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Division of Gastrointestinal Pathology,
Department of Pathology, and
Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, and
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The Harvard Digestive Diseases Center, Boston, MA 02115
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recent studies show that S. typhimurium contact with the apical pole of intestinal epithelial cells generates signal(s) that may be responsible for directing the trafficking of neutrophils across the intestinal epithelium. For example, we have previously demonstrated, using in vitro models of intestinal inflammation, that apical attachment of S. typhimurium to intestinal epithelial monolayers spontaneously stimulates physiologically directed PMN transepithelial migration. The signals responsible for orchestration of this inflammatory response do not utilize PMN n-formyl peptide receptor-directed migration (6), the best-understood, bacterial-derived, receptor-mediated pathway for directing PMN to a bacterial target. Among the events stimulated by such pathogen-host interactions is the release of chemotaxins that might guide PMN to the site of bacterial-epithelial contact (5, 6, 7, 8). For example, S. typhimurium-intestinal epithelial cell interactions induce the epithelial synthesis and polarized basolateral release of the potent neutrophil chemotactic peptide IL-8 (4, 5, 6, 7). However, while such basolateral secretion of IL-8 can direct PMN through the underlying matrix and to a subepithelial position via matrix imprinting (4), basolateral release of this chemokine is insufficient to induce migration across the epithelium (6). This later finding is not surprising given that IL-8 released from the basolateral surface of the epithelium results in an inappropriately directed (basolateral > apical) gradient to support transepithelial movement of PMN. Additionally, we recently provided evidence that transepithelial signaling events elicited by S. typhimurium-apical membrane contact represents a key virulence mechanism by which enteritis occurs in vivo (5). Here we show that S. typhimurium interactions with the apical plasma membrane of intestinal epithelial cells also elicit apically polarized secretion of a unique bioactivity with potent PMN chemotactic activity. Transfer studies indicate that this apically secreted chemoattractant is highly efficient in driving PMN migration across the epithelium (the final step of transepithelial migration).
In this study we examine the physical/biologic characteristics of PEEC
(pathogen-elicited epithelial chemoattractant). The
distinguishing features of PEEC indicate that it is relatively small
(nominally 1–3 kDa; unlike many known chemokines), stable, not highly
hydrophobic, and does not appear to signal via the n-formyl
peptide or IL-8 receptors. Like most neutrophil chemoattractants, PEEC
induces a PMN signal transduction cascade involving a GTP binding
protein (G
i) that also elicits a rise in
[Ca2+]i. In addition, bioassays reveal that
PEEC directly signals PMN but, also unlike known chemoattractants, PMN
respond to PEEC with essentially a purely chemotactic response in which
degranulation or superoxide generation is virtually not detectable,
even at saturating concentrations of PEEC bioactivity. Importantly,
these data predicate that epithelial cells can respond to surface
pathogens such as Salmonella by releasing distinctive
chemoattractants that sequentially orchestrate movement of PMN into the
subepithelial compartment by matrix imprinting (i.e., IL-8), and as
shown in this report, subsequently guide PMN across epithelia into the
luminal compartment (i.e., PEEC).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T84 intestinal epithelial cells (passages 70–95) were grown and maintained as confluent monolayers on collagen-coated permeable supports (9) with recently detailed modifications (10). Monolayers were grown on 5-cm2 and 0.33-cm2 suspended polycarbonate filters (Costar Corp., Cambridge, MA) and utilized 7 to 14 days after plating, as described previously (10). A steady-state resistance (approximately 1500 ohm cm2) is reached in 5 days with variability largely related to cell passage number. Inverted monolayers used to study transmigration of neutrophils in the physiologic basolateral-to-apical direction were constructed as described before (10, 11, 12).
Bacterial strains and growth conditions.
S. typhimurium 
3306 is a naladixic acid-resistant
(gyrA1816) strain derived from S.
typhimurium strain SR-11 (13). Luria broth was made as previously
described by Revel (14). L agar is Luria broth containing 12 g of
Bacto Agar (Difco Laboratories, Detroit, MI) per liter. Bacterial
growth conditions were as follows: nonagitated microaerophilic
bacterial cultures were prepared by inoculating 10 ml of Luria broth
with 0.01 ml of a stationary-phase culture followed by overnight
incubation (approximately 18 h) at 37°C, as previously
detailed (6, 15).
Transcellular chemotactic factor (PEEC) collection.
S. typhimurium interactions with polarized T84 apical
surfaces were as follows: to harvest PEEC bioactivity, polarized T84
monolayers grown on 5-cm2 inserts were subsequently
infected by S. typhimurium suspended in HBSS+
buffer, performed by the method of McCormick et al. (6), as shown in
Figure 1
A, with a slight
modification. Briefly, T84 monolayers (10, 11, 12) were extensively rinsed
in HBSS+ (containing Ca2+ and Mg2+,
with 10 mM HEPES, pH 7.4 (Sigma Chemical Co., St. Louis, MO)) to remove
residual media and serum components. Approximately 5 x
108 bacteria in 1.0 ml HBSS+ was gently
distributed onto the apical surface and incubated for 45 min at 37°C.
Nonadherent bacteria were subsequently removed from the monolayers by
extensive washing (four times with 4.0 ml of buffer/wash) and were then
transferred into the six-well tissue culture tray containing
HBSS+ buffer in each lower (basolateral) and upper (apical)
reservoir (apical surface now colonized with S.
typhimurium). Following an incubation of 2 h at
37°C, both the apical and basolateral supernatants were collected,
filtered through a 0.2-µ filter, and stored at -80°C until further
use. In the subset of experiments to determine the time course of PEEC
secretion, T84 monolayers were colonized by S. typhimurium,
and subsequent to the removal of nonadherent bacteria, were incubated
in HBSS+ buffer for 2 h and the apical supernatant was
removed. Fresh buffer was then added to these same monolayers, and
incubated for an additional 2 h (4 h total). The apical
supernatants were collected, filtered through a 0.2-µm filter, and
stored at -80°C until further use. This cycle was repeated for an
additional 2 h, corresponding to the 6-h time interval.
