Introduction

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Cell adhesion molecule (CAM) expression on the vascular endothelium facilitates leukocyte recruitment to sites of inflammation (1). Leukocytes bind to intercellular adhesion molecule-1 (ICAM-1), E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) expressed on endothelial cells via leukocyte counter-ligands: (1) lymphocyte function-associated antigen-1 (LFA-1; CD11a/CD18) and macrophage antigen-1 (Mac-1; CD11b/CD18); (2) L-selectin, sialyl-Lewis X and related carbohydrate structures; and (3) very late antigen-4 (VLA-4; ₄₁) or Act-1 (₄₇), respectively (2). VCAM-1, in particular, is thought to play a key role in the manifestation of airway inflammation associated with asthma. Endothelial VCAM-1 expression in bronchial mucosa biopsies from asthmatics has been correlated with eosinophil migration into the airways (5, 6). In vivo studies, using blocking antibodies against VCAM-1 and/or VLA-4 also show the importance of the VCAM-1/ VLA-4 pathway in eosinophil or lymphocyte recruitment into the lung or airway lumen of rats and mice following antigen challenge (7, 8). In vitro studies show that VCAM-1 mediates, in part, adhesion to cytokine-activated endothelial cells (EC) of eosinophils, basophils, lymphocytes, or monocytes but not neutrophils since the latter do not express VLA-4 (9).

VCAM-1, a 110-kD member of the immunoglobulin gene superfamily (17), is not constitutively expressed on EC. The contribution of VCAM-1 to the inflammatory process is thought to result from a distinct pattern of induction following exposure to inflammatory cytokines such as interleukin (IL)-1 or tumor necrosis factor- (TNF-) and also lipopolysaccharide (LPS) (17, 18). Increased concentrations of IL-1 and TNF- have been measured in the bronchiolar lavage fluid (BALF) of symptomatic asthmatics (19, 20). Although similar concentrations of LPS have been detected in asthmatic and nonasthmatic BALF, levels of two LPS accessory molecules, LPS-binding protein (LBP) and soluble CD14 (sCD14) were increased in post-antigen challenge asthmatic BALF (21). Moreover, asthma severity in patients exposed to house dust mite has been shown to be related to LPS, rather than allergen, concentration in house dust (22). IL-4, a 20-kD glycoprotein secreted by activated T lymphocytes and mast cells (23, 24) has also been detected in increased levels in BALF of allergic asthmatics and implicated in the pathogenesis of asthma (6, 24). It is known that IL-4 induces VCAM-1 expression on EC derived from several vascular sites, including umbilical or saphenous vein, dermal microvessels, or lymph node (9, 15, 25), and increases IL-1-, TNF--, or LPS-induced VCAM-1 expression (9, 25). Unlike IL-1, TNF-, or LPS, IL-4 had little or no effect on ICAM-1 or E-selectin expression (15, 25, 28, 30).

Much of the extensive work, to date, examining the effects of cytokines or LPS on endothelial CAM expression and/or leukocyte adhesion has been carried out using EC derived from human umbilical vein or microvessels of skin or intestine, as detailed above. There is evidence, however, for heterogeneity of EC between different vascular sites (30). Thus, it may be necessary to use EC derived from the microvasculature of the lung in in vitro studies that seek to investigate the role of endothelial CAM in the pathogenesis of inflammatory conditions within this organ. Previous studies have suggested cytokine modulation of CAM expression in human lung microvascular endothelial cells (HLMVEC) and murine LMVEC (33); however, specific analysis of the effects of agents present in allergic airways on VCAM-1 induction in HLMVEC has not, to our knowledge, been performed. Thus, the aim of the present study was to investigate the capacity of IL-4 alone and in combination with LPS to induce VCAM-1 expression on HLMVEC and enhance eosinophil adhesion.

	Materials and Methods

Top Abstract Introduction Materials & Methods Results Discussion References

Cell Culture Reagents

HLMVEC, prepared by Clonetics (San Diego, CA), were obtained from three donors, as cryopreserved third passage (3°C) cultures from TCS Biologicals Ltd. (Buckingham, UK). Microvascular endothelial growth medium (EGM-MV), 0.025% trypsin + 0.01% EDTA, Hanks' balanced salt solution (HBSS), and trypsin neutralizing solution were also obtained from TCS Biologicals Ltd. Dulbecco's phosphate-buffered saline (PBS) (with or without Ca²⁺/ Mg²⁺) was purchased from Gibco Laboratories (Paisley, UK).

Cytokines and Other Reagents

Human recombinant (hr) TNF- and IL-1 were obtained from Boehringer Mannheim UK (Lewes, East Sussex, UK; specific activity > 1 × 10⁸ or 5 × 10⁷ U/mg, respectively). Hr IL-4 was obtained from R&D Systems (Oxford, UK; specific activity > 2.9 × 10⁷ U/mg). The following products were purchased from Sigma Chemical Co. Ltd. (Poole, Dorset, UK): 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), gelatin, goat serum, horse serum, fetal calf serum (FCS), hydrogen peroxide (H₂O₂), dimethyl sulfoxide, LPS from Escherichia coli (055:B5), potassium chloride (KCl), calcium chloride (CaCl₂), magnesium sulfate (MgSO₄). Percoll and dextran were obtained from Pharmacia Biotech Ltd. (St. Albans, UK), sterile normal saline (0.9%) from FL (Manufacturing) Ltd., Fresenius Health Care Group (Basingstoke, Hampshire, UK) and very low endotoxin bovine serum albumin (BSA) from Bayer Ltd. (Basingstoke, Hampshire, UK). Citric acid, EDTA, di- sodium hydrogen orthophosphate (Na₂HPO₄), sodium dihydrogen orthophosphate (NaH₂PO₄), and D-glucose were obtained from BDH Chemicals Ltd. (Poole, Dorset, UK). MACS CS separation columns and CD16 microbeads were purchased from Miltenyi Biotech (Camberley, Surrey, UK). Calcein-AM was obtained from Cambridge Bioscience (Cambridge, UK).

