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(Journal of Leukocyte Biology. 2002;72:503-511.)
© 2002 by Society for Leukocyte Biology

Interleukin-10 inhibits proinflammatory activation of endothelium in response to Borrelia burgdorferi or lipopolysaccharide but not interleukin-1ß or tumor necrosis factor {alpha}

Tracy J. Lisinski and Martha B. Furie

Center for Infectious Diseases and Department of Pathology, State University of New York at Stony Brook

Correspondence: Tracy J. Lisinski, Center for Infectious Diseases/CMM, SUNY at Stony Brook, Stony Brook, NY 11794-5120. E-mail: tlisinsk{at}notes.cc.sunysb.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-10 is generally regarded as an anti-inflammatory cytokine, since it acts on a variety of cell types to suppress production of proinflammatory mediators. In inflammation, endothelial cells (EC) play a crucial role in recruiting leukocytes to sites of injury or infection. In this study, the actions of IL-10 on human umbilical vein EC were investigated. IL-10 reduced migration of monocytes and T lymphocytes across endothelium stimulated by lipopolysaccharide and decreased endothelial production of chemokines in response to lipopolysaccharide and Borrelia burgdorferi, the agent of Lyme disease. However, IL-10 did not affect these responses when EC were activated by the host proinflammatory cytokines IL-lß or tumor necrosis factor {alpha}. Moreover, IL-10 did not prevent up-regulation of the adhesion molecules E-selectin and intercellular adhesion molecule-1 by EC exposed to any of these activating agents. IL-10 therefore inhibits proinflammatory activation of EC in a manner that is selective with respect to stimulus and effector response.

Key Words: chemokines • leukocytes • adhesion molecules


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-10 was first identified as a product of T helper (TH) 2 lymphocytes but is now known to be synthesized by several other cell types as well, including TH0 and TH1 cells, B lymphocytes, monocytes, and macrophages. Although IL-10 was originally characterized by its ability to diminish the production of interferon-{gamma} (IFN-{gamma}), IL-2, and tumor necrosis factor ß (TNF-ß) by TH1 cells, it subsequently has been shown to have a broad range of actions on many different target cells. In general, its effects on the inflammatory response are suppressive. IL-10 inhibits production of a variety of proinflammatory cytokines by monocytes, neutrophils, and TH1 cells. In addition, IL-10 induces expression of IL-1 receptor antagonist by monocytes and neutrophils [1 2 3 ].

In inflammation, endothelial cells (EC) play a crucial role in recruiting leukocytes to sites of injury or infection. This recruitment involves up-regulation of adhesion molecules for leukocytes and secretion of chemokines [4 , 5 ]. Individual members of the chemokine family attract specific subsets of leukocytes. The CC chemokine CCL2 (monocyte chemoattractant protein-1) recruits monocytes, lymphocytes, and basophils, whereas the CXC chemokine CXCL8 (IL-8) is most active toward neutrophils [6 ]. Human umbilical vein EC (HUVEC), when stimulated by proinflammatory agents such as IL-1ß, TNF-{alpha}, or lipopolysaccharide (LPS), produce a number of chemokines, including CXCL8 and CCL2 [7 ].

Borrelia burgdorferi, the causative agent of Lyme disease, is also a proinflammatory agent that results in increased production of adhesion molecules [8 , 9 ] and chemokines [9 10 11 ] by endothelium. As a consequence, neutrophils [8 , 10 ], monocytes [11 ], and T lymphocytes [11 , 12 ] migrate avidly across endothelium that has been exposed to this bacterium. Activation of endothelium by B. burgdorferi appears to be mediated by its lipidated outer surface proteins [13 ]. Such activation of endothelium may contribute to the inflammatory lesions that typify this disease [14 ].

Despite its central role in inflammation, endothelium has received little attention as a potential target for IL-10. In this study, we have investigated four stimuli that activate endothelium (IL-1ß, TNF-{alpha}, LPS, and B. burgdorferi) and six endothelial effector responses [transmigration of monocytes and T lymphocytes, secretion of CCL2 and CXCL8, and up-regulation of the adhesion molecules E-selectin and intercellular adhesion molecule 1 (ICAM-1)] for their sensitivity to IL-10. None of these effector responses was inhibited by IL-10 when IL-1ß or TNF-{alpha} was used as a stimulus. In contrast, when HUVEC were activated by B. burgdorferi or LPS, IL-10 suppressed transendothelial migration of leukocytes and production of chemokines, but expression of adhesion molecules was unaffected. The ability of IL-10 to inhibit activation of HUVEC in response to B. burgdorferi or LPS, but not IL-1ß or TNF-{alpha}, suggests that bacterial agents and host-derived proinflammatory cytokines stimulate endothelium by different mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies and cytokines
Monoclonal antibody (mAb) BB11, directed against E-selectin [15 ], was provided by Roy R. Lobb (Biogen Inc., Cambridge, MA). mAb R6.5, directed against ICAM-1 [16 ], was provided by C. Wayne Smith (Baylor College of Medicine, Houston, TX). Recombinant human (rh) IL-1ß was supplied by Collaborative Biomedical Products (Bedford, MA) and Becton Dickinson Co. (Lincoln Park, NJ). The specific activities of the two preparations of IL-1ß used in these studies were 3.73 x 106 U/mg and 2.2 x 107 U/mg, respectively. rhTNF-{alpha} and IL-10 were obtained from R&D Systems, Inc. (Minneapolis, MN). LPS (Escherichia coli serotype 0111:B4) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis, MO).

