeruginosa-induced MC supernatant responsible for induction of PMN TEM, neutralizing Abs to IL-1{alpha}, IL-1{beta}, or TNF-{alpha} were used to pretreat the supernatants before addition to HUVEC monolayers. Alternatively, HUVEC were pretreated with IL-1RA before addition of supernatants. IL-1RA or anti-IL-1{alpha} Ab together with anti-IL-1{beta} Ab completely blocked the stimulatory effects of P. aeruginosa-induced MC supernatant for PMN TEM (Fig. 3Go). Anti-IL-1{beta} Ab alone showed partial inhibition, while anti-IL-1{alpha} Ab alone had no significant effect, suggesting synergistic roles of IL-1{alpha} and IL-1{beta} in MC-mediated PMN TEM. The neutralizing activity of anti-IL-1{alpha} Ab was confirmed in our experiment. IL-1{alpha} (1 ng/ml)-induced PMN TEM (% migration: 18.6 ± 0.2) was markedly inhibited by anti-IL-1{alpha} Ab at the dose of 20 µg/ml used with MC supernatant (% migration: 6.1 ± 0.5).



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  		FIGURE 3. MC-induced PMN TEM is dependent on IL-1{alpha} and IL-1{beta}. a, HUVEC monolayers were treated for 4 h with medium or with P. aeruginosa-induced MC supernatants in the absence (sham treated) or presence of IL-1RA, anti-IL-1{alpha} Ab, anti-IL-1{beta} Ab, or anti-IL-1{alpha} Ab + anti-IL-1{beta} Ab. PMN TEM was determined as in Fig. 2Go. Results are means ± SD of one representative experiment of three performed with triplicates. b–f, P. aeruginosa-induced MC supernatants were treated without or with anti-TNF-{alpha} Ab (or anti-IL-1{beta} Ab + anti-TNF-{alpha} Ab, f) for 60 min and used to stimulate HUVEC for 4 h. PMN and HUVEC from four different donors were used. Results are means ± SD of triplicate determinations.

 
In mice, MC-derived TNF-{alpha} contributes to PMN infiltration in several models (11, 12, 13). Therefore, we examined the role of TNF-{alpha} in the MC supernatant in inducing PMN TEM. Interestingly, treatment of supernatant with anti-TNF-{alpha} Ab had little or no effect on MC-dependent PMN TEM using PMN and HUVEC from different individuals (Fig. 3Go). Moreover, anti-TNF-{alpha} Ab did not alter the partial inhibition of PMN TEM by the anti-IL-1{beta} Ab (Fig. 3Go). The neutralizing activity of anti-TNF-{alpha} Ab was confirmed using rTNF-{alpha} as a stimulant for the HUVEC (data not shown). Thus, TNF-{alpha} was not a component in the MC supernatant responsible for PMN TEM.

Investigation of adhesion molecule mechanisms involved in the MC-induced PMN TEM

The fact that treatment of HUVEC with P. aeruginosa-induced MC supernatant stimulated PMN TEM suggests a role for adhesion molecules. Therefore, we examined the expression of ICAM-1 and E-selectin by HUVEC following stimulation with the P. aeruginosa-induced MC supernatant. As shown in Fig. 4Go, the supernatant from P. aeruginosa-stimulated CBMC, but not control supernatants (P. aeruginosa alone or MC alone), up-regulated the expression of ICAM-1 and E-selectin. Moreover, the contribution of these two molecules to PMN TEM induced by MC supernatant was determined by using mAb (F(ab')2) to each of these molecules to pretreat HUVEC after stimulation with MC supernatant. Treatment of the HUVEC with mAb to ICAM-1 inhibited TEM by 53% (control migrated = 19.6 ± 0.6%; anti-ICAM-1 = 10.6 ± 1.0%), with anti-ICAM-1 + ICAM-2 mAbs by 63% (8.9 ± 0.9% migrated). With anti-ICAM-1 + ICAM-2 + E-selectin mAbs, migration was inhibited by 87.1% (4.8 ± 0.5% migrated; mean ± SD of triplicates) (unstimulated migration = 2.7%).