Antibodies

Affinity isolated goat anti-mouse peroxidase conjugate gamma and light chain specific was obtained from TCS Biologicals Ltd. Mouse anti-human ICAM-1 (RR1/1) IgG₁ mAb (36) was provided by Dr. R. Rothlein (Boehringer Ingelheim Pharmaceuticals, Ridgefield, CT). Mouse IgG_1-k (MOPC21) and mouse anti-human CD18 (6.5E) IgG₁ monoclonal antibody (mAb) were gifts from Dr. M. Robinson (Celltech, Slough, UK). Mouse anti-human IgG₁ mAbs against E-selectin (1D2), VCAM-1 (4B2), and VLA-4 (2B4) were generous gifts from Dr. R. Pigott (British Biotech, Oxford, UK) and against the  chain of the IL-4 receptor (IL-4R; CDw124) was obtained from Coulter Electronics (Luton, Bedfordshire, UK). Mouse IgG₁ isotype-matched control mAb and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse polyclonal antibody were purchased from Dako (Buckinghamshire, UK).

Molecular Biology Reagents

Formamide, RNase-free water, salmon sperm DNA, socium dodecyl sulfate (SDS), Tris-hydrochloric acid (HCl), Ficoll, Polaroid Film Type 667, and guanidinium isothiocyanate were obtained from Sigma Chemical Co. Ltd. Propan-2-ol, hydrochloric acid, chloroform, polyvinylpyrrolidone, EDTA, phenol, and formaldehyde were obtained from BDH Chemicals Ltd. and ethanol from Hayman Limited (Whitham, Essex, UK). Hybond N membranes, [-³²P]- labeled deoxycytidine 5'-triphosphate (dCTP: > 3,000 Ci/mM), Multiprime DNA labeling system kits were obtained from Amersham (Amersham, Bucks, UK). Agarose was obtained from Promega (Southampton, Hampshire, UK). XOMAT-S Kodak autoradiography film was obtained from Keith Johnson and Pelling (London, UK). Plasmid vector containing the VCAM-1 cDNA insert was a kind gift from Dr. D. Simmons (Imperial Cancer Research Fund, Institute of Molecular Medicine, Oxford, UK), and the 1-kb probe was initially liberated from the plasmid vector by use of the digestion enzymes HindIII and Pst1 and purified using Gen Clean (Bio 101, Luton, Bedfordshire, UK). A 598-bp cDNA fragment of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was obtained as previously described (37).

Cell Culture

HLMVEC isolated from peripheral lung tissue (avoiding pleura and large blood vessels) have been shown to retain a number of properties of EC, including the production of human factor VIII-related antigen, uptake of acetylated LDL, and expression of CD31 (33). We have also confirmed that these cells were positive for factor VIII-related antigen and CD31 expression. HLMVEC were maintained in EGM-MV medium, a modification of MCDB 131, supplemented with 10 ng/ml hr epidermal growth factor, 1 µg/ml hydrocortisone, 5% heated-inactivated FCS, 50 µg/ml gentamycin, 50 ng/ml amphotericin-B, and bovine brain extract containing 12 µg/ml protein and 10 µg/ml heparin. Basal, TNF--, IL-1-, or LPS-induced CAM expression did not change as HLMVEC were passaged. In contrast, IL-4-induced VCAM-1 expression was only detected in HLMVEC less than passage 12; therefore, monolayers of passage 12 or less were used in these experiments. Hydrocortisone, in the culture medium, also had no discernible effect on CAM expression. Confluent cells were washed with HBSS, trypsinized using 0.025% trypsin + 0.01% EDTA and collected into trypsin neutralizing solution. Cells were seeded at a density of 3.2 × 10³ cells per well onto 1% gelatin-coated flat-bottomed Nunclon 96-well microtiter plates. Four days after seeding confluent monolayers were used for enzyme-linked immunosorbent assays (ELISA) or adhesion assays. The confluent cell density was approximately 4 × 10⁴ cells per well, and this was consistent between replicate wells and plates. HLMVEC were also grown on 24- or 6-well plates for IL-4R analysis or mRNA detection, respectively, and seeding densities for these plates are given in the relevant MATERIALS AND METHODS section.