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B, the
PEEC bioactivity of the collected supernatants was tested by placing
harvested material (300–500 µl) in the lower (apical) compartment.
Positive control transmigration assays were performed by imposing
chemoattractant gradients (10-7 M FMLP) favoring
basolateral-to-apical migration across inverted monolayers. At the end
of each experiment PMN transmigration was quantified (as cell
equivalents; CE) by assaying for the PMN-specific azurophilic granule
marker myeloperoxidase as described previously (18). PMN (CE), were
assessed as the number of PMN that had completely traversed the
transepithelial monolayer (into the lower reservoir). Underagarose chemotaxis assay. The underagarose assay was used to assess chemotaxis in the absence of an epithelial monolayer as detailed previously (19) using human peripheral blood PMN. Briefly, a mixture of 1% agarose (Sigma Chemical Corp.) and 0.5% gelatin (Life Technologies, Grand Island, NY) was solidified on 60-mm petri dishes (6 ml/plate). Eight sets of three 3-mm wells were punched in the plates with wells spaced 3 mm apart. Plates were preincubated with chemoattractant for 45 min at 37°C. Subsequently, addition of fresh chemoattractant, buffer controls, and purified neutrophils (10 µl at 108/ml) were added to the corresponding wells of the migration plates. Plates were incubated for 120 min at 37°C in a humid chamber and fixed with 5% glutaraldehyde overnight. After removal of agarose gels, plates were rinsed and air dried. PMN migration was measured microscopically by the leading front of migrating PMN (18) utilizing an ocular micrometer at x400. From each plate, directed migration and random migration were measured. Data are presented as a ratio (chemotactic index).
Analysis of PEEC physical characteristics. To estimate the nominal m.w. of PEEC, the supernatants were passed through a series of filters with defined m.w. exclusion (10,000, 3,000, and 1,000; YM10, YM3, and YM1, respectively) using an Amicon filtration unit (model 8400; Amicon Inc., Beverly, MA). Each filter retentate (supernatant remaining above the filter) and filtrate (supernatant that passed through the filter) was collected and tested for the ability to elicit PMN transepithelial migration. Unless otherwise indicated, PEEC retained in the 1000-retentate fraction was concentrated 20-fold by volume. This volume concentration step was necessary since a larger amount of HBSS+/cm2 is required to accommodate the transwell system.
PEEC stability. 1) Temperature sensitivity: to determine whether the PEEC bioactivity was heat stable, PEEC was heated to 100°C for 20 min, and subsequently examined for the ability to induce PMN transepithelial migration. 2) Freeze/thaw: the factor was subjected to three freeze/thaw cycles and then tested for the ability to elicit PMN transepithelial migration. In addition, supernatants were vacuumed dried, resuspended to the initial volume from which they were obtained in HBSS+, and then judged for the ability to promote PMN transepithelial migration. 3) Trypsin/proteinase K sensitivity: PEEC was treated with 40 µg/ml of trypsin (Sigma Chemical Corp.) for 2 h at 37°C, and then boiled for 15 min to inactivate the enzyme. The vehicle control included PEEC in the absence of trypsin but was treated for 2 h at 37°C, followed by 15 min at 100°C. Further, PEEC was treated with 10 U proteinase K (Sigma Chemical Corp.) at 37°C for 60 min. The enzyme was deactivated by boiling for 15 min (a manipulation that does not effect PEEC activity; see Results). The vehicle control consisted of PEEC in the absence of proteinase K treatment, but included heat treatment at 37°C for 60 min followed by boiling for 15 min. Both PEEC-trypsin- and proteinase K-treated samples were tested for their ability to elicit PMN transepithelial migration.
Phase separation/solubility studies. In subsets of experiments, PEEC was precipitated with two parts of absolute ethanol at 4°C for 30 min, and centrifuged at 10,000 x g for 20 min (11). The resulting supernatant and precipitate was individually processed to determine which fraction contained the bulk of bioactivity when tested for neutrophil transepithelial migration. The precipitate and the supernatant, following vacuum drying, were resuspended to the initial volume from which they were obtained in HBSS+.
Hydrophobic extraction. To determine whether PEEC was highly hydrophobic, we employed a Sep-Pak extraction procedure (Waters Corp., Milford, MA). Concentrated (50-fold) PEEC was added to two parts of absolute ethanol and placed at 4°C for 30 min. After centrifugation (2000 rpm), the supernatant was collected in a round-bottom flask, and was dried down in a rotary evaporation device. The residue was next resuspended in 200 µl of methanol followed by 10 ml of distilled water. The sample was passed through an activated Sep-Pak column (Waters Corp.) and washed with distilled water. Any hydrophobic compounds within the PEEC fraction were extracted with hexane followed by recovery of residual components from the PEEC fraction with less hydrophobic solvents. The solvents obtained were dried under CO2 and vacuum via rotary evaporation; the dried sample was resuspended in 300 µl of absolute ethanol, buffer exchanged into HBSS+, and stored at -80°C for subsequent bioassay of PEEC activity.
Receptor sensitivity. 1) Neutrophil IL-8 receptor A: neutralizing mAbs against the PMN IL-8 receptor (IL-8 receptor A) (a kind gift of J. Kim, Genentech, San Francisco, CA) were utilized via a slight modification by Chuntharapai (20) and described in McCormick et al. (4). Briefly, 50 µg/ml of the Ab was added to 5 x 107 PMN and the neutrophil-mAb mixture was gently rotated at 4°C for 90 min to prevent settling of the PMN. PEEC was added to the lower (apical) reservoir 15 min before the addition of 1 x 106 neutrophils to the upper reservoir of polarized T84 epithelial monolayers. Following a 120-min incubation period at 37°C, neutrophils were assessed for their ability to migrate across the epithelial monolayer. 2) Neutrophil FMLP receptor: N-t-BOC-1-methionyl-1-leucyl-1-phenylalanine (tBOC-FMLP), an n-formyl peptide receptor antagonist, was purchased from Sigma Chemical Corp. Human peripheral blood neutrophils (5 x 107/ml) were preincubated in the presence or absence of 300 µM t-BOC-FMLP for 5 to 10 min at 37°C. PEEC was added to the lower reservoir 15 min before the addition of PMN (40 µl or 106), which were added to the basolateral (upper) reservoir of T84 cell inverted monolayers. Positive controls included addition of imposed gradients of 10-7 M FMLP placed in the lower reservoir.