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  		FIGURE 4. Effect of P. aeruginosa-induced MC released mediators on endothelial ICAM-1 and E-selectin expression. HUVEC monolayers were incubated with medium or stimulated MC supernatants or IL-1{alpha} (0.5 ng/ml) for 4 h. The HUVEC were washed, and mAb to ICAM-1 (R6.5) or E-selectin (BB11) or control mAb (3H11 B9 anti-pertussis toxin) were used in a whole cell ELISA, as in Materials and Methods. Values are absorbance units ± SD of triplicates, minus value for control mAb (0.06). One representative experiment of two.

 
The counterligand on PMN for endothelial ICAM-1, ICAM-2, and possibly in part also for E-selectin is CD11/CD18 (5, 23). Therefore, PMN were treated with blocking mAb (IB-4) to CD18 of the CD11/CD18 heterodimer to assess the importance of this interaction. The anti-CD18 mAb blocked MC supernatant-induced PMN TEM by 85% (control = 27.2 ± 5.2% vs anti-CD18 = 3.6 ± 2.0%; n = 3), indicating that the CD11/CD18 complex is essential for MC-induced PMN recruitment in vitro.

IL-1{alpha}, IL-1{beta}, and TNF-{alpha} production by human MC after P. aeruginosa stimulation

The complete elimination of MC supernatant-induced PMN TEM by IL-1RA or by anti-IL-1{alpha} Ab plus anti-IL-1{beta} Ab indicates that IL-1{alpha} and IL-1{beta} are responsible for the activity. Thus, IL-1{alpha} and IL-1{beta} protein levels in the MC supernatants were determined by ELISA. The supernatant with the highest titer for inducing PMN TEM contained 311.7 pg/ml IL-1{alpha} and 398.8 pg/ml IL-1{beta}. As described above, this MC supernatant induced PMN TEM even after dilutions of 1/300, suggesting that MC-derived IL-1{alpha} and IL-1{beta} at ~1–1.3 pg/ml are able to induce PMN TEM. Such a potent effect led us to determine the minimum concentrations of rIL-1{alpha} and rIL-1{beta} required to induce PMN TEM. rIL-1{alpha} at concentrations of 50, 10, and 2 pg/ml induced 41.9, 11.0, and 1.3% of PMN TEM, respectively. rIL-1{beta} at 50, 10, and 2 pg/ml induced 37.5, 36.7, and 6.9% of PMN TEM, respectively. Baseline PMN TEM was 1.7%. Thus, the minimum concentration required to activate HUVEC for PMN TEM in our system is ~10 pg/ml (or between 10 and 2 pg/ml) for rIL-1{alpha}, a concentration slightly higher than that in MC supernatant after 1/300 dilution (1 pg/ml). The minimum concentration for IL-1{beta} for eliciting PMN TEM is 2 pg/ml, a concentration similar to that in the MC supernatant at 1/300 dilution (1.3 pg/ml). The following factors may likely contribute to a higher activity of IL-1{alpha} and IL-1{beta} in MC supernatant for PMN TEM: 1) other cytokines contained in this MC supernatant such as GM-CSF (549.6 pg/ml) and IL-6 (2428.0 pg/ml) may potentiate IL-1-dependent PMN TEM; 2) MC-derived IL-1{alpha} (and IL-1{beta}) vs recombinant proteins may likely possess different activities; and 3) potentially there may be a synergistic or additive effect of IL-1{alpha} and IL-1{beta} on stimulation of endothelium for PMN TEM.

To examine the time course of IL-1{alpha} and IL-1{beta} production by human MC after P. aeruginosa stimulation, we treated CBMC with P. aeruginosa strain 8821 for 3–48 h. As shown in Fig. 5Go, human MC produced both IL-1{alpha} and IL-1{beta} in a time course-dependent manner. Little or no IL-1{alpha} and IL-1{beta} were produced by unstimulated MC (<5 pg/ml).



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  		FIGURE 5. Production of IL-1{alpha} and IL-1{beta} by human MC in response to P. aeruginosa stimulation. a–d, CBMC (5 x 105 cells/ml) from two donors were stimulated with P. aeruginosa strain 8821 (1:50 cell-bacteria ratio) for various times. IL-1{alpha} and IL-1{beta} levels in cell-free supernatants were determined using ELISA. Error bars represent replicate. e–h, CBMC (5 x 105 cells/ml) after treatment with P. aeruginosa strain 8821 for 24 h were stained with mouse anti-human IL-1{alpha} Ab (e), control mouse IgG2a (f), mouse anti-human IL-1{beta} (g), or mouse IgG1 (h), as described in Materials and Methods. MC containing IL-1{alpha} or IL-1{beta} were stained purple in cytoplasm (e and g). Original magnification: x40. f, Insert is a flow cytometry analysis of CBMC stained with anti-c-kit FITC or mouse IgG1 FITC.