ELISA for ICAM-1, VCAM-1, and E-selectin Expression

ICAM-1, VCAM-1, and E-selectin were detected by an ELISA method (38) using mouse anti-human ICAM-1 (RR1/1), VCAM-1 (4B2), or E-selectin (1D2) primary mAbs, and a peroxidase-linked goat anti-mouse secondary antibody. Briefly, confluent HLMVEC monolayers in Nunclon 96-well plates were incubated with: (1) TNF-, IL-1, or LPS alone (for 6, 16, 24, 48, or 72 h) or in combination with IL-4 (for 6, 24, 48, or 72 h), with concentrations given in the RESULTS section; (2) LPS (1 µg/ml) or IL-4 (100 ng/ml) for 1 h, after which the monolayers were washed twice with PBS without Ca²⁺ and Mg²⁺ and then treated with IL-4 or LPS, respectively, for 23 h. Cytokines or LPS were diluted in serum-supplemented complete culture media. After removal of stimuli, cells were washed three times with PBS containing Ca²⁺, Mg²⁺, and 0.1% BSA and incubated for 45 min with 1 µg/ml RR1/1, 4B2, or 1D2 or MOPC21 diluted in complete medium. Primary antibody was removed by washing, and HLMVEC monolayers were incubated (45 min) with a 1:1,000 dilution (in PBS + 10% goat serum) of goat anti-mouse peroxidase conjugate followed by incubation with a peroxidase-sensitive substrate, ABTS (1 mg/ml, in 0.2 M citrate/phosphate buffer, pH 5, containing 0.1% H₂O₂) for 30 min. The reaction was terminated by addition of 0.2 M citrate. All incubations were carried out at room temperature. Chromophore development was determined by measuring optical density (OD) at 405 nm (OD₄₀₅) using a Titretec MCC/340 Multiscan microplate reader. Background absorbance was determined from monolayers incubated without primary antibody and this value was then subtracted from the absorbance readings. Reported data are derived from OD readings which fall along the linear portion of the development curve. Adhesion molecule expression is given as OD.

Flow Cytometric Analysis of EC IL-4R Subunit

Confluent HLMVEC monolayers in Nunclon 24-well plates (seeded at a density of 2 × 10⁴ cells/well) were incubated with culture medium, LPS (1 µg/ml), IL-4 (100 ng/ml), IL-4/ LPS, or TNF- (1 ng/ml) for 24 h. Monolayers were washed once with HBSS and detached by incubation with 0.025% trypsin + 0.01% EDTA for 5 min. One hundred microliters of HLMVEC, resuspended at 1 × 10⁶ cells/ml in PBS containing BSA (1% wt/vol), were incubated with anti-IL-4R mAb (50 µg/ml) for 45 min at room temperature. A mouse IgG₁ isotype-matched mAb was substituted for anti-IL-4R mAb in control experiments to determine background fluorescence. HLMVEC were washed twice with PBS/BSA (1%), incubated with a 1:20 dilution of goat anti-mouse IgG-FITC for 30 min, washed twice, and resuspended in FACSFlow. FITC fluorescence was determined using a FACScan flow cytometer (Becton Dickinson, Oxford, UK) and analyzed using Chalcocite software. Results were expressed as fold increases in geometric mean fluorescence intensity from that for the isotype-matched control mAb.

RNA Isolation and Northern Analysis

HLMVEC monolayers grown in 6-well plates (1 × 10⁶ cells/well) were incubated with culture medium, LPS (1 µg/ml), IL-4 (100 ng/ml), or IL-4/LPS. Total cellular RNA was extracted at time zero (t = 0) from untreated HLMVEC or at 2, 4, or 8 h from untreated, LPS-, IL-4-, or LPS/IL-4-treated HLMVEC, using the guanidinium isothiocyanate method as described by Chomzcynski and Sacchi (39). Briefly, media was removed and the cells were lysed with 800 µl of guanidinium isothiocyanate. Phenol (800 µl) and chloroform (200 µl) were added to each sample and then shaken vigorously, incubated at 4°C (15 min), and centrifuged (12,000 × g for 10 min, 4°C). RNA in the upper aqueous phase was precipitated using an equal volume of propan-2-ol (overnight at 70°C) and centrifugation (12,000 × g for 30 min, 4°C). The pellets were washed once with 75% ethanol (1 ml of 75% ethanol/sample; centrifugation for 30 min at 12,000 × g, 4°C). At the end of the procedure, the pellets were freeze-dried and dissolved in 10 µl of RNase-free water and total RNA was quantified by measuring absorbance at 260 nm (Gene Quant Pharmacia, St. Albans, UK). RNA samples (8 µg/lane) were electrophoresed on denaturing 1% formaldehyde-agarose gels, photographed using Polaroid film (Type 667), and transferred with 20× standard saline citrate (SSC) buffer (3 M NaCl, 0.3 M sodium citrate; pH 7.0) onto Hybond N nylon membranes and fixed by exposure to ultraviolet radiation (using an XL-1000 UV Crosslinker; Spectronics Corporation, New York, NY).

Filters were prehybridized for 4 h at 42°C in buffer consisting of 4× SSC, 50% formamide, 50 mM Tris-HCl (pH 7.5), 5× Denhardt's solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), 0.1% SDS, 5 mM EDTA, and 250 µg/ml sonicated denatured salmon sperm DNA. VCAM-1 cDNA probe (50 ng) was labeled with [-³²P]dCTP (> 3,000 Ci/mmol) by random priming, using a Multiprime DNA labeling system, and added to the prehybridization chambers at a final strength of 1 × 10⁶ cpm/ ml and incubated for 12 to 16 h at 42°C. After hybridization, the filters were washed to a stringency of 0.1× SSC/ 0.1% SDS for 30 min at 55°C. The membranes were autoradiographed at 70°C by exposure to Kodak XOMAT-S film for 1 to 2 d. After exposure, blots were stripped in 50% formamide, 10 mM NaH₂PO₄ for 1 h at 65°C before subsequent rehybridization. To account for difference in loading or transfer of the RNA, hybridization was performed with ³²P-labeled GAPDH cDNA probe (housekeeping gene whose expression is not altered by cytokine/ LPS treatment). Quantification of GAPDH and VCAM-1 mRNA was made using a scanning laser densitometer, attached to a computer with image analysis software (Quantity One Software, PDI, New York, NY), which was calibrated before use and gave linear readings over 4 OD units.

