Enteropathogenic Escherichia coli: Stimulating Neutrophil Migration Across a Cultured Intestinal Epithelium Without Altering Transepithelial Conductance

Abstract

Introduction 

Migration of neutrophils across the intestinal epithelium is the hallmark of inflammatory conditions of the bowel. In cultured intestinal epithelial monolayer models, neutrophils can be induced to migrate along a chemotactic gradient such as n-formyl-methionyl-leucyl-phenylalanine (fMLP). Physical passage of the neutrophils across the epithelium could disrupt the tight-junctions, possibly leading to a large increase in the transepithelial conductance (G_t). The goal of this study is to determine whether transepithelial migration of neutrophils induced by enteropathogenic Escherichia coli (EPEC) causes changes in G_t comparable with those seen with fMLP.

Methods 

The apical side of T84 monolayers were rapidly infected with EPEC E2348/69 or exposed to 1μM fMLP. A third group of monolayers exposed to neither EPEC nor fMLP served as control. Indium-labeled neutrophils were added to the serosal side of monolayers grown on a cell culture insert membrane (12 μm pores). G_t was measured at fixed intervals up to 4 hours. After a 150-minute incubation, radioactivity of the neutrophils that migrated to the apical side was assayed and the number of migrating neutrophils was calculated.

Results 

At 150 minutes, EPEC induced similar neutrophil chemotactic capability compared to fMLP (231 ± 34 · 10³ and 193 ± 48 · 10³, respectively, n = 13, P > 0.05). However, EPEC-induced neutrophil migration was not associated with significant increase in G_t, 1.13 ± 0.16 fold of baseline G_t, in distinction with fMLP groups, 13.3 ± 0.48 fold, n = 7 (P < 0.05). G_t changes with EPEC were seen after 4 hours of infection, but were not different in the presence or absence of neutrophil migration (1.37 ± 0.12 fold and 1.42 ± 0.17 fold of baseline G_t, respectively).

Conclusions 

The results indicate that EPEC-induced neutrophil migration can occur without significant disruption of barrier function. In addition, the chemo-attractant recruiting neutrophils during EPEC infection is unlikely to be fMLP; and, the G_t increase seen with fMLP-driven recruitment may indicate a discretionary compromise of barrier function during neutrophil migration.

INTRODUCTION

Intestinal inflammation is characterized by the recruitment of inflammatory cells into the mucosa. During inflammation, neutrophils actively migrate from the vascular space into the intestinal mucosa and can ultimately cross the epithelium (¹). Transepithelial migration of neutrophils has been examined in vitro using cultured intestinal epithelial monolayers and the potent chemoattractant n-formyl-methionyl-leucyl-phenylalanine (fMLP) (^2–4). This stimulated transmigration is associated with a large (4 to 10 fold) increase of transepithelial conductance (G_t), which occurs in a roughly proportional manner (^4–7). The increase in G_t is suggested to result from neutrophil disruption of the epithelial tight-junctions to gain passage through the epithelium (^4,6). Such a disruption would constitute a severe compromise in epithelial barrier function. However, all increases of G_t in this in vitro model may not reflect physical transmigration. For example, blocking the cell adhesive protein integrin CD18 with an antibody can eliminate transmigration but still produces a transient increase in G_t (²). This increase in G_t occurs together with stimulation of transient Cl secretion that requires a neutrophil-epithelial interaction (²). Moreover, when neutrophils interact with cultured intestinal epithelium without physically transmigrating, due to filters with small (0.45μm) pores, G_t still increases (⁸). The increase in G_t depends critically on the number of transmigrating neutrophils and the direction of transit, whether in the pathophysiologic direction from basal to apical side or from apical to basal side (³). Other studies suggest that neutrophil transmigration alone is not sufficient to change epithelial permeability (^9,10).