 
To test whether IL-1{alpha} and IL-1{beta} secretion can be induced by Fc{epsilon}RI-dependent mechanisms, CBMC were sensitized by human IgE (50 µg/ml; Chemicon International, Temecula, CA) overnight and stimulated for 24 h by anti-human IgE (rabbit serum, 1/200; DAKO Diagnostics Canada, Mississauga, Ontario, Canada). Interestingly, IL-1{alpha} was undetectable in CBMC supernatants from both anti-IgE-stimulated and normal rabbit serum (NRS, as control) samples. IL-1{beta} content was 7.8 and 9.0 pg/ml in anti-IgE and NRS samples, respectively, suggesting no induction of IL-1{alpha} and IL-1{beta} secretion from CBMC by anti-IgE stimulation, although significant GM-CSF secretion (158.9 pg/ml) was induced by anti-IgE (NRS control = 20 pg/ml). The mechanisms of differential IL-1 secretion induced by IgE and P. aeruginosa are unclear.

Production of IL-1{alpha} protein by human MC has not been reported previously. To verify that this cytokine detected is derived from MC, CBMC after P. aeruginosa treatment for 24 h were stained for IL-1{alpha} by immunocytochemistry. As shown in Fig. 5Goe, IL-1{alpha} was localized in human MC (39.5% positive). Similarly, IL-1{beta} was also seen in CBMC (Fig. 5Gog, 73.3% positive).

As shown above in the experiment using anti-TNF-{alpha} Ab, TNF-{alpha} in MC supernatant did not contribute to PMN TEM. However, a significant amount of TNF-{alpha} protein (120 pg/ml) was detected in the P. aeruginosa-MC supernatant used in Fig. 3Go. This led us to determine whether TNF-{alpha} in the supernatant is biologically active using L929 bioassay. The MC supernatant, which contained 120 pg/ml TNF-{alpha} as tested by ELISA, showed no cytotoxicity (data not shown), suggesting that the MC-derived TNF-{alpha} was biologically inactive. Furthermore, addition of this supernatant to rTNF-{alpha} to a level of 20 pg/ml (rTNF-{alpha} 10 pg/ml induced 50% L929 cell lysis) did not cause L929 cytotoxicity, indicating the presence of TNF-{alpha} inhibitor(s) in the MC supernatant.

Regulation of P. aeruginosa-induced cytokine production by IL-10 and dexamethasone

To examine whether IL-1{alpha}, IL-1{beta}, and TNF-{alpha} produced by P. aeruginosa-stimulated MC can be regulated, CBMC were incubated with various concentrations of IL-10 or dexamethasone during P. aeruginosa stimulation. As shown in Fig. 6Go, IL-10 dramatically abrogated P. aeruginosa-induced IL-1{alpha} and TNF-{alpha} production. At a concentration as low as 5 ng/ml, IL-10 significantly inhibited IL-1{alpha} and TNF-{alpha} production. Interestingly, P. aeruginosa-induced IL-1{beta} production was considerably more resistant to inhibition by IL-10 treatment. At the dose of 500 ng/ml, IL-10 showed 46–63% inhibition of IL-1{beta} production (Fig. 6Go).



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  		FIGURE 6. Effects of IL-10 on P. aeruginosa-induced production of IL-1{alpha}, IL-1{beta}, and TNF-{alpha} by human MC. CBMC (5 x 105 cells/ml) from two donors were stimulated with P. aeruginosa strain 8821 (1:50 cell-bacteria ratio) for 24 h in presence or absence of IL-10 (5, 50, or 500 ng/ml). Cell-free supernatants were harvested for determination of IL-1{alpha}, IL-1{beta}, and TNF-{alpha} by ELISA. Error bars represent replicate.