Culture of EC
EC were isolated from human umbilical veins by perfusion with collagenase [17 ]. Cell cultures were maintained at 37°C in Medium 199 (M199; Life Technologies, Inc., Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 µg/ml amphotericin B [8 ]. After 3–5 days, cells from confluent cultures were trypsinized, pooled, and passaged onto 6-well plates, 48-well plates, 96-well Falcon Primaria® microtiter plates (all from Becton Dickinson), or amnion for use in experiments. Human amniotic tissue was fastened to Teflon rings and prepared for use as an acellular culture substrate as previously described [8 ]. Confluent monolayers of second-passage HUVEC were used in all experiments.

Culture of B. burgdorferi
B. burgdorferi strain HBD1, originally isolated from human blood [18 ], was cultured at 33°C in Barbour-Stoenner-Kelly medium modified to minimize the content of LPS [8 ]. HBD1 spirochetes at passages 40–56 were used in all experiments. Spirochetes were harvested during late log-phase growth, centrifuged, and resuspended in M199 containing 20% heat-inactivated FBS (HIFBS; 30 min at 56°C). In conditioned media and enzyme-linked immunosorbent assay (ELISA) experiments, 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.2, was also added [8 ]. Some experiments included a sham preparation of bacteria, which consisted of a volume of uninoculated, spirochetal growth medium equal to the largest volume of spirochete culture used in each experiment and subjected to the same manipulations as the spirochetes themselves.

Quantitation of CCL2, CXCL8, E-selectin, and ICAM-1
To assess production of chemokines, HUVEC were plated at 1.2 x 105 cells/well in 48-well tissue-culture plates and grown to confluence. Cultures were incubated for the indicated times at 37°C with 0.5 ml M199–20% HIFBS containing various test preparations. Conditioned media were collected and centrifuged at 13,300 g for 5 min. Amounts of CCL2 or CXCL8 in the supernatants were measured using commercial ELISA kits (Antigenix America, Inc., Franklin Square, NY).

Expression of adhesion molecules on intact HUVEC monolayers was determined using mAb BB11 for E-selectin and R6.5 for ICAM-1 in a whole cell ELISA as previously described [8 ]. E-selectin and ICAM-1 were assayed after 4 h and 12 h of stimulation, respectively, when their up-regulation in response to B. burgdorferi and IL-1ß is maximal [8 ]. To ensure that the HUVEC monolayers remained intact and viable throughout the whole cell ELISA, duplicate plates were processed in parallel through addition of a streptavidin-horseradish peroxidase conjugate. After this step, o-phenylenediamine was added to plates in which expression of adhesion molecules was measured, whereas MTT (500 µg/ml culture medium) was added to the duplicate plates for assessment of viability. Plates containing MTT were incubated for 3.5 h at 37°C, medium was removed, and 10 µl dimethylsulfoxide was placed in each well for 3 min at room temperature. Isopropanol containing 0.04 N HCl (90 µl per well) was then added to the dimethylsulfoxide, and the plate was placed on a rotatory shaker for 5 min at room temperature to dissolve precipitated formazan. Absorbance was measured at 570 nm. Results from this assay showed that regardless of the stimulus used, the EC remained viable and constant in number throughout the ELISA (data not shown).

Isolation of total RNA and quantitation of specific mRNA
HUVEC were plated at 1.2 x 106 cells/well in six-well tissue culture plates and grown to confluence. EC were incubated with B. burgdorferi at a ratio of 10 organisms per EC or 0.05 U/ml IL-1ß in the presence or absence of 20 ng/ml IL-10 for 4 or 24 h. Total RNA was then isolated using TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) according to the manufacturer’s instructions. Levels of CXCL8, CCL2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were quantitated using Quantikine® kits (R&D Systems), specific for each mRNA, again following the manufacturer’s instructions.