The influence of transmigrating neutrophils on epithelial permeability is central to the progression of many inflammatory states. If the physical mechanism for neutrophil transmigration during high rates of exit involves disruption of epithelial barrier function, then moderating this transmigration rate may improve resolution of infection. Studies of induced neutrophil transmigration have used either fMLP as a chemoattractant (^2,3,4,9,11) or bacterial infection of monolayers (^12–14). Using different inducers of neutrophil transmigration provides additional details on the interactions between epithelial cells and neutrophils. Monolayer infection with enteropathogenic Escherichia coli (EPEC) (¹³), Shiga-toxin producing Escherichia coli (STEC) (³²) or Salmonella typhimurium (^12,14) stimulates neutrophil transmigration. Bacterial infection alone can increase G_t by 40 to 100% after 2 to 6 hours (^14–17); however, changes in G_t due to bacteria-induced neutrophil transmigration have not been reported (^12–14,18). Infection with STEC increased inulin permeability by 60% over a 4-hour period of neutrophil transmigration, also consistent with induction of some paracellular leaking (¹⁸). If high-rate neutrophil transmigration requires disruption of epithelial barrier function, disruption should occur with any stimulus that induces similar rates of transmigration. In the study reported here, EPEC infection did not increase G_t during high-rate neutrophil transmigration in the pathophysiologic direction from basal to apical side of the epithelium. Thus, massive neutrophil transmigration can occur without significant disruption of barrier function. The G_t increase seen with fMLP-driven recruitment may represent a specific interaction between host epithelial cells and neutrophils rather than a required physical disruption of tight-junctions during neutrophil transmigration.

METHODS

Preparation of Bacteria

Enteropathogenic Escherichia coli (EPEC) strain E2348/69 were a generous gift from Dr. T. Jilling (Northwestern University, Evanston, IL). EPEC were maintained on trypticase soy agar and grown overnight in static non-aerated Pennassay broth (Difco Laboratories, Detroit, MI) centrifuged and washed (3X). Mid log phase EPEC were suspended PBS (Life Technologies, Gaithersburg, MD) to a final concentration of 10⁸/100μL. Quantification of bacteria was performed using a standard curve comparing visible light absorbance, O.D. 600nm in Beckman DU-50 spectrophotometer (Beckman Instruments, Fullerton, CA), of bacterial suspensions to colony forming units (data not shown).

Preparation of Monolayers

T84 cells (American Tissue Culture Collection, Rockville, MD) were maintained in DMEM (Life Technologies), MEM, Fetal Bovine Serum (Atlanta Biologicals, Atlanta, GA), Penicillin, Streptomycin, and amphotericin (Life Technologies) in a humidified atmosphere with 5% CO₂. Approximately 1 · 10⁵ cells were plated on Costar (Corning Costar, Cambridge, MA) cell culture inserts, 1 cm² in surface area with a 12 μm pore size. The cells were allowed to become confluent, an average of 7 to 9 days after plating. Confluent density was 5 · 10⁵ cells/cm².

Preparation of Neutrophils

Neutrophils were isolated from fresh whole citrated blood from healthy volunteers in accordance with a protocol approved by the Institutional Review Board. Histopaque was added and the sample was centrifuged. The granulocyte layer was aspirated and washed. Any red blood cells collected with the neutrophils were lysed in distilled water (¹⁹). Neutrophils were then placed in RPMI media containing granulocyte colony stimulating factor GmCSF at10ng/ml (R&D Systems, Minneapolis, MN). Indium(III) oxine (Mallinckrodt, Dayton, OH) was used to label the neutrophils (²⁰). Briefly, 0.5 μCi of radioactive indium was added for every 10⁶ neutrophils and allowed to incubate for 15 minutes at 37°C. The neutrophils were washed three times to remove any free indium and resuspended in RPMI with GmCSF.

Infection of Monolayers

Monolayers were washed (3X) with Hank's Balanced Salt Solution (HBSS) to remove media containing antibiotics. The inserts were placed in 50 ml centrifuge tubes and the apical side of the monolayer was rapidly infected with EPEC strain E2348/69 by applying 100μL of DMEM containing 10⁸ mid-log-phase bacteria followed by centrifugation at 500 g for 4 minutes (Beckman T-J6 centrifuge). After centrifugation, monolayers were washed (3X) with HBSS to remove any non-adherent bacteria. Uninfected T-84 (control) monolayers were subjected to the same procedures except exposure to bacteria.