 
Similar to IL-10, dexamethasone at the concentration of 0.1, 1, and 10 µM strongly inhibited P. aeruginosa-induced IL-1{alpha} and TNF-{alpha} production (Fig. 7Go). P. aeruginosa-induced IL-1{beta} production was also significantly inhibited by dexamethasone at 1 and 10 µM, but at the lower dose (0.1 µM) effect on IL-1{beta} production varied between donors, suggesting that CBMC from different individuals may have different glucocorticoid sensitivity.



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  		FIGURE 7. Effects of dexamethasone on P. aeruginosa-induced production of IL-1{alpha}, IL-1{beta}, and TNF-{alpha} by human MC. CBMC (5 x 105 cells/ml) from two individual donors were stimulated with P. aeruginosa strain 8821 (1:50 cell-bacteria ratio) for 24 h in presence or absence of dexamethasone (0.1, 1, 10 µM). Cell-free supernatants were harvested for determination of IL-1{alpha}, IL-1{beta}, and TNF-{alpha} by ELISA. Error bars represent replicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
MC are well known for their heterogeneity in morphology and functions. Human MC differ from rodent MC in many features, including mediator composition and responsiveness to stimuli (14). To choose a model relevant to human diseases, human CBMC were used because these cells have a phenotype similar to that of human MC in the lung mucosa (24). Given that a direct interaction between human MC and P. aeruginosa has not been investigated previously, the phagocytosis of P. aeruginosa by human MC was examined. Electron microscopy showed that MC formed filapod-like structures and attached to bacteria, suggesting an active role of MC during these interactions. Bacterial colony-counting assay showed a progressive internalization of P. aeruginosa, but not L. pneumophila, suggesting a specific interaction between MC and P. aeruginosa.

P. aeruginosa infection is characterized by excessive PMN influx (4). To examine whether a direct MC-P. aeruginosa interaction would initiate a PMN-dominant inflammatory response, effects of P. aeruginosa-induced MC mediators on human PMN TEM were examined. Strikingly, supernatants harvested from CBMC following P. aeruginosa stimulation demonstrated potent stimulatory effects on human PMN TEM by activation of endothelium. Significant stimulation of PMN TEM was observed in P. aeruginosa-induced MC supernatant even after 300 times dilutions. Such a potent effect suggests a pathophysiologically important role of the MC during P. aeruginosa infection. This concept is supported by the finding that P. aeruginosa lung infection induces an increase in the number of MC in the airway (25, 26).

MC are able to produce a plethora of mediators, including those preformed such as histamine and those newly synthesized such as lipid mediators, cytokines, and chemokines. In human MC, a broad spectrum of cytokines (up to 25 of them) has been identified, including TNF-{alpha} and IL-1{beta} (15, 27). Although there is no direct evidence demonstrating the production of IL-1{alpha} by human MC, this cytokine has been closely associated with human MC activation (28). In this study, by using immunocytochemistry staining, IL-1{alpha} protein was localized in P. aeruginosa-treated human MC. It is important to recognize that MC do not produce all these factors simultaneously upon activation. The specific role of MC depends on the selective production of specific MC mediators in certain conditions. In rodents, MC secrete histamine after P. aeruginosa stimulation (29, 30). However, current knowledge of MC cytokine production in the context of bacterial infection is extremely limited. In humans, TNF-{alpha} and IL-6 are the only previously reported cytokines produced by human MC after bacterial infection (17, 31, 32). We attempted to determine those P. aeruginosa-induced MC mediators responsible for enhanced human PMN transmigration across endothelium.

Given that IL-1{alpha}, IL-1{beta}, and TNF-{alpha} are produced during P. aeruginosa infection and that these cytokines can induce expression of adhesion molecules on endothelium to enhance PMN transmigration, their role in MC-induced PMN TEM was evaluated. The results of IL-1R blockade, using IL-1RA treatment of HUVEC as well as the Ab neutralization of IL-1{alpha} and IL-1{beta} in the supernatant of P. aeruginosa-stimulated CBMC, clearly showed that IL-1{alpha} and IL-1{beta} are the cytokines activating HUVEC for supporting the PMN TEM. Because the partial inhibition of PMN TEM by Ab to IL-1{beta} was further enhanced to a complete inhibition by adding Ab to IL-1{alpha}, these two cytokines produced by MC may have a synergistic role in mediating PMN TEM. These data are supported by a recent report that prolonged elevation of IL-1 expression in P. aeruginosa infection contributes to tissue destruction by continued stimulation of PMN infiltration (33). Consistent with this concept, IL-1R knockout mice demonstrated diminished PMN infiltration in the lung after P. aeruginosa infection (34). Production of IL-1{alpha} and IL-1{beta} proteins by human CBMC following P. aeruginosa stimulation was demonstrated in a time course-dependent manner. Although human MC have been reported to be able to produce IL-1{beta} (15, 27) and have been associated with IL-1{alpha} (28), this is the first direct demonstration of the biologically relevant functional role of MC-derived IL-1{alpha} and IL-1{beta} in contributing to the response to the P. aeruginosa pathogen.

