Endothelium-derived Toll-like receptor-4 is the key molecule in LPS-induced neutrophil sequestration into lungs (original) (raw)

Mice. C3H/HeJ and TLR4 KO mice were purchased from The Jackson Laboratory (Bar Harbor, Maine, USA), and C3H/HeN and C57BL/6 mice were purchased from Charles River Laboratories (Montreal, Quebec, Canada). All mice were maintained in a pathogen-free facility until they weighed 20–35 g and were 6–10 weeks old, at which time they were used. To ensure that our model of pulmonary neutrophil recruitment was not simply a selectin- and integrin-dependent (adhesion-independent) model, we obtained E/P-selectin KO and CD18 KO mice, a generous gift from R.G. Collins and A.L. Beaudet (Baylor College of Medicine, Houston, Texas, USA), and subjected them to the same model of endotoxemia as we did the TLR4 chimeric mice.

Bone marrow transplantation. Briefly, bone marrow chimeras were generated following a standard protocol previously described by researchers from our laboratory (8, 9). Two sets of chimeras were generated for this study. The first set used C3H/HeN and C3H/HeJ mice. The second set used C57BL/6 and TLR4 KO mice to ensure that the spontaneously occurring mutants and generated knockouts had similar phenotypes. Bone marrow was isolated from mice euthanized by spinal cord displacement. Recipient mice were irradiated with 2 doses of 5 Gy (Gammacell 40 137Cs γ-irradiation source; Nordion International, Kanata, Ontario, Canada). An interval of 3 hours was allowed to pass between the first and second irradiations. Next, 8 × 106 donor bone marrow cells were injected into the tail vein of recipient irradiated mice. The mice were kept in microisolator cages for 8 weeks to allow full humoral reconstitution. This protocol previously confirmed that about 99% of leukocytes in Thy1.1 and Thy1.2 congenic recipient mice were from donor bone marrow (8).

Determination of lung myeloperoxidase activity. Lung myeloperoxidase (MPO) activity was used as a biochemical index of neutrophil recruitment into lungs. MPO is an enzyme found in cells of myeloid origin and has been used extensively as a biochemical marker of granulocyte (mainly neutrophil) infiltration into the lung (10). At the end of each experiment, lung samples were weighed, frozen, and processed for determination of MPO activity. The samples were stored at –20°C for no more than 1 week before the MPO assay was performed as previously described (10, 11), with the volumes of each reagent modified for use in 96-well microtiter plates. Change in absorbance at 450 nm over a 60-second period was determined using a kinetic microplate reader (Molecular Devices Corp., Sunnyvale, California, USA).

Lung histology. Untreated mice or mice treated for 30 minutes, 4 hours, or 12 hours with LPS (Escherichia coli 0111:B4; Calbiochem-Novabiochem Corp., San Diego, California, USA) were sacrificed, and lungs were fixed with 10% formalin via tracheal injection for 1 hour, harvested, and resuspended in 10% formalin. Formalin-fixed tissues were embedded in paraffin. Four-micrometer-thick sections were stained with H&E and chloroacetate esterase staining (Leder stain) for neutrophils. The sections were analyzed by light microscopy in a blinded fashion by a pathologist (F. Green). Neutrophil numbers were determined by counting the number of positive-stained cells over 20 fields at a magnification of ×40. The mean number of positive cells per high-power field was then calculated.

Lung electron microscopy. Untreated mice or mice treated for 30 minutes, 4 hours, or 12 hours with LPS were sacrificed, and lungs were harvested as above using 2.5% glutaraldehyde and processed for electron microscopy as previously described (12). Briefly, samples were postfixed for 2 hours at 4°C with 1% osmium tetroxide and subsequently dehydrated in a graded series of acetone solutions. Tissues were then embedded in Epon 812, and ultrathin sections were obtained using an ultramicrotome equipped with a diamond knife (Ultracut E; Reichert-Jung, Vienna, Austria). Sections were stained with uranyl acetate and lead citrate and then viewed with a Hitachi H-7000 electron microscope (TEMHitachi H-7000, Tokyo, Japan).

Quantification of P-selectin expression. Expression of P-selectin was determined as a measure of endothelial activation using a modified dual-radiolabeled Ab technique (13, 14). The Ab’s RB40.34 (against P-selectin; Pharmingen, San Diego, California, USA) and A110-1 (a rat IgG1, λ isotype control; Pharmingen) were labeled with 125I or 131I, respectively, using the Iodogen (Pierce Chemical Co., Rockford, Illinois, USA) method as previously described (13, 14). A110-1 was used to control for nonspecific binding in the murine system.