Transmigration Assay

After infecting the monolayers, the inserts were inverted and indium-labeled neutrophils (1.5 · 10⁶) added to the upper chambers (serosal side of the monolayer separated by a 12 μm-pore cell culture insert membrane) (Fig. 1). This represents a neutrophil to T84 cell ratio of approximately 3:1. To compare the effect of EPEC with that of fMLP on neutrophil migration, another set of monolayers was centrifuged without EPEC and exposed to 1μM fMLP in the lower chamber bathing the apical side of the cells. All monolayers were centrifuged in a similar manner to eliminate centrifugation as a variable that could potentially affect neutrophil migration or epithelial barrier function. The neutrophils were incubated on the serosal side of the monolayers for 150 minutes. At the conclusion of the incubation time, all migrated neutrophils across the membrane were counted (within monolayer + the apical bathing solution). The radioactivity of the neutrophils was assayed using a gamma counter (Beckman gamma 5500) and the number of neutrophils migrated calculated. The calculation was done individually for each experiment based on the radioactivity of a known number of neutrophils (counted in a hemocytometer).

FIG. 1.: EPEC-induced transmigration experimental layout: Indium-labeled neutrophils were added to the serosal side of confluent, inverted, and infected T-84 monolayers separated by a 12-μm pore insert membrane.

Measurement of Monolayer Conductance

Transepithelial electrical conductance (G_t) was measured across the monolayer using STX-II Ag-AgCl electrodes (World Precision Instruments, Sarasota, FL) and a current clamp (VCC MC dual channel voltage/current clamp, Physiologic Instruments, San Diego, CA) to determine the voltage responses. Transepithelial conductance was calculated using Ohm's law for voltages produced in response to passage of 30μA current (G = I/V, where V is voltage, I is current and G is conductance).

Neutrophil Migration in Response to EPEC Growth Solution

After overnight growth of EPEC, EPEC bacteria were removed by centrifugation followed by filtration through a 0.22-μm filter (Fisher Scientific, Pittsburgh, PA). The growth solution equivalent to 10⁸ bacteria (100 μL) was applied to the apical side of monolayers and neutrophil migration was determined.

Quantification of Neutrophils Adherent to Apical Surface of Monolayer

This method was adapted from Reeves et al (²¹). After neutrophil migration was completed, the monolayer was centrifuged at low speed (50 g) for 5 minutes. The apical surface of the monolayer was washed (2X) gently to allow the number of adherent neutrophils to be calculated. This number represents the migrated neutrophils that remained attached to the apical surface of the T84 cells.

EPEC Binding to Monolayers

To determine if rapid infection of T-84 monolayers is a valid method for EPEC binding, the monolayers were infected as above and allowed to incubate at 37°C for 60 minutes. After the incubation, non-adherent bacteria were removed by washing (3X). Trypsin EDTA was used to detach the infected monolayer from the insert. Infected T84 cells were plated on MacConkey agar (Difco). Colony-forming units were counted after overnight incubation.

All reagents were obtained from Sigma (St. Louis, MO) unless otherwise stated.

Statistical Methods

A paired Student t test was used to identify any statistically significant difference in neutrophil migration between groups. One-way repeated measures ANOVA was used to determine significant changes in conductance in a particular group in relation to time. Bonferroni multiple comparison tests were used to compare conductance data within each group. Data are expressed as mean ± standard error of mean, and considered significant at P < 0.05.

RESULTS

Rapid Infection of Monolayers with EPEC

To determine if rapid infection of EPEC is a valid method for the binding of EPEC to the T84 cells, the monolayer was centrifuged with EPEC as described in the methods section. Immediately following centrifugation, monolayers were washed and allowed to incubate for 60 minutes, then washed again to remove non-adherent bacteria. The T84 cells were removed from the cell culture insert and colony-forming units (CFU) of EPEC were counted. Significant EPEC attachment was seen with an average of 530 ± 50 · 10³ bacteria attached to each monolayer (n = 5). This infection corresponds to roughly one EPEC/T84-cell, which is similar to the lowest-level infection in a previous study of EPEC-induced neutrophil transmigration (¹³). Thus, rapid infection of monolayers through centrifugation appears to be an effective method for producing EPEC binding to the monolayers.

Neutrophil Migration Induced by EPEC Infection

After infecting T84 monolayers, neutrophils were added on the serosal side for 150min to allow migration in the physiological direction from the serosal to the mucosal side. EPEC infection induced migration of 231 ± 34 · 10³ neutrophils (Fig. 2), corresponding to 15% of the added neutrophils. This EPEC-induced migration was larger than previously reported migrations induced with 100-EPEC/T84-cell (¹³) or 80-Salmonella-typhimurium/T84-cell (¹⁴), further supporting the effectiveness of centrifugation as an infection method. fMLP induced an average migration of 193 ± 48 · 10³ neutrophils (Fig. 2), 13% of those added, which was not significantly different from the EPEC group (P < 0.05, n = 13). Thus, migration induced by EPEC infection was comparable to that induced by fMLP.