Transendothelial migration assay
To isolate leukocytes, venous blood of healthy donors was collected in syringes containing a final concentration of 0.12% disodium ethylenediaminetetraacetate. After removal of erythrocytes by sedimentation with 6% dextran (Pharmacia, Piscataway, NJ), monocytes were isolated using a hyperosmotic gradient medium (Accudenz; Accurate Chemical Co., Westbury, NY) [19 ]. This method routinely yielded preparations that were greater than 90% pure, as judged by the number of cells that ingested latex beads. To isolate T lymphocytes, anticoagulated blood was diluted with an equal volume of Dulbecco’s phosphate-buffered saline (DPBS) without Mg++ or Ca++, layered over Accu-Prep Lymphocytes gradient medium (Accurate Chemical), and centrifuged at 675 g for 20 min to collect peripheral blood mononuclear cells (PBMC) at the interface. PBMC were washed three times in DPBS without Mg++ or Ca++ to remove platelets, and the T lymphocytes were then purified by negative selection using a MACS Pan T cell isolation kit (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. The T lymphocytes were greater than 97% pure as determined by immunostaining and flow cytometry to detect CD3.

HUVEC monolayers, seeded on acellular, amniotic tissue at a density of 1.5 x 105 cells/cm2 and cultured for 7 days [17 ], were washed twice with M199-20% HIFBS containing 25 mM HEPES, pH 7.2, and incubated with 0.5 ml control medium or various test preparations at 37°C for 8 h, a time that was previously determined to stimulate maximal transmigration of leukocytes in this model system [8 , 12 ]. After incubation, HUVEC were washed three times with M199-20% HIFBS containing HEPES. Monocytes (1x105 cells in 0.4 ml assay medium) were added to the HUVEC-amnion monolayers and incubated for 20 min at 37°C, whereas T lymphocytes (1x106 cells in 0.5 ml assay medium) were allowed to incubate for 4 h. Unbound leukocytes were removed by aspiration, and the tissues were fixed overnight in 10% buffered formalin at 4°C. Tissues were removed from their holders with a cork borer, stained with Wright stain (Sigma Chemical Co.), and viewed en face by light microscopy. The number of leukocytes in nine randomly chosen fields was counted at 400x magnification for each sample, and this information was used to calculate the total number of leukocytes, adherent and migrated, that were associated with the tissues. To distinguish between leukocytes that migrated beneath the endothelium and those that adhered to the apical surface, tissues were embedded in glycol methacrylate and sectioned perpendicularly to the plane of the HUVEC monolayer. Corrections were made for loss of adherent cells during the embedding procedure as previously described [20 ].

Statistics
Data from all experimental groups were subjected to an unpaired ANOVA with the Tukey-Kramer multiple-comparison test using GraphPad InStat version 3.01 (GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Previously, we observed that IL-10 attenuates some responses of endothelium to B. burgdorferi, but not to IL-1ß [11 , 12 ]. To extend these studies, we investigated the effect of IL-10 on secretion of the chemokine CXCL8 by endothelium exposed to a variety of stimuli. As summarized in Table 1 , the amounts of CXCL8 in conditioned media collected from HUVEC that had been incubated for 24 h with control medium, IL-1ß, TNF-{alpha}, LPS, or B. burgdorferi in the absence or presence of 20 ng/ml IL-10 were measured in several separate experiments. IL-10 inhibited the production of CXCL8 by HUVEC activated with various amounts of B. burgdorferi or with 1 or 2 ng/ml LPS by approximately 40%. However, when EC were treated with a higher concentration of LPS (4 ng/ml), IL-10 significantly decreased production of CXCL8 (by ~20%) in only two of four experiments. Based on studies of endotoxemia in humans [21 ], amounts of LPS greater than 4 ng/ml were deemed unlikely to be physiologically relevant and so were not tested. Similarly, B. burgdorferi was not used at more than 100 bacteria per EC, as relatively few organisms are seen in the lesions or blood of patients with Lyme disease [22 , 23 ]. In contrast to our results with bacterial agents, no diminution in production of CXCL8 by IL-10 was noted when HUVEC were stimulated with various concentrations of IL-1ß or TNF-{alpha}. Notably, this observation held true even when the amounts of IL-1ß or TNF-{alpha} that were used provoked responses similar in magnitude to those induced by B. burgdorferi (Table 1) .