In contrast to the potent induction of PMN TEM by IL-1{alpha} and IL-1{beta} in the MC supernatant, the MC-derived TNF-{alpha} seems not to contribute to the induction of PMN TEM, although CBMC did produce this cytokine after P. aeruginosa stimulation. A separate bioassay for TNF-{alpha} activity (L929 cytotoxicity) demonstrated that MC-derived TNF-{alpha} was biologically inactive and that the activity of rTNF-{alpha} was inhibited upon addition of MC supernatant. Thus, human MC after P. aeruginosa stimulation may also produce TNF-{alpha} inhibitors, most likely soluble TNF-{alpha} receptors. This was unexpected because in mice, MC-derived TNF-{alpha} was shown to be essential for PMN recruitment in some conditions (11, 12, 13). However, a role of MC-derived TNF-{alpha} has not been tested in P. aeruginosa-induced inflammation in mice. In addition, MC receptors involved in P. aeruginosa-induced activation have not been characterized. It is possible that different bacterial strains may elicit distinct inflammatory responses through different host receptors on MC. For example, bacterial-induced activation of Toll-like receptor (TLR) 2, which plays a major role in Gram-positive bacterial recognition (35), induced a distinct cytokine profile from MC, including IL-4, IL-5, TNF-{alpha}, but not IL-1{beta} (36). In contrast, TLR4, which has a significant role in Gram-negative bacterial recognition, mediated a separate cytokine profile from murine MC, including IL-1{beta} and TNF-{alpha}, but not IL-4 and IL-5 (36). Whether different MC phenotypes express different TLRs and produce distinct cytokine profiles (such as different TNF-{alpha}, IL-1{alpha}, and IL-1{beta} production) is unclear. Thus, the MC receptors involved in P. aeruginosa recognition and the in vivo significance of MC-derived TNF-{alpha}, IL-1{alpha}, and IL-1{beta} during P. aeruginosa lung infection require further study.

Adhesion molecules involved in MC-dependent human neutrophil transendothelial migration have not been investigated previously. In this study, the supernatants from P. aeruginosa-stimulated MC markedly up-regulated ICAM-1 and E-selectin on HUVEC. The essential role of these adhesion molecules in the MC-induced PMN TEM is shown by significant inhibition (87%) of TEM by mAb blocking on the HUVEC of ICAM-1, ICAM-2, and E-selectin. ICAM-1 is an endothelial ligand for PMN CD11/CD18 integrins (5, 23), and E-selectin under static conditions may also bind CD11/CD18 (37) and contribute to CD11/CD18-dependent PMN TEM (20, 38). In keeping with the ICAM-1 and E-selectin dependency of observed PMN TEM, blockade of CD11/CD18 on PMN with mAb dramatically reduced MC-induced PMN TEM, suggesting a major role for CD11/CD18 in this migration, as seen in acute P. aeruginosa lung infection (39). Depending upon the nature of inflammation and the tissues, distinct mechanisms of adhesion molecule utilization may be involved in PMN recruitment (5, 21). For example, PMN accumulation during acute Escherichia coli- and P. aeruginosa-involved pneumonia is CD11/CD18 dependent (39, 40), but not during Streptococcus pneumoniae infection (5, 6, 41). Our findings are consistent with the hypothesis that P. aeruginosa-activated MC produce IL-1{alpha} and IL-1{beta}, which stimulate endothelial cell ICAM-1 and E-selectin expression required for PMN TEM.