FIG. 2.: Neutrophil transmigration after 150 minutes: EPEC caused significant neutrophil transmigration compared to non infected controls, n = 13, P < 0.05. fMLP caused significant transmigration, compared to control (n = 13, P < 0.05). Neutrophil transmigration induced by EPEC and fMLP was not significantly different (P > 0.05). EPEC growth solution did not induce significant transmigration (n = 3, P > 0.05).

Locating the Migrated Neutrophils

Migrated neutrophils responding to EPEC were mostly found in association with the monolayer rather than in the apical bathing fluid, which contrasts with fMLP induction: 89.5 ± 13.0% with the monolayer after EPEC infection, 37.4 ± 5.1% with the monolayer in response to fMLP. The neutrophils found in the monolayer may not have migrated and still be on the basal side of the tight-junctions or may have completed migration and be attached to the apical surface. Since fMLP is present in the apical bathing solution, the transmigrated neutrophils could be expected to detach into this bathing fluid. In contrast, the neutrophils would likely migrate toward attached EPEC so that in this case the transmigrated neutrophils also would be found attached to the apical surface. The number of neutrophils attached to the apical surface was measured by the method of Reaves et al. (²¹), adapted to calculate the percentage of apically adherent neutrophils. The majority of the transmigrated neutrophils with EPEC infection were adherent to the apical surface of the monolayer, 57.7 ± 8.8% of all neutrophils in response to EPEC and 11.1 ± 1.5% in response to fMLP (Fig. 3). Thus, the total percentage of neutrophils that completed transmigration across the tight-junction was 68.2 ± 10.9% (10.5% + 57.7%) for EPEC and 73.7 ± 8.7% (62.6% + 11.1%) for fMLP. Using the measure of neutrophil migration (Fig. 2) together with the percentage of transit through the tight-junctions (Fig. 3), transmigration was 157 ± 34 · 10³ neutrophils for EPEC infection and 142 ± 39 · 10³ neutrophils for fMLP recruitment.

FIG. 3.: Distribution of migrated neutrophils: Neutrophil transmigration in response to EPEC and fMLP was allowed to proceed for 150 minutes. The locations of the transmigrated neutrophils were identified in 3 areas; the apical bathing solution (reservoir), attached to the apical surface of the monolayer (apical surface), or within the monolayer itself (monolayer). The percent of neutrophils that completely crossed the tight junctions (reservoir + apical surface) was comparable in both groups (P > 0.05, n = 5).

Neutrophil Transmigration and G_t

Neutrophil transmigration induced by fMLP was associated with a significant increase in G_t (Fig. 4). This increase was detectable as early as 30 minutes after the addition of neutrophils and increased further by 150 minutes (13.3 ± 0.5 fold of baseline G_t, n = 7 (P < 0.05)). If G_t changes were due to the physical migration of neutrophils, then a similar increase in G_t after EPEC-induced migration might be expected, since chemotactic migrations induced by EPEC and fMLP were comparable (Figures 2 and 3). However, EPEC-induced neutrophil transmigration was not associated with a significant change in G_t (1.13 ± 0.16 fold of baseline G_t).

FIG. 4.: Changes in conductance (mS/cm²) in the different experimental groups: Monolayers exposed to the following: EPEC alone (EPEC), neutrophil transmigration secondary to EPEC (EPEC + Neut), neutrophils without chemoattractant (Neut), fMLP only (fMLP), monolayer alone (control), did not induce significant changes in conductance, n = 6–9, up to 150 minutes. However, neutrophil transmigration across an fMLP gradient (Fmlp + Neut) was associated with significant increase in conductance (n = 6, P < 0.05).

Source of EPEC-induced Chemotaxis

EPEC growth solution was obtained as described in the methods section. The growth solution was applied to the apical surface of monolayers and neutrophils were added to the serosal side of the inverted monolayer. After 150 minutes, the EPEC growth solution did not result in significant neutrophil migration (Fig. 2) indicating that EPEC did not induce neutrophil migration through a secreted substance, but instead more likely through a bacteria-induced signal generated by the T84 cells (¹⁴).