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  		Table 1. Effect of IL-10 on Production of CXCL8 by HUVEC

 
As the production of CXCL8 by B. burgdorferi-stimulated HUVEC was reproducibly and substantially inhibited by IL-10, we used this stimulus and effector response to examine the actions of IL-10 on endothelium in further detail. Dose-response experiments showed that as little as 0.2–2 ng/ml IL-10 produced maximal inhibition of CXCL8 secretion (Fig. 1 ). In contrast, even at the highest concentration of IL-10 tested, 50 ng/ml, there was no diminution in production of CXCL8 when HUVEC were activated by IL-1ß (data not shown). IL-10 effectively reduced production of CXCL8 in response to B. burgdorferi over a time period of at least 48 h but did not affect production in response to IL-1ß (Fig. 2 ). Moreover, IL-10 was still able to suppress secretion of CXCL8 even when added up to 4 h after exposure of HUVEC to B. burgdorferi had begun (Fig. 3 ). In two experiments, HUVEC were pretreated with 20 ng/ml IL-10 for 1–24 h, in addition to including the IL-10 during stimulation with B. burgdorferi. The pretreatment step did not produce any greater inhibition of secretion of CXCL8 than when the IL-10 and bacteria were simply added simultaneously (data not shown). Furthermore, experiments in which IL-10 was added and removed before activation of HUVEC indicated that IL-10 must be present during stimulation with B. burgdorferi to produce a significant decrease in CXCL8 (Fig. 4 ).



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  		Figure 1. IL-10 inhibits production of CXCL8 by B. burgdorferi-stimulated HUVEC in a dose-dependent manner. HUVEC were incubated for 24 h with control medium or B. burgdorferi at a ratio of 10 bacteria/EC in the absence of IL-10 or with IL-10 at the indicated concentrations. The amounts of CXCL8 in the conditioned media were determined by ELISA. Bars represent the means ± SD of three replicate samples. *, Significantly different from B. burgdorferi-treated samples that did not receive IL-10 (P<0.001). In a repetition of this experiment, maximum inhibition was observed using 2 rather than 0.2 ng/ml IL-10.

 


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  		Figure 2. IL-10 reduces production of CXCL8 by B. burgdorferi-stimulated HUVEC for at least 48 h. HUVEC were incubated with control medium (Unstim), B. burgdorferi at a ratio of 10 bacteria/EC (Bb), or 0.05 U/ml IL-1ß in the absence or presence of 20 ng/ml IL-10. Conditioned media were collected at the indicated time points, and the amounts of CXCL8 were determined by ELISA. Data represent the means ± SD of three replicate samples. IL-10 produced significant (P<0.01) inhibition of B. burgdorferi-stimulated CXCL8 secretion at all time points, whereas no inhibition of IL-1ß-induced secretion was observed. This experiment was repeated three times with similar results.

 


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  		Figure 3. IL-10 decreases production of CXCL8 by B. burgdorferi-stimulated HUVEC even when added after stimulation is initiated. HUVEC were incubated with control medium (Unstim), B. burgdorferi at a ratio of 10 bacteria/EC, or a sham preparation of bacteria in the absence (No Addition) or presence of 20 ng/ml IL-10. The IL-10 was added at the same time as (0 h) or 0.5, 1, 2, or 4 h after the bacteria. All samples were then incubated for a total of 24 h. Conditioned media were collected, and the amounts of CXCL8 were determined by ELISA. Bars represent the means ± SD of three replicate samples. *, Significantly different from B. burgdorferi-stimulated CXCL8 secretion in the absence of IL-10 (P<0.001).

 


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  		Figure 4. IL-10 must be present during stimulation to exert its inhibitory effects. HUVEC were incubated with control medium (No Addition) or with 20 ng/ml IL-10, which was added 1, 4, or 24 h before the start of stimulation. In some groups, the IL-10 was then removed (+ IL-10, pretreatment only). In others, IL-10 was present only during the period of stimulation (+ IL-10). HUVEC were stimulated with B. burgdorferi at a ratio of 10 bacteria per EC, a sham preparation of bacteria, or control medium (Unstim). After 24 h of stimulation, conditioned media were collected, and the amounts of CXCL8 were determined by ELISA. Bars represent the means ± SD of three replicate samples. *, Significantly different from B. burgdorferi-stimulated CXCL8 secretion in the absence of IL-10 (P<0.01). A repetition of this experiment yielded similar results.

 
Previously, Burns and Furie [11 ] observed that IL-10 decreases the production of another chemokine, CCL2, by 55% ± 11% when HUVEC are stimulated by B. burgdorferi. We repeated this experiment three times and observed a similar reduction of 53% ± 9% (data not shown). With respect to LPS, results were more variable. IL-10 significantly decreased production of CCL2 in two of three experiments (by 23% and 27%) when 1.0 ng/ml LPS was used as a stimulus and in two of four experiments (by 32% and 53%) when 0.5 ng/ml LPS was added. At an even lower concentration of LPS, 0.1 ng/ml, only one of four experiments resulted in a significant diminution (33%) of CCL2 production by IL-10. With higher amounts of LPS (2 ng/ml), IL-10 did not inhibit secretion of CCL2 in three experiments. IL-10 also did not reduce secretion of CCL2 by HUVEC treated with 0.01 (data not shown), 0.1, or 5.0 U/ml [11 ] IL-1ß. Similarly, in five separate experiments, IL-10 did not diminish the amount of CCL2 produced by HUVEC stimulated with concentrations of TNF-{alpha} ranging from 0.01 to 1.0 ng/ml. The only exception was a small (13%) but significant decrease in one of three experiments performed using 0.01 ng/ml TNF-{alpha} (data not shown).