To determine whether P. aeruginosa-induced MC IL-1{alpha} and IL-1{beta} can be regulated, dexamethasone and IL-10 were chosen because corticosteroids and IL-10 have been directly or indirectly implicated in P. aeruginosa infection (42, 43, 44, 45, 46). We demonstrated that dexamethasone markedly inhibited P. aeruginosa-induced IL-1{alpha}, IL-1{beta}, as well as TNF-{alpha} production by human MC. Similarly, IL-10 dramatically blocked P. aeruginosa-induced IL-1{alpha} and TNF-{alpha} production. Although IL-10 resembles many effects of dexamethasone in other systems (47, 48), it had limited effect on P. aeruginosa-induced IL-1{beta} production by MC. The differential effects of IL-10 on IL-1{alpha}, TNF-{alpha}, and IL-1{beta} suggest a potential selective modulation of MC cytokine production. However, the mechanisms of dexamethasone- and IL-10-induced inhibition of P. aeruginosa-stimulated cytokine production by CBMC remain to be determined. Although dexamethasone is able to induce apoptosis in certain cell types and indeed dexamethasone-mediated decrease of MC numbers in vivo is most likely via induction of MC apoptosis (49), it is not clear whether dexamethasone-induced inhibition of MC cytokine production involves an apoptotic process.

Dysregulation of IL-10 has been implicated in host response to a range of infectious pathogens including P. aeruginosa (43, 44, 45). Several animal studies indicated that endogenous IL-10 deficiency is associated with an increased inflammatory response to P. aeruginosa (43, 45). Exogenous IL-10 attenuated lung injury and improved lung function and survival in P. aeruginosa pneumonia in mice (43). The inhibitory effects of IL-10 on IL-1{alpha} and TNF-{alpha} production in this study are consistent with the concept that exogenous IL-10 may be beneficial to the host during P. aeruginosa infection (43, 45). This is the first demonstration using a human system that P. aeruginosa-induced proinflammatory cytokine production is down-regulated by IL-10. Thus, it may be feasible to use anti-inflammatory agents such as IL-10 or dexamethasone to improve P. aeruginosa-associated inflammation in humans. Indeed, corticosteroids have demonstrated a beneficial effect on pulmonary function in CF patients with chronic P. aeruginosa infection (46). However, the multiple targets other than MC of IL-10 and dexamethasone may not always be beneficial should the IL-10 or dexamethasone overly suppress normal host defenses (42, 44). The adverse effects of corticosteroids due to their nonspecific immunosuppressive actions limit their application in CF patients (50). Therapeutic alternatives are sought to reduce inflammation in CF (50). IL-1RA has been used for the treatment of patients with rheumatoid arthritis (51). The importance of IL-1 in MC-dependent PMN TEM in our study, together with the fact that corticosteroid-induced decrease of IL-1 levels in P. aeruginosa-infected CF patients is associated with improved lung functions (52), suggest that IL-RA may potentially be used to reduce P. aeruginosa-induced inflammation in CF. Our findings support a novel concept of focusing on the MC as a potential therapeutic target for regulation of P. aeruginosa-induced adverse inflammation.


   Acknowledgments
 
We thank Patricia Colp for her excellent technical assistance in immunohistochemical staining, and Elizabeth Garduno for her skillful help in preparation of bacterial cultures.


   Footnotes
 
1 This work was supported by grants from Canadian Cystic Fibrosis Foundation, Canadian Institutes of Health Research (ROP 44944), and Izaak Walton Killam Health Center (to T.-J.L.), and Grant MOP-7684 from Canadian Institutes of Health Research (to A.C.I.). T.-J.L. is the recipient of an Investigatorship Award from the Izaak Walton Killam Health Center. R.T.M.B. is the recipient of a trainee award from Cancer Care Nova Scotia. Back

2 Address correspondence and reprint requests to Dr. Tong-Jun Lin, Izaak Walton Killam Health Center, Department of Pediatrics, 5850 University Avenue, Halifax, Nova Scotia, Canada, B3J 3G9. E-mail address: tong-jun.lin{at}dal.ca Back

3 Abbreviations used in this paper: CF, cystic fibrosis; CBMC, human umbilical cord blood-derived MC; HSA, human serum albumin; IL-1RA, IL-1R antagonist; MC, mast cell; NRS, normal rabbit serum; PMN, neutrophil; TEM, transendothelial migration; TLR, Toll-like receptor. Back

Received for publication April 12, 2002. Accepted for publication August 15, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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