Time Course of Neutrophil Transmigration During EPEC Infection

Neutrophil transmigration induced by EPEC infection was measured at various time intervals to determine the onset and rate of migration. Neutrophil migration was detectable as early as 30 minutes after addition of neutrophils to the monolayers (Fig. 5). The rate of transmigration at this early time point ∼1600neutrophils/min) was similar to that measured in a previous study using fMLP as a chemo-attractant (⁴).

FIG. 5.: Time course of neutrophil transmigration during EPEC infection: Neutrophil transmigration in response to EPEC infection was significantly higher than non-infected controls. Significant transmigration was seen as early as 30 minutes after infection (n = 3, P < 0.05).

EPEC Infection and G_t

Measurements of G_t after addition of neutrophils to EPEC-infected T84 monolayers were extended beyond 150 minutes to 240 minutes. After 240 minutes of EPEC infection, G_t increased 1.42 ± 0.17 fold from baseline for control monolayers without neutrophils, similar to previous reports (^15,16,22,23). Neutrophils were added to a second set of monolayers and transmigration was allowed to proceed. Monolayers with neutrophils present had a G_t increase of 1.37 ± 0.12 fold only after 240 minutes. Infection of monolayers with EPEC, with or without neutrophils, did not change short-circuit current from the control value of near 0.1μA/cm² (data not shown), suggesting a lack of electrogenic Cl secretion, consistent with previous observations with T84 monolayers (²⁴). Thus, neutrophil transmigration induced by EPEC infection did not significantly alter G_t, indicating that barrier function was not seriously compromised due specifically to massive neutrophil movement through the tight-junctions onto the apical surface of the epithelium.

DISCUSSION

Enteropathogenic Escherichia coli is a gram-negative bacillus with strong and specific interaction with the host cell. Upon initial encounter, the EPEC bacterium can translocate its own protein receptor, the translocated intimi receptor (Tir), into the host membrane. The Tir then becomes phosphorylated and ultimately causes cytoskeletal changes leading to the formation of actin rich pedestals, which are termed attachment and effacement lesions (A/E-lesions) (^24,25).

In spite of increased knowledge of the cellular changes induced by EPEC on the host cell, the exact mechanism by which this bacteria causes diarrhea is still unknown. One hypothesis explaining the diarrhea relates it to the A/E-lesions seen with EPEC infection. It is suggested that diarrhea can occur secondary to malabsorption from the loss of the brush border surface area. However, in human volunteers, the diarrhea develops within a few hours, so early in the process of infection (²⁶) that malabsorption, which can take several days to develop, seems unlikely.

An inflammatory infiltrate has been found in the intestine after infection with this enteric pathogen, composed mainly of neutrophils (^27,28). The presence of a chemotactic agent is necessary for the neutrophils to migrate from the vascular space to the mucosa and finally cross the tight-junctions of the epithelium. EPEC has been shown to induce this chemotactic behavior in monolayers of T84 cells (¹³). Neutrophil transmigration across epithelial monolayers (^4,9,10) also is induced using an E-coli product, n-formyl-methionyl-leucyl-phenylalanine (fMLP). This fMLP-induced neutrophil transmigration across epithelial monolayers is associated with diminished barrier function, which is suggested to be secondary to disruption of the tight-junctions as the neutrophils transmigrate (^4,11). However, neutrophils maintain close cell-to-cell contact with epithelial cells during transmigration across MDCK monolayers, which prevents focally enhanced permeability (⁹). Maintenance of low permeability at the site of neutrophil passage through tight-junctions (⁹) is similar to the low permeability maintained during the physiologic extrusion of epithelial cells from intestinal epithelia (²⁹). Thus, these two phenomena, neutrophil transmigration and increased tight-junction permeability, may occur through separate mechanisms.