To determine whether IL-10 inhibits production of CXCL8 and CCL2 through regulation of mRNA expression, total RNA was isolated from untreated HUVEC or HUVEC that were treated for 24 h with B. burgdorferi (10 Bb/EC) or IL-1ß (0.05 U/ml) in the absence or presence of 20 ng/ml IL-10. Amounts of CXCL8 and CCL2 mRNA were determined and normalized to levels of mRNA for GAPDH. As shown in Tables 2 and 3, exposure of HUVEC to B. burgdorferi markedly increased levels of CXCL8 and CCL2 mRNA, and this increase was inhibited by IL-10. In sharp contrast, IL-10 had no effect on expression of CXCL8 or CCL2 mRNA in HUVEC stimulated by IL-1ß. Although CXCL8 mRNA was significantly up-regulated in HUVEC exposed to IL-1ß for 24 h compared with untreated cells, the amounts were relatively low. To determine whether the kinetics of up-regulation of chemokine mRNA might be different in response to IL-1ß versus B. burgdorferi, experiments were performed using a shorter period of stimulation, 4 h. At this time point, B. burgdorferi induced little to no increase in mRNA encoding CXCL8 and CCL2, but IL-1ß up-regulated both more robustly than at 24 h. IL-10 did not diminish amounts of CXCL8 mRNA (Table 2) or CCL2 mRNA (Table 3) in HUVEC stimulated with IL-1ß for 4 h, consistent with our observations at 24 h.


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  		Table 2. Effect of IL-10 on Levels of CXCL8 mRNA

 

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  		Table 3. Effect of IL-10 on Levels of CCL2 mRNA

 
Endothelium that is activated by proinflammatory agents not only produces chemokines, but also up-regulates expression of adhesion molecules for leukocytes. Therefore, the effects of IL-10 on expression of endothelial E-selectin and ICAM-1 were examined. A whole cell ELISA was used to measure up-regulation of these adhesion molecules on intact, living monolayers of HUVEC in response to B. burgdorferi, LPS, IL-1ß, and TNF-{alpha} in the absence or presence of 20 ng/ml IL-10. IL-10 did not inhibit the up-regulation of E-selectin (Fig. 5 ) or ICAM-1 (data not shown) by HUVEC in response to any of the stimuli used.



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  		Figure 5. IL-10 does not affect the expression of E-selectin by HUVEC regardless of stimulus. HUVEC were incubated with control medium (Unstim), B. burgdorferi at a ratio of 10 bacteria/EC (Bb), 1 ng/ml LPS, 0.01 U/ml IL-1ß, or 0.1 ng/ml TNF-{alpha} in the absence (No Addition) or presence of 20 ng/ml IL-10. Expression of E-selectin was measured by whole cell ELISA with mAb BB11 after 4 h of incubation. Bars represent the means ± SD of four to eight replicate samples per experimental group. Similar results were obtained in three additional experiments using IL-1ß and B. burgdorferi as stimuli and five experiments using LPS and TNF-{alpha}.

 
As endothelial-derived chemokines are involved in recruitment of leukocytes [10 , 11 , 20 ], the effect of IL-10 on transendothelial migration of leukocytes in vitro was studied. Monolayers of HUVEC cultured on acellular, amniotic tissue mimic authentic endothelium with respect to morphology and permeability properties and thus serve as in vitro models of the microvascular wall [17 ]. Previous work from our laboratory has shown that stimulation of HUVEC-amnion cultures with IL-1ß or B. burgdorferi increases the rate at which monocytes subsequently migrate across the endothelial monolayers [11 ]. Inclusion of IL-10 during the stimulation period inhibits migration of monocytes across HUVEC exposed to B. burgdorferi, but not IL-1ß [11 ].

A possible shortcoming of our previous study, however, is that the concentration of IL-1ß that was used (5.0 U/ml) was far greater than needed to provoke maximal activation of the EC. Potentially, then, sensitivity of IL-1ß to IL-10 could have been masked by excessive amounts of stimulus used. Using the same HUVEC-amnion model system, we therefore repeated this experiment using only 0.05 U/ml IL-1ß, a concentration that induces submaximal activation of endothelium, as assessed by up-regulation of E-selectin (Fig. 5) . As seen in Figure 6 , IL-10 again had no effect on the number of monocytes that migrated across HUVEC that had been pretreated with IL-1ß, even using this much lower concentration of stimulus. This result supports our previous conclusion that activation of endothelium by B. burgdorferi, but not IL-1ß, is suppressed by IL-10.