Pertussis toxin sensitivity. A total of 1 x 107 PMN were treated with 2 µg/ml of pertussis toxin (BioMol, Plymouth Meeting, PA) for 90 min at 37°C under static (nonagitated) conditions. Vehicle control conditions incorporated treatment of PMN at 37°C for 90 min in the absence of 2 µg/ml of pertussis toxin. PEEC was added to the lower reservoir 15 min before the addition of pertussis-treated or control PMN (20 µl or 106/well), which were added to the basolateral (upper) reservoir of T84 cell inverted monolayers. Positive control conditions included addition of imposed gradients of 500 ng/ml IL-8 placed in the lower reservoir, which were established 15 min before the addition of pertussis-treated or control PMN.
Bacterial and epithelial protein synthesis inhibition. After apical colonization of S. typhimurium (5 x 108/ml/well; 45 min, 37°C) to T84 intestinal epithelial cell monolayers grown on collagen-coated 5-cm2 inserts (Costar), the monolayers were washed free of nonadherent bacteria. To inhibit bacterial protein synthesis, S. typhimurium-colonized monolayers were treated with 100 µg/ml of chloramphenicol (CAM), a bacteriostatic protein inhibitor that does not similarly effect eukaryotic cells, for 1 h at 37°C (21, 6). Monolayers were then gently washed twice in HBSS+ buffer and added to fresh plates containing physiologic HBSS+ buffer at 37°C. Collection of PEEC was performed as described above, following a 2-h and 4-h time course. In a similar subset of experiments, following apical colonization of S. typhimurium to polarized T84 cell monolayers, the monolayers were treated with 2 µg/ml of cycloheximide (CHX; Sigma Chemical Corp.) for 1 h at 37°C. CHX, an inhibitor of eukaryotic but not prokaryotic protein synthesis, was prepared in a 2-mg/ml stock in 95% ethanol and diluted to 2 µg/ml in HBSS+ buffer. This concentration has been found to inhibit 75% of radiolabeled leucine incorporation into T84 cell-precipitable protein (22). Monolayers were then gently washed twice and added to a fresh plate containing HBSS+ physiologic buffer at 37°C. Collection for PEEC, as described above, was performed following a 2-h and 4-h time course. Addition of either protein synthesis inhibitor, CAM or CHX, to T84-polarized monolayers did not influence monolayer barrier function as assessed by transepithelial cell resistances, or agonist-induced Cl-secretion (6).
Direct effects of PEEC on PMN function.
Calcium influx: human peripheral blood neutrophils at a density of
1.5 x 106 (30 µl of 5 x
107/ml stock) were attached to acetone-washed glass
coverslips for 15 min at room temperature. Nonadherent PMN were then
washed three times with normal buffer solution (140 mM NaCl, 5 mM KCl,
2.5 mM CaCl2, 1.1 mM MgCl2, 2.6 mM dextrose, 10
mM HEPES, pH 7.4) and loaded with fura 2-AM (final concentration 7.5
µM) for 1 h at room temperature. The fura-loaded PMN were washed
once in normal buffer solution, cooled on ice, and challenged with
saturating concentrations (determined by initial peak of dose-response
curve) of various chemoattractants that included n-FMLP
(10-6 M; Sigma), IL-8 (500 ng/ml; R&D systems), and the
test sample, PEEC (200-fold concentrated by volume representing the
saturating concentration of this chemoattractant; see
Results). Control buffer in this experiment is
HBSS+-conditioned with T84 cells for 2 h at 37°C.
Measurements were performed in a microscopic spectrofluorometer (Photon
Technology International, model 4000; S. Brunswick, NJ).
[Ca2+]i (in nM) was calculated according to
the following equation:
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Neutrophil superoxide assays. Superoxide generation from human PMN was measured as the superoxide dismutase-inhibitable reduction of cytochrome c as previously described (24), with some modifications. Assays were performed in a 96-well microtiter plate with each well containing 0.2 ml final volume. To each well was added a small volume of agonist including FMLP (2 µl of a 10-µM stock in HBSS; final concentration 100 nM), PMA (1 µl of a 1-mg/ml stock in DMSO; final concentration 500 ng/ml), IL-8 (10 µl of a 14-µM stock in HBSS; final concentration 0.7 µM), PEEC (100 µl of a 200x filtrate in HBSS), or conditioned HBSS (control; 100 µl). The volume of each well was adjusted to 100 µl with HBSS followed by the addition of 100 µl of cytochrome c buffer (1.25 mg cytochrome c (Sigma; horse heart type VI) per ml of HBSS) containing 1 x 106/ml PMN. Matching wells were prepared that also contained superoxide dismutase and catalase at a final concentration of 125 µg/ml (Calbiochem (La Jolla, CA) and Sigma, respectively). For cytochalasin preactivation, PMN were resuspended in cytochrome c buffer, and were treated with 7 µg/ml of dihydrocytochalasin B for 3 min (20°C) before initiation of the assay. Assays were initiated by the addition of PMN in cytochrome c buffer (20°C) and the OD at 550 monitored kinetically in a Molecular Devices Ultra Violet (UV; Menlo Park, CA) max microtiter plate reader for 10 min. From the reduced minus oxidized difference absorbance values, superoxide was quantified assuming an extinction coefficient of 1.85 x 104 L · M-1·cm-1 for cytochrome c and a path length of 0.5 cm.