Separation of Human Peripheral Blood Eosinophils or Neutrophils

Granulocytes were isolated from peripheral blood of normal or mildly atopic adult donors, for neutrophils or eosinophils, respectively, by the method of Haslett and colleagues (40) as described by Burke-Gaffney and Hellewell (41). Briefly, blood was collected to a total volume of 40 ml into 3.8% citrate and spun for 20 min at 300 × g. The platelet-rich plasma was removed, underlaid with 90% Percoll, and spun at 2,000 × g for 20 min at room temperature (temperature used unless otherwise stated) to produce platelet-poor plasma (PPP). To the lower buffy coat produced by the first spin, 5 ml of 6% dextran was added and the volume made up to 50 ml with 0.9% saline. This was allowed to stand for 30 min for erythrocyte sedimentation to occur. The leukocyte-rich supernatant was removed and centrifuged at 300 × g for 8 min. The pellet was resuspended in PPP and layered onto freshly prepared discontinuous Percoll-plasma gradients (42 and 51% Percoll in PPP) and centrifuged for 10 min at 260 × g. The granulocyte band was collected, washed in PPP, and resuspended in Krebs Ringer phosphate dextrose (KRPD) buffer (4.8 mM KCl; 3.1 mM NaH₂PO₄; 12.5 Na₂HPO₄: 5% vol/vol glucose; 2% vol/vol FCS). Granulocytes obtained from normal donors were > 98% neutrophils. Granulocytes obtained from mildly atopic donors were used to purify eosinophils and were incubated with anti-CD16 microbeads to remove neutrophils (1:10 dilution when 40 µl of beads were added per 10⁸ granulocytes for 45 min). A Type-CS MACS separation column was set up in a magnetic separator and microbead-labeled granulocytes applied to the top of the column. Eosinophils (> 97% pure) were eluted in 35 ml KRPD buffer. Eosinophils or neutrophils (5 × 10⁶ cells/ml) were labeled (30 min, 37°C) with a fluorescent dye, calcein-AM (10 µM dissolved in 1% dimethyl sulfoxide in KRPD without FCS). Cells were washed twice in KRPD without FCS and resuspended at 1.25 × 10⁶ cells/ml in KRPD containing 2.5% FCS, 0.93 mM CaCl₂, and 1.2 mM MgSO₄ for the adhesion assay.

Measurement of Eosinophil or Neutrophil Adhesion to Lung Microvascular Endothelial Cells

HLMVEC monolayers grown on 96-well plates were pre-treated with LPS (1 µg/ml), IL-4 (100 ng/ml), or LPS/IL-4 for 24 h. Monolayers were washed three times with PBS (containing Ca²⁺/Mg⁺) to remove stimuli before carrying out the adhesion assay. One hundred microliters of 2× final concentration 6.5E (60 µg/ml), 2B4 (60 µg/ml), MOPC21 (60 µg/ml) or KRPD with Ca²⁺/Mg²⁺ were added per well, followed by 100 µl of calcein-AM-labeled granulocytes and the plate was incubated at 37°C for 30 min (42). Fluorescence was measured using a Biolite F1 plate reader (excitation wavelength of 485 ± 25 nm and emission wavelength of 530 ± 25 nm) before and after washing the plate to remove nonadherent cells by two gentle washes with PBS containing 1% horse serum. Percent adhesion was expressed as fluorescence after washing the plate minus the background fluorescence, divided by fluorescence before washing plate minus background ×100.

Statistics

Results are expressed as mean ± SEM of n experiments. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls multiple comparison test which compares all values to each other unless otherwise stated. Instat GraphPad software was used to perform statistical analysis. Results were deemed significant if P < 0.05.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

TNF-, IL-1, or LPS Induce VCAM-1 and E-selectin Expression on HLMVEC

TNF, IL-1, or LPS induced VCAM-1 and E-selectin in a time- and concentration-dependent manner, although neither CAM was detected on resting HLMVEC (Figures 1A through 1D). TNF- (10 ng/ml) or IL-1 (10 ng/ml) induced significant VCAM-1 expression at 6 h (0.50 ± 0.04 or 0.30 ± 0.02, respectively; n = 5, P < 0.01) and maximum expression at 24 or 16 h, respectively (Figure 1A). LPS (10 µg/ml)-induced VCAM-1 was maximal at 6 h (0.24 ± 0.03, n = 5). VCAM-1 expression remained elevated at 48 or 72 h on TNF--treated but not IL-1- or LPS-treated HLMVEC (Figure 1A). VCAM-1 induction (16 h) was concentration dependent, and significant induction was detected with concentrations of or greater than 0.01 (P < 0.05) or 0.1 ng/ml (P < 0.01) of IL-1 or TNF-, respectively, or 10 ng/ml (P < 0.05) of LPS (Figure 1B). TNF- (10 ng/ml), IL-1 (10 ng/ml), or LPS (10 µg/ml) induced maximal E-selectin expression at 6 h that was reduced at 16 or 24 h and not detected at 48 or 72 h (Figure 1C). Significant increases (P < 0.01) in E-selectin expression (6 h) were detected with concentrations of or greater than 0.01, 0.1, or 1 ng/ml IL-1, TNF-, or LPS, respectively (Figure 1D).