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  		Figure 6. IL-10 does not inhibit the migration of monocytes across HUVEC stimulated by IL-1ß. HUVEC-amnion cultures were treated for 8 h with control medium (Unstim) or 0.05 U/ml IL-1ß in the absence or presence of 20 ng/ml IL-10. Cultures were washed, and monocytes were added to the apical sides of the cultures for 20 min. The total height of each bar represents the number of monocytes associated with each culture as a percentage of the total added. The lower, hatched portion of each bar represents the percentage that migrated beneath the endothelium; the upper, open portion represents the percentage adherent to the apical surface of the endothelium. Bars represent the means ± SD of five replicate samples.

 
We next tested TNF-{alpha} and LPS at concentrations that provoked migratory responses that were similar in magnitude to those produced by IL-1ß in the study described above. Migration of monocytes was not diminished when HUVEC were activated with TNF-{alpha} in the presence of IL-10 compared with control cultures incubated with TNF-{alpha} only. In contrast, the number of monocytes that migrated across HUVEC activated with LPS was decreased in two separate experiments, by 67% and 77%, when IL-10 was included during stimulation (Fig. 7 ).



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  		Figure 7. IL-10 inhibits the migration of monocytes across HUVEC stimulated by LPS but not TNF-{alpha}. HUVEC-amnion cultures were incubated for 8 h with control medium (Unstim), 4 ng/ml LPS, or 0.2 ng/ml TNF-{alpha} in the absence (No Addition) or presence of 20 ng/ml IL-10. Cultures were washed, and monocytes were added to the apical sides of the cultures for 20 min. The total height of each bar represents the number of monocytes associated with each culture as a percentage of the total added. The lower, hatched or cross-hatched portion of each bar represents the percentage that migrated beneath the endothelium; the upper, open portion represents the percentage adherent to the apical surface. Bars represent the means ± SD of five replicate samples. *, Significantly different from the LPS-stimulated group in the absence of IL-10 (P<0.001). This experiment was repeated once, yielding similar results.

 
Recently, we reported that IL-10 also inhibits (by 70%±21%) migration of T lymphocytes across HUVEC stimulated with B. burgdorferi but not IL-1ß [12 ]. To further examine the specificity of IL-10, HUVEC were treated with LPS or TNF-{alpha} in the absence or presence of IL-10. Similar to results obtained with monocytes, migration of T lymphocytes was suppressed (by 71% and 57%) when HUVEC were stimulated with LPS in the presence of IL-10 in two separate experiments. However, IL-10 did not inhibit migration of T lymphocytes across TNF-{alpha}-stimulated endothelium (Fig. 8 ).



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  		Figure 8. IL-10 inhibits the migration of T lymphocytes across HUVEC stimulated by LPS but not TNF-{alpha}. HUVEC-amnion cultures were treated for 8 h with control medium (Unstim), 4 ng/ml LPS, or 0.2 ng/ml TNF-{alpha} in the absence (No Addition) or presence of 20 ng/ml IL-10. Cultures were washed, and T lymphocytes were added to the apical sides of the cultures for 4 h. The total height of each bar represents the number of T lymphocytes associated with each culture as a percentage of the total added. The lower, hatched or cross-hatched portion of each bar represents the percentage that migrated beneath the endothelium; the upper, open portion represents the percentage adherent to the apical surface. Bars represent the means ± SD of three to five replicate samples. *, Significantly different from the LPS-stimulated group in the absence of IL-10 (P<0.001). This experiment was repeated once, yielding similar results.

 
Finally, migration of neutrophils across HUVEC monolayers stimulated by the four proinflammatory agents in the absence or presence of IL-10 was measured. IL-10 significantly reduced migration (by 20%) in only one of five experiments when B. burgdorferi was used as the stimulus. There was no significant decrease when HUVEC were stimulated with IL-1ß, TNF-{alpha}, or LPS in the presence of IL-10 (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The anti-inflammatory properties of IL-10 have been investigated primarily using mononuclear leukocytes; only a limited number of published studies have examined the effects of this cytokine on EC [11 , 12 , 24 25 26 27 28 29 30 31 32 ]. We previously found that IL-10 reduces migration of monocytes [11 ] and T lymphocytes [12 ] across B. burgdorferi-stimulated endothelium. In contrast, migration of these leukocytes across HUVEC activated by IL-1ß is not affected by IL-10. In this report, we extend these findings to show that IL-10 diminished migration of monocytes and T lymphocytes across monolayers of EC stimulated by LPS, but not TNF-{alpha}. Moreover, IL-10 decreased endothelial production of chemokines in response to B. burgdorferi and LPS, but not to IL-lß or TNF-{alpha}. In addition, amounts of CXCL8 and CCL2 mRNA in HUVEC exposed to B. burgdorferi but not IL-1ß were decreased by IL-10. Interestingly, IL-10 did not prevent up-regulation of adhesion molecules by EC exposed to any of these activating agents. The actions of IL-10 on endothelium thus appear to be specific with respect to effector function and inciting stimulus. This selective inhibition by IL-10 suggests that bacterial stimuli and host cytokines use different mechanisms to activate EC.