Neutrophil degranulation assays. Effects of PEEC on primary (myeloperoxidase) and secondary (lactoferrin) granule release were studied as follows. To separate microcentrifuge tubes were added a small volume of agonist including FMLP (1 µl of a 100-µM stock in HBSS+; final concentration 1 µM), PMA (10 µl of a 10-µg/ml stock in HBSS+; final concentration 500 ng/ml), IL-8 (10 µl of a 14-µM stock in HBSS+; final concentration 0.7 µM), LTB4 (leukotriene B4; 1 µl of a 10-4-M stock in DMSO; final concentration 0.5 µM), PEEC (100 µl of a 200x filtrate in HBSS+), or conditioned HBSS+ (control; 100 µl). The volume of each well was adjusted to 100 µl with HBSS+. Assays were initiated by the addition of 100 µl of HBSS containing 6 x 106/ml PMN. For cytochalasin preactivation, PMN at 6 x 106/ml in HBSS+ were treated with 7 µg/ml of dihydrocytochalasin B for 3 min (20°C) before initiation of the assay. After 20 min (20°C), assays were terminated by microcentrifugation and cellfree supernatants were harvested. Supernatants were then assayed for lactoferrin (ELISA) and myeloperoxidase (enzymatic) exactly as previously described (24). The total myeloperoxidase content of PMN was estimated by solubilizing an equivalent number of PMN in HBSS containing 0.5% Triton X-100.
Data presentation. Neutrophil isolation was restricted to 10 different donors (repetitive donations) over the course of these studies. Myeloperoxidase assay (i.e., neutrophil transmigration) data were compared by Student’s t test. Neutrophil transmigration results are represented as neutrophil CE derived from a daily standard neutrophil dilution curve. Neutrophils that completely traverse the filter are represented as the number of neutrophils CE/ml. Values are expressed as the mean and SD of an individual experiment done in triplicate repeated n times.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As depicted in Figure 2, apical
attachment of S. typhimurium to T84 intestinal epithelial
monolayers elicits lumenally directed PMN transepithelial migration
(6). However, unlike transepithelial migration driven by imposed
gradients of the model n-formyl peptide FMLP or by human
rIL-8, Salmonella-elicited transepithelial migration is not
affected by antagonists of the n-formyl peptide or IL-8
receptors. These observations suggested that this model pathogen-host
interaction may trigger the release of a PMN chemoattractant that
signals via an alternative receptor. Given the physiologic
basolateral-to-apical direction of induced PMN migration, the simplest
explanation would be apical release of a chemoattractant, thus
resulting in an appropriate transepithelial gradient.
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We next sought to determine whether a bioactivity with properties
that would stimulate basal-to-apically directed PMN transepithelial
migration was released apically as a result of
Salmonella-epithelial apical membrane interactions.
Supernatants were collected from either the apical (lumenal) or
basolateral (subepithelial) compartments of polarized epithelial
monolayers with apically attached S. typhimurium (unattached
bacteria removed before assay). To test for bioactivities contained in
these conditioned supernatants that might explain the PMN
transmigration described above, these supernatants were transferred to
the apical or basolateral compartments of unmanipulated epithelial
monolayers. Spontaneous basolateral-to-apical PMN transmigration was
then assessed to determine whether a soluble chemoattractant
bioactivity was contained in the supernatants and, if so, to assess the
sidedness of release of this bioactivity. As shown in Figure 3, supernatants isolated from the apical
compartment of Salmonella-colonized monolayers induced
transepithelial migration of PMN when transferred to the apical
compartment of unmanipulated monolayers. In contrast, other transfers
(basolateral supernatant to apical compartment, apical supernatant to
basolateral compartment, basolateral supernatant to basolateral
compartment) did not result in PMN transepithelial migration.
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We next tested whether this soluble, transferable PEEC was, as found
for the spontaneous PMN migration across
Salmonella-colonized monolayers (Fig. 2), also independent
of the PMN n-formyl peptide and IL-8A receptor signaling
pathways. As shown in the inset to Figure 3, antagonists for these
receptors (that were effective in inhibiting transepithelial migration
by imposed n-formyl peptide or human rIL-8 gradients FMLP,
as shown in Fig. 2), did not affect transmigration in response to
apical transfer of PEEC. Similar findings occurred when neutralizing
polyclonal Abs to IL-8 (30 µg/ml, sufficient to inhibit responses to
imposed gradients of human rIL-8) were utilized (data not shown). Taken
together, these data indicate that, as a result of S.
typhimurium-apical epithelial associations, a soluble bioactivity
is released into the apical compartment. This bioactivity is sufficient
to induce spontaneous transepithelial migration in the physiologic
direction and, like transmigration in Salmonella-colonized
monolayers, is independent of PMN IL-8A and n-formyl peptide
receptors. Furthermore, although the size of the PMN transmigration
response varied considerably with individual donors, for any given
experiment the apically transferred signal generally reproduced 20 to
40% of the transmigration response.
To determine whether Salmonella-derived LPS could influence the bioactivity defined as PEEC, we measured the levels of LPS in the apical supernatants of monolayers that were surface colonized by Salmonella. Samples containing PEEC had LPS levels of <1.25 endotoxin units/ml. This level of endotoxin is not dissimilar from that trace amount found in HBSS+ buffer controls (<1.25 endotoxin units/ml). Moreover, purified LPS from S. typhimurium failed to stimulate neutrophil transepithelial migration when examined across a three-log dose response, 0.1 µg/ml to 10 µg/ml (0.37 ± 0.027, 0.383 ± 0.028, 0.374 ± 0.021, vs 0.421 ± 0.350 and 19.83 ± 3.0 for 0.10 µg/ml, 1.0 µg/ml, 10.0 µg/ml LPS vs negative buffer control and 10-7 M FMLP, respectively). The dose range of LPS utilized represents the average concentration of LPS estimated from bacterial densities corresponding to conditions that the monolayers are exposed to during infection, and include both a one-log higher and one-log lower density than that that occurs at the monolayers. Likewise, isolated S. typhimurium outermembranes were also ineffective at inducing neutrophil transepithelial migration (data not shown). Thus, LPS does not appear to account for the bioactivity observed. Additionally, as will be shown below, PEEC bioactivity can be captured in a low molecular mass filtrate that excludes LPS (a large macromolecular structure with a weight >100,000 kDa). Thus, LPS is not required to obtain transferable PEEC bioactivity.