Neutrophil transmigration induced by EPEC infection (Fig. 5) was not associated with increased G_t (Fig. 4). This lack of G_t increase was not a result of slow transmigration, since the number of neutrophils transmigrating was similar to the number induced by fMLP (Fig. 2–3) when G_t did increase (Fig. 4). The rate of transmigration ∼1600neutrophils/min, Fig. 5) also was comparable to previous studies of fMLP-induced transmigration across T84 monolayers (⁴). These results using a different chemotactic inducer (EPEC infection) indicate that massive neutrophil transmigration can be stimulated without increasing G_t. Based on these results, neutrophils can transmigrate through an epithelium at high rates without inducing changes in tight-junctions that increase epithelial permeability and consequent loss of barrier function. Extension of these results to the intact mucosa suggests that increased permeability observed with EPEC infection does not result simply from the arrival of neutrophils through the epithelium. Thus, compromise of the mucosal barrier must result from other processes.

Neutrophils also respond to fMLP by stimulating a respiratory burst that aids killing of bacteria with reactive oxygen species. Stimulation of a respiratory burst near tight-junctions of an epithelium could possibly lead to degraded sealing properties and loss of barrier function. However, addition of oxygen radical scavengers did not blunt the increase of G_t observed during fMLP-stimulated neutrophil transmigration (⁵). Furthermore, neutrophils from patients with chronic granulomatous disease increased G_t during fMLP-stimulated transmigration even thought these cells are incapable of generating superoxide radicals (⁵). Thus, the increased G_t that distinguishes fMLP stimulation of neutrophil transmigration from EPEC stimulation is not due to damage caused by products of respiratory bursts.

The increased G_t observed during neutrophil transmigration induced by fMLP appears to require contact between epithelial cells and neutrophils. Inducing neutrophils to first enter MDCK monolayers in an apical-to-basal direction using basal solution fMLP followed by reversal of the fMLP gradient to cause exit in the basal-to-apical direction, demonstrated that G_t returned toward control as neutrophils detached from the monolayer and floated into the apical solution (¹¹). In addition, preventing neutrophils from transmigrating by using filters with pores too small for transmigration but large enough for pseudopodia extension leads to increased monolayer G_t (⁸). Apparently, fMLP-stimulated neutrophils interact with host epithelial cells to induce changes in tight-junctions that increase G_t without physical migration across the epithelium.

Neutrophils transmigrating under the influence of the EPEC were mostly found adhering to the apical surface of the monolayer as expected since EPEC were localized to the brush border. This neutrophil action was targeted towards the apical surface presumably to attack the bacteria as part of the host defense system (Fig. 3 and 6). The relative lack of neutrophils in the apical solution suggests that transmigration was not stimulated by a soluble chemo-attractant released by EPEC. Since EPEC-induced transmigration was not associated with increased G_t (Fig. 4), fMLP is unlikely to be a key factor during EPEC infection. In addition, an fMLP antagonist blocks fMLP-induced transmigration, but not EPEC-induced transmigration (¹³). Specifically, EPEC growth solution failed to induce neutrophil transmigration (Fig. 2), supporting an EPEC-induced epithelial signal for neutrophil transmigration (¹³) rather than a secreted bacterial substance.

FIG. 6.: Scanning electron micrograph (1250 magnification) of the apical surface of infected T-84 monolayer. Two densely white structures are seen on the apical surface of the cells that represent two neutrophils that completely migrated in the physiological direction from the basolateral to the apical side of the T-84 cells.

The ability of EPEC to recruit neutrophils to the intestinal epithelium is consistent with the microscopic picture of inflammatory infiltrates seen in the infected intestine (^27,28). In this study, EPEC were capable of inducing neutrophil migration across the epithelium very early during the infection process, as early as 30 minutes (Fig. 5). It is possible that the migrating neutrophils contribute towards the development of the diarrhea seen during EPEC infection, but compromise of barrier function was not apparent until 4hr after infection. The early timing of the neutrophil migration is consistent with the early onset of EPEC-induced diarrhea. In general, intestinal mucosal inflammation is frequently associated with clinical symptoms of diarrhea (³⁰) and neutrophil recruitment to the mucosa can promote gastrointestinal injury (^31,32); however the act of transmigration does not appear to be the source of barrier dysfunction. Even though neutrophil migration in this in vitro experimental model was not associated with electrical evidence of Cl secretion to explain the development of diarrhea, in an in vivo environment, several other variables are present within the intact intestinal mucosa such as neurovascular and other immune-related systems. These variables could be crucial in the development of the diarrhea seen during this enteric infection.

Acknowledgment:

The statistical analysis of the data by Adrienne Stolfi, M.S.P.H., is appreciated.