Chen and Manning [26 ] have observed that IL-10 reduces production of CXCL8 by 39%, 33%, and 40% when HUVEC are activated with IL-1ß, TNF-{alpha}, or LPS, respectively. Although their data conflict with our observation that IL-10 has no effect on proinflammatory activation of endothelium in response to IL-1ß or TNF-{alpha}, we found similar results with regard to LPS. In addition, other studies support our data. Specifically, it has been reported that IL-10 does not diminish production of CCL2, CXCL8, CXCL1 (GRO{alpha}), or CCL5 (RANTES) [30 ] by endothelium treated with IL-1ß nor does it inhibit secretion of CCL5 in response to TNF-{alpha} and IFN-{gamma} [25 ]. In contrast to our results, De Beaux et al. [24 ] have observed that IL-10 enhances secretion of CXCL8 by HUVEC exposed to LPS. It is possible that differences in EC culture conditions account for this discrepancy, as De Beaux et al. [24 ] grew HUVEC in medium supplemented with growth factors and hydrocortisone, which we did not use, and stimulated the cells in the presence of human rather than bovine serum. In our studies, IL-10 alone did not induce secretion of CXCL8 (Fig. 4) or CCL2 (data not shown) by HUVEC. In contrast, IL-10 has been reported to increase production of CXCL8 by HMEC-1, a transformed line of human dermal microvascular EC [31 ].

Others have demonstrated that IL-10 diminishes levels of mRNA encoding various cytokines in a number of cell types [33 34 35 36 37 38 39 40 41 ], so we examined whether IL-10 reduced the amounts of mRNA for CXCL8 and CCL2 in HUVEC stimulated with B. burgdorferi. Indeed, levels of CXCL8 and CCL2 mRNA were decreased by IL-10 when HUVEC were exposed to B. burgdorferi but not IL-1ß (Tables 2 and 3) . This decrease could be a result of transcriptional [33 , 34 , 37 , 39 , 40 ] or post-transcriptional [33 , 35 , 36 , 38 , 41 ] mechanisms, as both have been observed in IL-10-mediated reduction of cytokines in other cell types. In several types of cells other than endothelial, IL-10 has been shown to reduce activation of the transcription factor nuclear factor {kappa}B (NF-{kappa}B) [42 43 44 45 46 47 ]. NF-{kappa}B is involved in the regulation of many proinflammatory genes such as CXCL8 and CCL2 [48 ]. IL-1ß [49 ], B. burgdorferi [9 ], TNF-{alpha} [49 ], and LPS [50 ] activate NF-{kappa}B in EC. If IL-10 is exerting its selective actions on endothelium through NF-{kappa}B, then the upstream signaling cascades induced by the bacterial agents and host cytokines must differ in their sensitivity to IL-10. Alternatively, bacterial stimuli and host cytokines may induce endothelial production of different factors that work in concert with NF-{kappa}B, and perhaps IL-10 inhibits only those cofactors activated in response to the bacterial agents.

When exposed to proinflammatory mediators, EC up-regulate expression of chemokines and adhesion molecules for leukocytes [4 ]. We expected that expression of these adhesion molecules would be reduced by IL-10 in a stimulus-specific manner, as we observed for chemokines. Surprisingly, however, IL-10 did not diminish expression of E-selectin or ICAM-1 in response to any of the four stimuli that we tested. This result indicates that at least in response to bacterial agents, identical pathways do not regulate the expression of chemokines and adhesion molecules. Consistent with our data, others have reported that IL-10 does not reduce levels of E-selectin, ICAM-1, or vascular cell adhesion molecule-1 (VCAM-1) on EC activated by IL-1, TNF-{alpha}, or a combination of the two cytokines [28 , 51 ]. In contrast, it has been noted that IL-10 decreases the amounts of ICAM-1 and VCAM-1 detected by ELISA in IL-1-treated HUVEC that have been fixed with ethanol [52 ]. At a relatively high concentration (100 ng/ml), IL-10 also reduces the percentage of transformed human microvascular EC that express ICAM-1 after stimulation with LPS, as assessed by flow cytometry [27 ].