Production of apical PEEC requires Salmonella-T84 epithelial contact
We next determined whether S. typhimurium-epithelial
apical contact, or merely apical exposure to soluble S.
typhimurium products was required for production of transferable
PEEC bioactivity. Exposure of monolayer apical membranes to S.
typhimurium-soluble products obtained from bacterial densities
ranging from 5 x 106 to 5 x 107
bacteria/ml is shown in Figure 4. Such
S. typhimurium-soluble products were obtained following a
2.5-h incubation in HBSS+ (conditions similar to those that
S. typhimurium encounters in the transmigration assay but
with the absence of T84 epithelial cell and neutrophils). Such ranges
in bacterial densities reflect the conditions to which monolayers are
exposed during colonization, and include at least a one-log higher
density than that that occurs at the monolayer surface once nonattached
bacteria are washed away (as is the standard for the assay). Thus,
utilizing such bacterial-soluble products under conditions that
prevented direct Salmonella-epithelial contact did not
result in spontaneous PMN transepithelial migration (0.255 ±
0.029 and 1.16 ± 1.06 CE x 104 vs 0.432 ±
0.20 for 5 x 106 and 5 x 107
bacteria/ml vs negative control, NS). In addition, attempts to isolate
PEEC from cultured S. typhimurium in Luria broth at the same
densities as above were unsuccessful (data not shown). Before the
addition of S. typhimurium to the T84 cell monolayers, the
bacteria are washed free of Luria broth and resuspended in
HBSS+. Thus, taken together, these data indicate that in
the absence of direct Salmonella-epithelial contact, release
of PEEC bioactivity into the apical reservoir does not occur.
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As shown in Figure 5A,
PEEC bioactivity passes through a 3000 nominal m.w. cut-off filter, but
is retained above a 1000-nominal-m.w. cut-off filter, strongly
suggesting that the m.w. characteristics of this soluble
chemoattractant are between 1000 and 3000 Da. That measurable amounts
of bioactivity were not found in the 3000-m.w. cut-off retentate was
not surprising given that neat apical supernatants similarly did not
elicit significant bioactivity and, additionally, greater than 95% of
the sample with m.w.s less than 3000 were passed through this filter
and concentrated in the 1000-m.w. cut-off retentate. Thus, only
concentrated sample from the 1000-m.w. cut-off retentate elicited the
ability to induce neutrophil transepithelial migration. Additionally,
subsequent dilutional analysis of such apical supernatants, as shown in
Figure 5B, failed to show a promotion of chemotactic
activity in the transepithelial migration assay, indicating that the
concentration of PEEC isolated (20x concentrated by volume) did not
induce tachyphylaxis. Moreover, the concentration of PEEC isolated by
this procedure is sufficiently low enough and pure enough that,
according to a 280-UV scan, no material was detected. Importantly,
these filtration characteristics also permit semipurified PEEC activity
to be isolated and thus concentrated for dose-response studies outlined
below and, additionally, permit buffer exchange and washing of PEEC
bioactivity. Subsequent experiments use this semipurified form of PEEC
concentrated 20x unless otherwise indicated.
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C, the resultant concentrated basolateral fractions were
again ineffective at inducing PMN transepithelial migration. Subsequent
dilutional analysis of basolateral supernatants also failed to elicit a
chemotactic activity in the transepithelial migration assay (data not
shown), indicating that the lack of a chemotactic bioactivity in the
20x-concentrated fraction was not explained by tachyphylaxis due to
high PEEC concentration. These results indicate that release of PEEC
bioactivity is highly apically polarized (at least 20:1), in contrast
to the release of chemokines such as IL-8, which are released in a
basolaterally polarized fashion.
As shown in Figure 6, PEEC activity is
stable to a variety of physical manipulations. It is not significantly
attenuated by either 40 µg/ml of trypsin (74 ± 17% of control)
or 10 U of proteinase K (76 ± 19% of control); is not lost after
20 min of boiling (111 ± 19% of control); and withstands vacuum
drying (70 ± 15% of control), repeated freeze/thaw cycles
(94 ± 13% of control), and shifts in pH ranging from less than
2.0 to greater than 13 (87 ± 10%, and 77 ± 7%,
respectively). In addition, 85% of the PEEC activity, which is defined
by the ability to induce PMN transepithelial migration, could be
recovered in the supernatant fraction of ethanol precipitates, while
only 16% was recovered from the pellet. PEEC bioactivity, however, was
shown to be sensitive to treatment of 95% acetonitrile. Since small
peptides can be resistant to proteolysis, and PEEC bioactivity is of
low m.w., these features are rather nonspecific in differentiating a
PEEC activity due to a polypeptide as opposed to another structure. To
further assure that the PEEC activity was not highly hydrophobic, the
activity was loaded onto a C-18 (Sep-Pak) column, and activity was
assayed in the hexane eluant. Hexane failed to solubilize PEEC
bioactivity (data not shown), indicating that PEEC is probably not a
lipid. Preliminary absorbance characterization of the PEEC-active
fraction (not shown) shows very little detectable material, consistent
with a bioactivity that is effective at an exceedingly low
concentration.