View larger version (24K): [in this window] [in a new window]   Figure 1.   ELISA determination of VCAM-1 (A, B), E-selectin (C, D), or ICAM-1 (E, F ) expression on HLMVEC following: (A, C, E) exposure to culture medium or for 6, 16, 24, 48, or 72 h to TNF- (10 ng/ml; open circles), IL-1 (10 ng/ml; filled circles), or LPS (104 ng/ ml; 10 µg/ml; open squares); (B, D, F  ) exposure to culture medium, 0.001 to 10 ng/ml TNF- or IL-1, or 0.01 to 104 ng/ml LPS for 16 (B), 6 (D), or 24 (F ) h, respectively. Results are shown as means ± SEM of three to five experiments. Statistical analysis of the results was performed using one-way ANOVA followed by Dunnett's test for comparison with control monolayer. To ensure clarity of the figure, statistics and SEM are given in the results section.

Constitutive ICAM-1 Expression on Resting HLMVEC and Upregulation by TNF-, IL-1, or LPS

Constitutive ICAM-1 expression was detected on resting HLMVEC monolayers, since the OD₄₀₅ measured after incubation of monolayers with anti-ICAM-1 mAb (0.23 ± 0.03, n = 5) was significantly greater (P < 0.001) than that measured when monolayers were incubated without primary mAb (0.08 ± 0.01, n = 5) or with a control antibody, MOPC21 (0.07 ± 0.01, n = 3). TNF- (10 ng/ml), IL-1 (10 ng/ml), or LPS (10 µg/ml) induced significant ICAM-1 expression (P < 0.01) at 6 h and maximal expression at 24 h (TNF- or IL-1) or 48 h (LPS); significant expression persisted for at least 72 h (Figure 1E). ICAM-1 induction (24 h) was concentration dependent and significant increases (P < 0.01) were detected with 0.1 to 10 ng/ml of TNF- or IL-1 or 0.01 to 10 µg/ml LPS (Figure 1F).

IL-4 Synergizes with LPS to Induce Expression of VCAM-1 but Not ICAM-1 or E-selectin in HLMVEC

IL-4 (100 ng/ml) caused a small but significant (P < 0.01) induction of VCAM-1 expression at 72 h but not at 6, 24, or 48 h (Figure 2A). LPS (1 µg/ml) induced significant VCAM-1 expression at 6 h only. To investigate effects of IL-4 (100 ng/ml) in combination with LPS, a concentration of LPS (1 µg/ml) was used that alone did not induce VCAM-1 expression at 24, 48 or 72 h. LPS incubated in combination with IL-4 significantly (P < 0.001) induced VCAM-1 at 24, 48, or 72 h (Figure 2A). Lower concentrations of IL-4 (1 or 10 ng/ml) also induced VCAM-1 expression at 72 h (data not shown) and synergized with LPS to induce expression at 24, 48, or 72 h; for example, at 24 h, VCAM-1 induced by LPS combined with 0.1, 1, or 10 ng/ml of IL-4, compared with 100 ng/ml IL-4, was 0.26 ± 0.01, 0.47 ± 0.07, 0.55 ± 0.05, or 0.49 ± 0.08 (OD₄₀₅, n = 4), respectively. IL-4 (100 ng/ml) did not induce E-selectin or ICAM-1 expression, alter LPS-induced expression of either CAM, or modify basal ICAM-1 expression (Figures 2B and 2C).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Effects of IL-4 (100 ng/ml; open squares), LPS (1 µg/ml; filled circles), or IL-4 in combination with LPS (open circles) on (A) VCAM-1, (B) E-selectin, or (C) ICAM-1 expression on HLMVEC at 6, 24, 48, or 72 h. Results show means ± SEM of four experiments. *P < 0.01 and **P < 0.001 denote significant VCAM-1 induction compared with treatment with culture medium. +P < 0.01 denotes a significant difference compared with OD405 measured following IL-4 treatment for 72 h.

Having shown a selective interaction between IL-4 and LPS in combination to induce VCAM-1 expression, we then examined whether preincubation with IL-4 or LPS followed by sequential exposure to the other agent was sufficient to result in enhanced expression. Exposure to LPS or IL-4 alone for 24 h did not result in significant VCAM-1 expression. In contrast, preincubation of HLMVEC monolayers with LPS for 1 h followed by 23 h of exposure to IL-4 induced VCAM-1, although preincubation with IL-4 for 1 h followed by LPS for 23 h was significantly more effective (P < 0.01; Figure 3).

View larger version (15K): [in this window] [in a new window]   Figure 3.   Effects of preincubating HLMVEC monolayers (1 h) with LPS or IL-4 on IL-4- or LPS-induced VCAM-1 expression, respectively, measured at 24 h. Monolayers were exposed for 24 h to culture medium, LPS (1 µg/ml), or IL-4 (100 ng/ml) or to LPS (1 h) followed by IL-4 (23 h) or IL-4 (1 h) followed by LPS (23 h), and VCAM-1 expression was determined by ELISA. Results show means ± SEM of four experiments. *P < 0.001 denotes significant VCAM-1 induction compared with treatment with culture medium. +P < 0.01 denotes a significant difference compared with OD405 measured following LPS (1 h)/IL-4 (23 h) treatment.