To determine P-selectin expression, animals were injected i.v. with a mixture of 10 μg 125I-RB40.34 and a variable dose of 131I-A110-1 calculated to achieve a total injected 131I activity of 400,000–600,000 cpm (total volume 200 μl). The Ab’s were allowed to circulate for 5 minutes; then the animals were treated with heparin. A blood sample was obtained from the carotid artery catheter, and the mice were exsanguinated. The lungs, heart, liver, mesentery, small intestine, large intestine, muscle, skin, and stomach were harvested and weighed. 131I and 125I were measured in plasma and tissue samples. P-selectin expression was calculated per gram of tissue, by subtraction of the accumulated activity of the nonspecific Ab (131I-A110-1) from the accumulated activity of the P-selectin Ab (125I-RB40.34). P-selectin data are represented as the percentage of the injected dose of Ab per gram of tissue. It has been previously demonstrated that this approach provides reliable quantitative values of adhesion molecule expression, and that radiolabeled binding Ab can be displaced specifically with sufficient amounts of unlabeled Ab. The technique is sufficiently sensitive that basal levels of P-selectin can be detected in WT mice relative to P-selectin–deficient mice (8, 13, 14).

Labeling of cells for flow cytometric analysis. To quantify the degree of neutrophil activation in the circulation in response to LPS, levels of L-selectin and CD11b expression were measured using flow cytometry. Briefly, whole blood was collected by cardiac puncture from mice treated with LPS for 4 hours using a 1-ml insulin syringe precoated with heparin. The blood (100 μl) was stained with 1 μg of mAb against L-selectin (MEL-14 rat anti-mouse; Pharmingen), CD11b (Mac-1 rat anti-mouse; Pharmingen), or a nonspecific isotype control (R35-95; Pharmingen) for 30 minutes at room temperature. Red blood cells were lysed with OptiLyse B (Immunotech, Marseille, France), and leukocytes were incubated with FITC-conjugated polyclonal goat anti-rat Ig (Pharmingen) for 30 minutes at room temperature. The cells were washed, resuspended in PBS/0.5% BSA/20 mM glucose solution, and read on a BD FACScan flow cytometer (BDBiosciences, Mountain View, California, USA) using CellQuest Pro software (Becton Dickinson Immunocytometry Systems). Data were compared with results from mice that did not receive LPS.

In a separate set of experiments, we examined in vitro direct LPS stimulation of neutrophils from chimeric mice. Using L-selectin shedding as an endpoint, this series of experiments also ensured that neutrophils of the chimeric mice were replaced by bone marrow transplantation and responded to LPS appropriately. From the chimeric mice, 100 μl of whole blood was incubated without or with LPS (100 μg/ml) for 30 minutes in a 37°C shaking water bath. OptiLyse B was used to lyse rbc’s, and leukocytes were then stained with L-selectin or isotype control Ab’s as described above.

Bronchoalveolar lavage and lung macrophage isolation. To determine the phenotype of lung macrophages (TLR4+/+ or TLR4–/–) in the chimeric mice, these cells were isolated and probed for responses to LPS. Mice were anesthetized by intraperitoneal (i.p.) injection of a mixture of 10 mg/kg xylazine (MTC Pharmaceuticals, Cambridge, Ontario, Canada) and 200 mg/kg ketamine hydrochloride (Rogar/STB, London, Ontario, Canada) and secured on a dissecting board, and the trachea was exposed. Bronchoalveolar lavage (BAL) was performed by slow delivery of up to 1 ml warm saline (∼37°C) into the mouse trachea. The fluid was slowly withdrawn by gentle suction immediately after delivery, and this procedure was repeated seven times. BAL was stored on ice until further processing. The lungs were filled with 1 ml Dispase (GIBCO BRL; Invitrogen Life Technologies Inc., Burlington, Ontario, Canada) via the tracheal catheter and allowed to collapse naturally, expelling part of the Dispase. Next, 450 μl 1% low-melting-temperature agarose (warmed to 45°C) was slowly infused via the catheter into the lungs that were covered with crushed ice and cooled for 2 minutes. The lungs were suspended in Dispase for 45 minutes at room temperature, then transferred into DMEM containing 0.01% DNaseI before the tissue was carefully teased away from the airways. The resulting cell suspension was filtered through 100-μm and 40-μm nylon mesh before the cells were pelleted and resuspended in 10 ml DMEM. Single-cell BAL and lung digest suspensions were seeded into 24-well plates at a concentration of 2 × 106 cells per ml for 60 minutes at 37°C. Lung macrophages were isolated by washing away the nonadherent cells in the wells with eight 1-ml rinses of DMEM. Adherent lung macrophages were then cultured with or without 10 μg/ml LPS for 18 hours. Culture supernatants were removed, and cells were lysed in TRIzol reagent (GIBCO BRL; Life Technologies Inc.).