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Although PEEC bioactivity displayed PMN chemotactic activity in
the transepithelial migration assay, this assay does not differentiate
a chemotactic activity that directly signals the PMN as a
transepithelial gradient from one that indirectly signals PMN via
influencing epithelial signaling cascades. Thus we next determined
whether PEEC exerted chemotactic influences by directly signaling to
PMN. For this purpose the underagarose chemotaxis assay was employed in
which PMN represents the only cell type present. To discern between
chemotactic vs chemokinetic responses, a chemotactic index was
evaluated for each sample tested (FMLP, PEEC, and
HBSS+) and compared with the negative (buffer) control
chemotatic index (21, 22). As shown in Table I, positive controls for directed
chemokine-mediated PMN transmigration (gradients established by
addition of FMLP 10-8-10-9 M to the
chemokine-containing wells) elicited the expected chemotactic index
(2.03 ± 0.23 vs 0.98 ± 0.25; for positive vs negative
control). Under similar conditions, 20-fold (by volume) concentrated
PEEC also induced a directed (chemotactic index of 1.98 ± 0.27 vs
0.95 ± 0.23 for PEEC vs the negative control) and dose-dependent
PMN migration response that quantitatively was not dissimilar to the
maximal migration elicited by the potent positive control FMLP
(10-8 M FMLP gradient = 
80% of maximal response
attainable with this potent model chemokine; see below). These data
indicate that PEEC bioactivity released apically in response to
Salmonella-epithelial apical membrane interactions
represents a potent chemoattractant activity that is capable of direct
signaling to the PMN.
|
To compare the phenotype of PEEC-elicited PMN activation with that
of PMN activation by other chemokines and agonists, the concentration
dependency of PEEC was obtained. For this purpose PEEC was concentrated
in HBSS+ by filtration (200 x volume) and a
dilutional assay of PEEC established in the transepithelial migration
assay. As shown in Figure 7A,
FMLP-elicited transepithelial migration becomes saturated at
10-7 M, as previously reported (25). PEEC responsiveness
saturated at a 200-fold concentration, and the response range was
defined as occurring over a two-log dilution (Figure 7B). Maximal PEEC-elicited PMN transmigration
responses ranged from 40 to 85% of maximal FMLP responses (variability
depending on donor PMN and age of T84 cell monolayers). In contrast to
FMLP-elicited PMN transmigration, the PEEC response exhibited a
slightly steeper slope (5.88 vs 7.70 for PEEC vs FMLP) indicative of a
more narrow range of concentration over which the response becomes
saturated. Thus, these data indicate that maximal PEEC activity can be
obtained by a 200-fold concentration step, and such activity once
concentrated is comparable to that exhibited by maximal concentrations
of a potent model chemokine (FMLP).
|
Apical PEEC secretion in response to apical Salmonellacolonization is dependent on protein synthesis by epithelial cells
We next sought to determine whether S. typhimurium or
epithelial cells were responsible for the secretion of PEEC into the
apical epithelial compartment by exploiting selective inhibitors of
eukaryotic (CHX) vs prokaryotic (CAM) protein synthesis. An initially
complicating feature of these studies was the fact that after
pathogen-epithelial apical contact, sequential series of new genes are
transcribed to permit the evolving pathogen-eukaryote relationship.
However, we have previously shown that pathogen-epithelial cell
interrelationships required for induction of epithelial proinflammatory
responses are fulfilled within 45 min of epithelial-pathogen contact
(4, 6). We show here (see above) that PEEC secretion is not limited to
a narrow time window and occurs continuously even after the first hour
of colonization. Thus, since bacterial-epithelial contact is required
for eliciting proinflammatory responses, we were able to permit
pathogen-epithelial relationships to evolve (45 min), treat them with
selective protein synthesis inhibitors (1 h), wash the monolayers, and
then collect apical supernatants to be analyzed for PEEC bioactivity.
As shown in Figure 8, treatment of
S. typhimurium-colonized T84 monolayers with the eukaryotic
protein synthesis inhibitor CHX completely inhibited apical secretion
of PEEC-like bioactivity (p < 0.001). Under
these conditions of CHX treatment, sensitive indicators of epithelial
monolayer function (resistance and Cl secretory responses to agonists)
were unaffected (not shown) (6). In contrast, following of S.
typhimurium-colonized T84 cell monolayers with CAM at
concentrations sufficient to selectively inhibit bacterial protein
synthesis (24), substantial PEEC activity continued to be secreted (no
statistical difference between the CAM vs the no treatment group).
These data indicate that, following established pathogen-epithelial
interactions, eukaryotic protein synthesis is required for PEEC
secretion and suggest that PEEC bioactivity is epithelially
derived.
|
A wide variety of neutrophil chemoattractants utilize signal
transduction pathways that route through a GTP-binding protein
exhibiting the pertussis toxin-sensitive Gi subunit
(26). Pertussis toxin-mediated ADP ribosylation of this subunit
effectively uncouples signal transduction mediated by Gi
containing heterotrimeric GTP-binding proteins. As shown in Figure 9A, PMN treated with either 2
µg/ml of pertussis toxin or vehicle alone (see Materials and
Methods) were subsequently examined in the transepithelial
transmigration assay for responses to imposed gradients of PEEC or IL-8
(500 ng/ml, the later serving as a positive control for pertussis toxin
effects). As shown, pertussis toxin diminished the PMN chemotactic
response to both IL-8 (40%) and PEEC (78%), indicating that
PEEC shares this common signal transduction pathway utilized by several
families of PMN chemoattractants that influence PMN movement via G
protein-coupled surface receptors.
|
B,
PEEC and IL-8 both stimulated rapid and transient increases (ranging
from 200–370 nM) in intracellular free calcium
[Ca2+]i. All responses reached a maximum
within approximately 10 s of stimulation and returned to basal
levels by 5 min.