Expression of IL-4 Receptors on HLMVEC

A possible mechanism for the synergistic effect of IL-4 and LPS on VCAM-1 expression may be an increase in IL-4 receptor expression on HLMVEC. A significant (P < 0.005, Student's t test) increase in binding of an anti-IL-4R mAb (geometric mean fluorescence = 17.5 ± 1.4, n = 6) compared with an IgG₁ isotype-matched control mAb (11.0 ± 1.1, n = 6) indicated constitutive expression of IL-4 receptor  chain on HLMVEC monolayers incubated with complete culture medium only (24 h). Figure 4 shows fluorescence histograms from a representative experiment (A) and a bar graph of mean values of four to six experiments showing fold increases in geometric mean fluorescence from control mAb (B). IL-4R expression was significantly (P < 0.001) increased following incubation (24 h) of HLMVEC monolayers with TNF- (1 ng/ml; 2.88 ± 0.38-fold increase from control mAb; Figure 4B). In contrast, incubation (24 h) with IL-4 (100 ng/ml), LPS (1 µg/ml), or IL-4/ LPS together did not significantly alter IL-4R expression (Figure 4B).

View larger version (16K): [in this window] [in a new window]   Figure 4.   Expression of IL-4 receptor  chain on HLMVEC following incubation (24 h) of monolayers with culture medium, IL-4 (100 ng/ml), LPS (1 µg/ml), IL-4/LPS (1 ng/ml), or TNF- (1 ng/ml). Expression of IL-4R was measured using a FACScan flow cytometer and is shown as fluorescence histograms from a representative experiment (A) and as fold increases in geometric mean fluorescence (GMF) from the isotype-matched control mAb (11.0 ± 1.1 GMF; B). The key for the histograms (A) are as follows: isotype-matched control mAb (thin solid line); culture medium (shaded histogram); LPS (dotted line); TNF- (thick solid line). Results (B) show means ± SEM of four to six experiments. *P < 0.001 denotes significant IL-4R induction compared with treatment with culture medium.

Synergistic Effect of LPS and IL-4 on VCAM-1 mRNA Induction in HLMVEC

Total RNA was isolated from HLMVEC before (t = 0) and after treatment for 2, 4, or 8 h with culture medium alone, LPS (1 µg/ml), IL-4 (100 ng/ml), or LPS/IL-4 and Northern blot analysis used to measure VCAM-1 or GAPDH mRNA. Induction was expressed as a ratio of VCAM-1/ GAPDH mRNA density, normalized so that the ratio for LPS induction was 100. LPS or LPS/ IL-4 caused induction of VCAM-1 mRNA that was detectable at 2 h, maximal at 4 h, and reduced at 8 h; IL-4 alone caused a lesser effect that was detected at 4 or 8 h only (data not shown). Figure 5 shows blots for GAPDH (A) or VCAM-1 mRNA (B) from a representative experiment and a bar graph (C) of mean VCAM-1/GAPDH ratios for three experiments at 4 h. The VCAM-1/GAPDH mRNA ratio for control, untreated HLMVEC at 4 h (1.9 ± 0.6) was not increased from t = 0 (1.7 ± 0.8; Figure 5). IL-4 alone caused a small increase in the VCAM-1/GAPDH ratio (13.7 ± 0.3) and increased the LPS-induced ratio by approximately twofold (Figure 5).

View larger version (30K): [in this window] [in a new window]   Figure 5.   Effects of IL-4 on LPS-induced VCAM-1 mRNA expression in HLMVEC. Total RNA was isolated from HLMVEC before (t = 0) and after treatment for 4 h with culture medium, LPS (1 µg/ml), IL-4 (100 ng/ml), or LPS/IL-4, and Northern blot analysis was used to measure VCAM-1 or GAPDH mRNA. Induction is expressed as a ratio of VCAM-1/GAPDH mRNA density, normalized so that the ratio for LPS induction was 100. Blots for GAPDH (A) or VCAM-1 mRNA (B) of a representative experiment and a bar graph (C) of the means ± SEM of VCAM-1/ GAPDH ratios for three experiments are shown.

IL-4 and LPS Treatment of HLMVEC Have Synergistic Effects on Eosinophil Adhesion

In these experiments, we investigated whether the synergistic effect of LPS and IL-4 on VCAM-1 induction measured at 24 h (Figure 2) supported eosinophil and/or neutrophil adhesion. Basal adhesion of eosinophils to untreated HLMVEC monolayers (9.6 ± 1.5%, n = 7) was not significantly increased when HLMVEC were pretreated with IL-4 (100 ng/ml; 11.7 ± 1.7%, n = 7) or LPS (1 µg/ml; 15.2 ± 2.5%, n = 6) for 24 h (Figure 6). Pretreatment (24 h) of HLMVEC with IL-4 and LPS in combination, however, caused a significant (P < 0.001) increase in adhesion (26.9 ± 3.3%, n = 6; Figure 6). Neutrophil adhesion to LPS- or LPS/IL-4-treated HLMVEC (15.3 ± 1.9% or 18.9 ± 2.2%, respectively; n = 4) was significantly greater than basal adhesion (4.3 ± 0.7%, n = 4) but not significantly different from each other (Figure 6), demonstrating that the effect of IL-4 and LPS was specific for eosinophils.