RT-PCR of iNOS gene expression was used as an index of responsiveness in isolated lung macrophages. Total RNA was isolated from the macrophages using the RNeasy Mini Kit (QIAGEN Inc., Valencia, California, USA). RT-PCR was performed using the OneStep RT-PCR kit (QIAGEN Inc.). The primer sequences used for amplifying iNOS cDNA were sense, 5′-TCACTGGGACAGCACAGAAT-3′, and antisense, 5′-TGAAGCCATGACCTTTCGCATTAGCATG-3′, with a PCR product size of 1,423 bp. GAPDH cDNA was coamplified as an internal control using the following primer sequences: sense, 5′-CGGAGTCAACGGATTTGGTCGTAT-3′, and antisense, 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′, with a final PCR product size of 302 bp. The RT-PCR conditions were optimized so that both iNOS and GAPDH mRNAs were expanded linearly for 35 cycles, as follows: 100 ng total RNA, 0.5 μM of each iNOS primer, 0.2 μM of each GAPDH primer. PCR products were electrophoresed through 2% agarose gel containing 0.5 μg/ml ethidium bromide. Bands were visualized and analyzed using a Fluor-S MAX MultiImager (Bio-Rad Laboratories Inc., Hercules, California, USA).

Murine pulmonary endothelial cell isolation. Lungs were harvested from control or TLR4 chimeric mice and finely minced with scalpels in HBSS. The tissue was suspended in 0.25% collagenase type II (250 U/mg; Worthington Biochemical Corp., Lakewood, New Jersey, USA) in HBSS with 2% FBS at 37°C for 45 minutes, with vortexing at 10-minute intervals. The resulting cell suspension was immediately filtered through a 100-μm mesh and washed in PBS. The dissociated cells were resuspended in 100 μl OptiLyse B solution as a fixative agent. Following a 10-minute incubation, 1 ml ddH2O was added to lyse the contaminating rbc’s. Cells were washed and stained using phycoerythrin:TLR4 (PE:TLR4) mAb (MTS510; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) and FITC:vascular endothelial–cadherin (VE-cadherin) polyclonal Ab (Bender MedSystems, Vienna, Austria) as a marker of endothelial cells, or appropriate isotype controls. Analysis was performed using a BD FACScan and CellQuest Pro software (BDBiosciences).

Intravital microscopy. Mice were anesthetized by i.p. injection of a mixture of 10 mg/kg xylazine and 200 mg/kg ketamine hydrochloride. The left jugular vein was cannulated to administer anesthetic. To view the cremaster muscle microcirculation, an incision was made to separate the scrotal skin from the associated fascia, and a second incision was made on the ventral surface of the cremaster muscle. The testicle and epididymis were separated from the underlying muscle and reintroduced into the abdominal cavity. The muscle was then spread out over an optically clear viewing pedestal and secured along the edges with 4-0 suture. The exposed tissue was superfused with warm bicarbonate-buffered saline (pH 7.4). The microcirculation was observed through an intravital microscope with a ×10 eyepiece and a ×25 objective lens (Axioskop; Carl Zeiss Canada Ltd., Don Mills, Ontario, Canada). The microcirculation was recorded using a video camera (Panasonic 5100 HS; Panasonic, Osaka, Japan). Images of the microcirculation were recorded for 1 hour in untreated mice and 4 hours after LPS stimulation. All experimental parameters were quantified at these time points.

Single unbranched venules (25–40 μm in diameter) were selected, and to minimize variability, the same section of the venule was observed throughout the experiment. Venular diameter was measured using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, Texas, USA). The number of rolling and adherent leukocytes was determined offline during video playback analysis. Rolling leukocytes were defined as those leukocytes that rolled at a velocity slower than that of erythrocytes within a given vessel. Leukocyte rolling velocity was measured for 20 leukocytes and was determined as the time required for a leukocyte to traverse a 100-μm length of venule.

Experimental protocol. In all experiments, mice received 0.5 mg/kg (approximately 12.5 μg/mouse) of purified LPS i.p. This dose was chosen to avoid any mortality during the study period, even following anesthesia. Initially, we used highly purified LPS (E. coli 0111:B4) with less than 0.0008% contaminating bacterial proteins, provided by S.M. Goyert (Division of Molecular Medicine, North Shore University/New York University School of Medicine, Manhasset, New York, USA) (15). As our data were identical with this LPS and with purified LPS from Calbiochem EMDBiosciences. (E. coli 0111:B4), we used the latter in most studies. At 4 hours, in separate groups of mice, tissue expression of P-selectin was quantified, lung tissue was harvested for MPO analysis, and pulmonary macrophages were assessed for TLR4 responsiveness. Since platelets can also be a source of P-selectin, in some P-selectin expression experiments EndotheliumTLR4–/– mice received 50 μl/mouse of rabbit anti–mouse thrombocyte serum (Accurate Chemical and Scientific Corp., Westbury, New York, USA) i.p. 1 hour before LPS treatment. The anti–mouse thrombocyte serum depleted circulating platelets by 96%. Finally, in some experiments, 4 hours after LPS administration, intravital microscopy was used to examine the peripheral microvasculature of the cremaster muscle.

Statistical analysis. All results are expressed as mean ± SEM. A Student’s t test with Bonferroni correction was used for multiple comparisons. Statistical significance was set at P < 0.05.