While many of the chemokines share Gi and intracellular
Ca2+ signal transduction pathways, they also influence
global PMN responses in a distinctive fashion and thus produce
activation patterns that further distinguish their unique nature. Thus,
we next sought to determine whether PEEC could elicit superoxide
production and/or release of primary and secondary PMN granules, and
compared the phenotype of this response to that elicited by other
chemokines or agonists that activate PMN. These studies were performed
at saturating concentration of agonist, and PMN responses were assessed
in both the presence or absence of dihydrocytochalasin B (the latter
condition amplifies PMN superoxide production and degranulation
responses (27)). As shown in Table II,
PEEC was similar to IL-8 in its failure to stimulate superoxide
production under any condition. However, FMLP, like PMA, stimulated
superoxide production that was enhanced by the addition of
dihydrocytochalasin B. Moreover, PEEC also did not stimulate either
primary (myeloperoxidase) or secondary granule (lactoferrin) release;
in fact, PEEC may show a down-regulation of this response. In contrast,
FMLP, and to a somewhat lesser extent, LTB4, elicited degranulation of
both primary and secondary granules that was further enhanced by the
addition of dihydrocytochalasin B. IL-8, in contrast, elicited
degranulation of primary granules but not secondary granules. The data
describing the distinctive phenotypes of the IL-8, PMA, LTB4, and FMLP
responses are consistent with that reported by others (28, 29, 26). In
contrast to these known PMN chemokines/agonists, PEEC exhibits
virtually no activation phenotype other than strong chemotaxis, while
sharing proximal signal transduction pathways
(Gi/Ca2+). These data show that PEEC
bioactivity is defined as purely chemotactic, distinguishing it from
many known chemokines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Such observations led us to speculate that, in addition to basolaterally secreted regulators of the acute inflammatory response, other epithelial signal(s) perhaps including apically secreted mediators may assist in the movement of PMN across the epithelium and into the lumenal compartment. In this study we now report the identification and initial characterization of a novel soluble bioactivity that is important for regulating this later step of transepithelial PMN migration. We demonstrate that model human intestinal epithelial cells, when apically colonized with S. typhimurium, release PEEC bioactivity that has properties of a PMN chemokine and is secreted in a polarized apical manner.
The PEEC bioactivity harbors properties that appear to set it apart
from the other known peptide- or lipid-based neutrophil
chemoattractants. For instance, ultrafiltration results indicate that
PEEC exhibits a nominal molecular mass in the range of 1000 to 3000 Da,
and behaves in a fashion inconsistent for C5a (16,000 m.w.; 32),
C-C, or C-X-C family member cytokines (>7000 m.w.; 28); or for the
hydrophobic PMN chemoattractants, including LTB4 (<500 Da molecular
mass; 33), 5-hydroxyeicosatetraenoic acid (34), or platelet activating
factor (<800 Da) (35, 36). Further PEEC bioactivity is not highly
lipophilic as determined by hexane extraction of PEEC-loaded C-18
columns.
Although subsets of bacterially derived n-formyl peptides could well be retained in the 1- to 3-kDa fraction, as is PEEC bioactivity, n-formyl peptides do not appear to account for the PEEC bioactivities observed. Given the numbers of bacteria present in the assay (3 x 106), known bacterial masses (10-14 gm/bacteria), and the known apical bath volume (1 ml), one can calculate whether n-formyl peptide-based signaling could possibly account for the transepithelial migration seen. Such analyses indicate that even if the entire bacterial mass present in the assay consisted of the potent, small m.w., n-formyl peptide, FMLP, the gradient across the monolayer would be insufficient to induce transepithelial migration (6, 25). Thus the observed transmigration would not be expected even if the entire dry weight of the bacteria present represented small, potent n-formyl peptides. Perhaps more importantly, inhibition of the PMN n-formyl peptide receptor does not influence PEEC responses, and PEEC-based patterns of PMN activation do not correspond to those elicited by activation of n-formyl peptide receptors.
PEEC bioactivity also appears to be derived from epithelia since it is dependent of continuous protein synthesis by epithelial cells (and not by bacteria), only after establishment of bacterial-epithelial contact. The above argument, derived from consideration of bacterial mass in the assay and apical volumes in which the assays are employed, also supports the explanation that PEEC is derived from epithelia. That is, given the likelihood that PEEC (like other chemokines) will signal in the nM to µM range, and considering the bacterial mass present, lumenal reservoir size of 100 µl, and size of PEEC, there is insufficient bacterial product to mount such a response.
The physical characteristics of PEEC activity, however, do not clearly identify its nature, given that PEEC readily signals PMN in buffer solution, is not hexane soluble, and can be easily reconstituted in buffer subsequent to drying on glass in a rotary evaporation device. Thus, it appears that PEEC exhibits at least moderate hydrophilicity. The simplest interpretation from the protein synthesis inhibition data is that PEEC is a peptide, although clearly other interpretations are possible (i.e., a nonpeptide structure that depends on induction of an enzyme for its synthesis). Bulk purification of PEEC for more detailed structural analyses has been confounded by the observation that epithelial cells must be highly polarized, and therefore grown on permeable supports. However, if it is a polypeptide, PEEC resistance to proteolysis and acid degradation is not surprising given its small size. Acid hydrolysis, a general approach in protein degradation, has been also applied to complex homogenates to isolate small, bioactive peptides such as defensins, guanylin, and vasoactive intestinal peptide (37, 38, 39).
PEEC-based activation of PMN appears to behave like other chemokine receptors in regard to its pertussis toxin sensitivity and intracellular Ca2+ signal. Important in distinguishing PEEC-based PMN activation from that of various other PMN chemoattractants is analysis of more global activation responses of PMN (40, 41). For example, while IL-8 elicits substantial neutrophil degranulation of secondary granules, this chemokine exhibits a weak degranulation of primary granules even in the presence of dihydrocytochalasin B. Moreover, as reported by others (42) and confirmed here, IL-8 essentially does not induce the respiratory burst. However, the ability of IL-8 to produce a respiratory burst is controversial and our data conflict with that of Baggiolini, Imboden, and Detmers, who report that a respiratory burst can be detected when preparations of human neutrophils that respond to FMLP are stimulated with IL-8 (43). Additionally, FMLP, when compared with IL-8, was much more potent in its ability to elicit the release of primary granules from neutrophils and likewise strongly stimulated degranulation of secondary granules and induced neutrophil respiratory burst. Similarly, the preferential release of secondary granules as opposed to primary granules contrasts the strongly chemotactic lipid mediator LTB4 to other chemokines (29). PEEC activity also has a unique profile in terms of global PMN activation, and of the chemokines examined has characteristics more like IL-8. Interestingly, while PEEC exhibits maximal chemotactic activity in the same range as the potent chemotactic peptide FMLP, in contrast to FMLP and other strong PMN chemokines, PEEC has either no detectable or a minimal effect on respiratory burst or degranulation of either primary or secondary granules. Since a PEEC dose-response curve was established by concentration of the PEEC bioactivity, and this activator was compared with all others at identical effective (i.e., saturating) concentrations, such results indicate a unique aspect of PEEC as a nearly "pure" chemoattractant with regard to activation of PMN. Members of the functionally defined family of pure chemoattractants include TGF-ß and substance P, and are characterized by the inability to provoke cell-cell aggregation, secretion of O2, or lysosomal enzymes at any concentration (44, 45). In contrast to PEEC bioactivity, these previously described chemoattractants activate G proteins without eliciting increments in cytosolic Ca2+ (44, 45). In addition, the molecular mass of TGF-ß (Mr 25,000) is another defining characteristic that separates PEEC from TGF-ß (44, 45). Thus, taken together, PEEC appears to exhibit characteristics that are distinct even from known "pure" neutrophil chemoattractants.