View larger version (28K): [in this window] [in a new window]   Figure 6.   Adhesion of eosinophils or neutrophils to HLMVEC pretreated for 24 h with culture medium, IL-4 (100 ng/ml), LPS (1 µg/ml), or IL-4/LPS. Results are expressed as means ± SEM of four to seven separate experiments. *P < 0.01 denotes a significant difference compared with eosinophil or neutrophil adhesion to HLMVEC pretreated with culture medium. +P < 0.01 or NS denote significant or nonsignificant differences, respectively, compared with adhesion to HLMVEC pretreated with LPS alone.

Figure 7 shows that eosinophil adhesion to LPS/IL-4-activated HLMVEC was reduced, compared with a control antibody (MOPC21, 30 µg/ml), by anti-VLA mAb (2B4, 30 µg/ml) but not anti-CD18 mAb (6.5E, 30 µg/ml) toward levels observed with eosinophil adhesion to untreated HLMVEC (Figure 7). In three similar experiments, 2B4 significantly (P < 0.05) reduced eosinophil adhesion to 24.2 ± 4.9% of control adhesion to LPS/IL-4-treated HLMVEC monolayers (100 ± 16%) when compared with MOPC21 (78.9 ± 11%), but 6.5E had no significant effect (84.4 ± 10.8%).

View larger version (28K): [in this window] [in a new window]   Figure 7.   Adhesion of eosinophils to HLMVEC pretreated for 24 h with culture medium, IL-4 (100 ng/ml), LPS (1 µg/ml), or LPS/IL-4 as indicated by bar fill pattern. Eosinophils were exposed during the adhesion assay (30 min) to KRPD buffer, a control antibody MOPC21 (30 µg/ml), anti-VLA-4 (2B4; 30 µg/ml), or anti-CD18 mAb (6.5E; 30 µg/ml). Data are means ± SD of three replicate measurements shown as a representative of three similar experiments.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

This study has shown that for effective induction of VCAM-1 expression in HLMVEC, IL-4 must act in concert with a second inflammatory stimulus, LPS, and that the resulting synergistic increase in VCAM-1 expression supports eosinophil, but not neutrophil, adhesion. To our knowledge, this is the first report to suggest that synergism with a second stimulus, such as LPS, may be necessary for IL-4 to induce VCAM-1 expression in lung microvascular endothelium and that alone IL-4 is an insufficient stimulus. These results contribute to our understanding of the factors responsible for VCAM-1 induction in allergic asthma and may have important implications for the development of new anti-asthma therapies.

In the present study, we first compared the effects of LPS with TNF- or IL-1 on CAM expression in HLMVEC. These stimuli were chosen because increased levels of LBP and sCD14, two LPS accessory molecules, and also TNF- or IL-1 have been detected in BALF of symptomatic asthmatics (19). Also, asthma severity in patients exposed to house dust mite has been shown to be related to LPS, rather than allergen, concentration in house dust (22). TNF-, IL-1, or LPS caused a transient induction of E-selectin that peaked at 6 h and an increase in ICAM-1 that was sustained at 72 h; TNF- was the most effective stimulus and LPS the least. Transient E-selectin induction and sustained ICAM-1 expression are characteristic of other EC, including human umbilical vein endothelial cells (HUVEC), murine LMVEC, and human intestinal microvascular (HIM) or human dermal microvascular (HDMV) EC, although the rate at which E-selectin is lost from the cell surface appears to depend on the source of EC and/or the induction stimuli (27, 30, 35, 43).

Kinetics of VCAM-1 induction in HLMVEC were also stimulus dependent. TNF--induced expression peaked at 24 h and was sustained at 72 h, whereas LPS- or IL-1-induced expression peaked at 6 or 24 h, respectively, and was not detected at 48 or 72 h. Studies with HUVEC and HDMVEC have also shown that TNF- induces sustained (48 to 72 h) expression of VCAM-1 (27, 30), which, together with the results of the present study, might suggest that microvascular and macrovascular EC respond in a similar manner to TNF-. In contrast, LPS induced sustained VCAM-1 expression in HUVEC but kinetics of expression in HIMVEC were similar to our findings with HLMVEC (30). This may suggest that mechanism(s) exist to prevent prolonged LPS-induced VCAM-1 expression in the microvascular endothelium of the lung and intestine, organs perhaps most likely to be exposed to LPS. These differences in VCAM-1 induction serve to illustrate the necessity of using EC derived from lung microvessels for in vitro studies that seek to investigate the role of endothelial CAM in the pathogenesis of inflammatory lung disease.

We next investigated the effects of IL-4 on CAM expression in HLMVEC. It is thought that the link between allergic asthma and increased BALF IL-4 levels may, in part, be due to selective induction of VCAM-1 in the microvascular endothelium of the airways/lung (6). This link has been suggested since IL-4, in contrast to TNF-, IL-1, or LPS, has been shown to induce VCAM-1 in cultured EC obtained from several vascular sites, with little or no effect on ICAM-1 or E-selectin expression (15, 25, 28, 30). A number of studies using HUVEC have reported initial expression of IL-4-induced VCAM-1 varying from 24 to 72 h; this may suggest that small differences in culture conditions could influence IL-4-induced VCAM-1 expression (15, 24, 27, 28). In contrast, IL-4-induced VCAM-1 induction reported for HUVEC, but not HIMVEC within the same study, may reflect differences between microvascular and macrovascular EC in addition to differences in culture conditions (30). In the present study, using HLMVEC, we have shown a selective effect on VCAM-1 expression following incubation with IL-4 for 72 but not 6, 24, or 48 h. Assuming that these results are indicative of VCAM-1 induction in the microvessels of the lung, they do not help to explain the increased VCAM-1 expression detected on bronchial mucosa biopsies from allergic asthmatics obtained 24 h after allergen challenge (44).