Although we saw no effect of IL-10 on expression of adhesion molecules by endothelium, we reasoned that reduced secretion of chemokines might diminish transendothelial migration of leukocytes. Indeed, in this report and previous work [11 , 12 ], we found that IL-10 consistently and markedly diminished migration of monocytes and T lymphocytes across HUVEC stimulated with bacterial agents, but not host cytokines. Studies by others have shown that IL-10 decreases adhesion of human monocytes [32 ], monocytic cell lines [52 , 53 ], or a lymphoblastic T cell line [52 ] to unstimulated endothelium [32 , 53 ] or endothelium treated with IL-1 [52 ]. However, it is not known whether this diminished adhesion would necessarily result in decreased transendothelial migration. In agreement with our results, IL-10 inhibits the migration of PBMC across monolayers of HUVEC stimulated with LPS [29 ]. Although IL-10 substantially reduced secretion of CXCL8 by HUVEC exposed to either B. burgdorferi, it did not diminish traversal of neutrophils across these monolayers or across endothelium exposed to any stimulus tested. As antibody to CXCL8 almost completely eliminates the migration of neutrophils across B. burgdorferi-stimulated endothelium [10 ], our results are not likely to be a result of production of other neutrophil attractants by pathways that are insensitive to IL-10. Rather, it may be that CXCL8 is sufficiently potent that even a 40% reduction does not lead to a decrease in the number of responding neutrophils. Whether such a reduction would have an effect in vivo, where CXCL8 would probably be subject to dilution by tissue fluids and flowing blood, remains an open question.

Although IL-10 reduced secretion of chemokines by EC exposed to either B. burgdorferi or LPS, inhibition was more profound and consistent when B. burgdorferi was the stimulus. This observation suggests that B. burgdorferi and LPS use different mechanisms to activate endothelium. Consistent with this idea, B. burgdorferi lacks LPS and instead stimulates HUVEC via its lipidated outer surface proteins [13 ]. These lipoproteins signal through toll-like receptor 2 (TLR2) [54 , 55 ], whereas LPS is a ligand for TLR4 [56 ]. The use of different receptors and subsequent formation of different complexes for signaling may explain why B. burgdorferi and LPS differ in their sensitivity to IL-10, at least with respect to their ability to induce production of chemokines in endothelium. In contrast, our present and previous [11 , 12 ] work shows that transendothelial migration of monocytes and T lymphocytes is strongly and consistently reduced by IL-10 when the endothelium is activated by B. burgdorferi or LPS. Interestingly, the amount of LPS used in these transmigration studies was outside the range in which we noted inhibition of secretion of CCL2 by IL-10. Moreover, CCL2 is not the major chemoattractant that mediates migration of monocytes across B. burgdorferi-stimulated HUVEC [11 ]. It is unlikely, then, that a reduction in CCL2 fully explains the ability of IL-10 to reduce migration of mononuclear leukocytes across endothelium exposed to bacterial agents. Rather, other as-yet-unidentified factors that are critical to the process must also be affected.

Our results indicate that IL-10 may play an especially important role in regulating inflammation during Lyme disease. This conclusion is supported by the observation that IL-10-deficient mice develop more severe arthritis than do their wild-type counterparts when infected with B. burgdorferi. The infected tissues of the IL-10-deficient mice also contain tenfold fewer bacteria than do those of wild-type mice [57 ]. Together, these data indicate that IL-10 normally serves to reduce recruitment of leukocytes to tissues infected with B. burgdorferi, which limits the severity of inflammation but impairs the ability of the host to eliminate the bacteria. Our observations raise the possibility that IL-10 may be suppressing extravasation of leukocytes, at least in part, by acting on the vascular endothelium. However, IL-10 may also regulate inflammation in Lyme disease through other mechanisms. For example, IL-10 reduces production of proinflammatory cytokines by monocytic cells stimulated with lipoproteins from B. burgdorferi [58 ].

The data presented herein provide more extensive support for the conclusion that IL-10 has selective actions on endothelium with regard to stimulus and effector responses. Specifically, IL-10 reduces proinflammatory activation of endothelium by B. burgdorferi and LPS, but not by host-derived cytokines. These results raise the possibility that IL-10 may be of particular therapeutic value in treating inflammation associated with bacterial infections.


   ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health grant AI47313 and research awards from the National Office and Long Island Chapter of the Arthritis Foundation. We thank Jorge L. Benach and Howard B. Fleit for critical review of the manuscript.

Received January 25, 2002; revised March 6, 2002; accepted April 11, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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