In summary, apical attachment of Salmonella to epithelial monolayers induces a host of epithelial responses that serve to coordinate the recruitment of neutrophils to the lumenal space in which the pathogen is positioned. Although not involved in transepithelial migration, polarized basolateral release of IL-8 can serve, as we have previously shown (4), to imprint subepithelial matrices with long-lived chemotactic gradients that resist the sweeping-away effects of large-scale water transport that characterize transporting mucosal surfaces. Thus, in the final step of transepithelial migration, subepithelial PMN would be ideally positioned to traffic across the monolayer in response to secreted PEEC activity. Such a presumption has recently been corroborated by the work of Rothenberg et al. (46), who demonstrate that intranasal or s.c. application of eotaxin selectively recruits large numbers of eosinophils into the mouse lung and skin, respectively. Therefore, given that apically directed PMN migration is by itself thought to contribute to epithelial dysfunction in a host of mucosal diseases (1, 2, 3, 47, 48, 49, 50, 51), it is possible that interference with the PEEC-based signaling pathway may provide a potentially important new therapeutic target for treatment of acute inflammatory diseases of intestinal and perhaps other mucosal surfaces.
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Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Beth McCormick, Department of Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital, 149 13th Street (1493404), Charlestown, MA 02129-2000.
3 Abbreviations used in this paper: PMN, polymorphonuclear leukocytes; PEEC, pathogen-elicited epithelial chemoattractant; CE, cell equivalents; tBOC-FMLP, N-t-BOC-1-methionyl-1-leucyl-1-phenylalanine; CAM, chloramphenicol; CHX, cycloheximide; LTB4, leukotriene B4.
Received for publication May 27, 1997. Accepted for publication September 22, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. W. Liang, R. A. DeMarco, R. J. Mrsny, A. Gurney, A. Gray, J. Hooley, H. L. Aaron, A. Huang, T. Klassen, D. B. Tumas, et al. Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation Am J Physiol Cell Physiol, December 1, 2000; 279(6): C1733 - C1743. [Abstract] [Full Text] [PDF]
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C. A. Lee, M. Silva, A. M. Siber, A. J. Kelly, E. Galyov, and B. A. McCormick A secreted Salmonella protein induces a proinflammatory response in epithelial cells, which promotes neutrophil migration PNAS, October 24, 2000; 97(22): 12283 - 12288. [Abstract] [Full Text] [PDF]
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V. Hofman, V. Ricci, A. Galmiche, P. Brest, P. Auberger, B. Rossi, P. Boquet, and P. Hofman Effect of Helicobacter pylori on Polymorphonuclear Leukocyte Migration across Polarized T84 Epithelial Cell Monolayers: Role of Vacuolating Toxin VacA and cag Pathogenicity Island Infect. Immun., September 1, 2000; 68(9): 5225 - 5233. [Abstract] [Full Text] [PDF]
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D Haller, C Bode, W P Hammes, A M A Pfeifer, E J Schiffrin, and S Blum Non-pathogenic bacteria elicit a differential cytokine response by intestinal epithelial cell/leucocyte co-cultures Gut, July 1, 2000; 47(1): 79 - 87. [Abstract] [Full Text] [PDF]
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A. G. Frick, T. D. Joseph, L. Pang, A. M. Rabe, J. W. St. Geme III, and D. C. Look Haemophilus influenzae Stimulates ICAM-1 Expression on Respiratory Epithelial Cells J. Immunol., April 15, 2000; 164(8): 4185 - 4196. [Abstract] [Full Text] [PDF]
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 M.-H. Coconnier, V. Liévin, M. Lorrot, and A. L. Servin Antagonistic Activity of Lactobacillus acidophilus LB against Intracellular Salmonella enterica Serovar Typhimurium Infecting Human Enterocyte-Like Caco-2/TC-7 Cells Appl. Envir. Microbiol., March 1, 2000; 66(3): 1152 - 1157. [Abstract] [Full Text]
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S. D. Li, D. Kersulyte, I. J. D. Lindley, B. Neelam, D. E. Berg, and J. E. Crabtree Multiple Genes in the Left Half of the cag Pathogenicity Island of Helicobacter pylori Are Required for Tyrosine Kinase-Dependent Transcription of Interleukin-8 in Gastric Epithelial Cells Infect. Immun., August 1, 1999; 67(8): 3893 - 3899. [Abstract] [Full Text] [PDF]
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 K. H. Darwin and V. L. Miller Molecular Basis of the Interaction of Salmonella with the Intestinal Mucosa Clin. Microbiol. Rev., July 1, 1999; 12(3): 405 - 428. [Abstract] [Full Text] [PDF]
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A. T. Gewirtz, A. M. Siber, J. L. Madara, and B. A. McCormick Orchestration of Neutrophil Movement by Intestinal Epithelial Cells in Response to Salmonella typhimurium Can Be Uncoupled from Bacterial Internalization Infect. Immun., February 1, 1999; 67(2): 608 - 617. [Abstract] [Full Text] [PDF]
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B. A. McCormick, A. M. Siber, and A. T. Maurelli Requirement of the Shigella flexneri Virulence Plasmid in the Ability To Induce Trafficking of Neutrophils across Polarized Monolayers of the Intestinal Epithelium Infect. Immun., September 1, 1998; 66(9): 4237 - 4243. [Abstract] [Full Text] [PDF]
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