We have hypothesized, therefore, that to induce effective VCAM-1 expression in microvascular endothelium of the airways/lung, IL-4 may require the presence of a co-stimulus and we have proposed that this may be LPS. Our results show that LPS incubated together with IL-4 caused significant VCAM-1 induction in HLMVEC at 24, 48, or 72 h; also, that LPS alone had no effect at these times and that the increase detected at 72 h with IL-4/LPS was greater than that with IL-4 alone. Our findings are supported by those of Kapiotis and associates (29), who, using HUVEC, also reported synergism between IL-4 and LPS on VCAM-1 induction. TNF- or IL-1 has also been shown to synergize with IL-4 to induce VCAM-1 in EC derived from several vascular sites (9, 15, 25), and we have seen similar effects with HLMVEC (Blease, Hellewell and Burke-Gaffney, unpublished observation). However, TNF- or IL-1 synergism with IL-4 may be secondary to synergism with LPS since LPS may, in part, trigger the increased TNF- or IL-1 detected in asthmatic BALF.

Therefore, with the proviso that pre-existing epithelial damage, characteristic of asthma, may be necessary for LPS to reach the lung microvessels, we speculate the following: the initial response to allergen may include IgE-dependent stimulation of IL-4 release from mast cells in the respiratory tract (45) which may synergize with LPS, inhaled together with allergen (22), to induce VCAM-1 expression. Synergism between IL-4 and LPS is further supported by our observation that pre-exposure to either stimuli, followed by subsequent exposure to the other, was sufficient to induce VCAM-1 expression. This might suggest a mechanism by which VCAM-1 expression and, as a consequence, airway inflammation, may be perpetuated. It is not clear why priming with IL-4 followed by LPS resulted in a greater increase in VCAM-1 expression than the reverse conditions and the mechanism(s) responsible for these priming effects is the subject of further investigation.

In the present study, we investigated two mechanisms that might contribute to the synergistic effect observed when IL-4 was co-incubated with LPS: (1) upregulation of IL-4R expression and (2) VCAM-1 mRNA induction. First, we showed that in experiments in which TNF- increased the expression of IL-4R, IL-4, LPS, or IL-4/LPS did not. Schnyder and co-workers (46) have previously shown that IL-4 transiently decreased IL-4 receptor expression in HUVEC although expression was upregulated on marmoset splenic lymphocytes (46). Our observation that TNF- upregulated IL-4 receptor expression in HLMVEC is supported by a similar finding with murine sarcoma cells (47). In addition, it has also been shown that endothelial IL-4R expression in bronchial mucosa biopsies from asthmatics was significantly increased compared with that of normal control subjects, although the mediator(s) responsible has not been identified (48). Our results would suggest, however, that upregulation of the IL-4 receptor  chain in HLMVEC is unlikely to contribute to the synergistic effect on VCAM-1 induction detected following co-incubation with IL-4 and LPS.

In contrast, we showed that IL-4 synergized with LPS to induce VCAM-1 mRNA, measured at 4 h, and we suggest that this may, in part, account for the increased VCAM-1 induction seen in HLMVEC. A similar synergistic effect on VCAM-1 mRNA has been shown in HUVEC with TNF- or IL-1 (28, 29) but not, to our knowledge, with LPS. TNF- has been shown to prolong the half-life of IL-4-induced VCAM-1 mRNA in HUVEC and a similar mechanism may contribute to the synergism between LPS and IL-4 in HLMVEC, in our study. Alternatively, or in addition, LPS and IL-4 may activate separate, but co-operative, transcription pathways in HLMVEC, both of which may be required for full activation of the VCAM-1 promoter. In support of this, LPS and IL-4 have been shown to induce VCAM-1 in HUVEC by NFB-dependent or -independent mechanisms, respectively (49).

Finally, we showed that pretreatment of HLMVEC monolayers with IL-4 in combination with LPS increased eosinophil adhesion and that this increase was inhibited with a mAb against the VCAM-1 ligand, VLA-4. Neutrophil adhesion was increased following treatment of HLMVEC with LPS alone but was not further increased in the presence of IL-4; also, eosinophil adhesion to LPS/IL-4-treated monolayers was greater than neutrophil adhesion to LPS-treated monolayers. These findings, together with those showing a reduction in airway eosinophilia following administration of mAb against VCAM-1 and/or VLA-4 in rat or murine models of asthma (5, 6), suggest that the VCAM-1/VLA-4-dependent pathway plays a role in selective migration of eosinophils into the airways in allergic asthma. The LPS-induced increase in neutrophil adhesion to HLMVEC may also explain why ragweed allergen, that was shown to be contaminated with LPS, caused increased neutrophil recruitment, at 24 h, in BALF of allergen-challenged patients (50). In conclusion, the present study has provided evidence that presence of a co-stimulus, LPS, is necessary for IL-4 to induce effective VCAM-1 expression in the microvascular endothelium of human airways/lung and that IL-4/LPS-induced VCAM-1 expression may mediate, in part, the selective recruitment of eosinophils into the airways of allergic asthmatics.