Methods in Molecular Biology TM VOLUME 239 Cell Migration in Inflammation and Immunity Methods and Protocols Edited by Daniele D’Ambrosio Francesco Sinigaglia Chemotaxis and Vascular or Lymphatic Endothelium 1 Chemotaxis and Interaction with Vascular or Lymphatic Endothelium Silvano Sozzani, Annunciata Vecchi, Paola Allavena, and Alberto Mantovani Introduction Leukocyte recruitment has been recognized as an early event in inflammatory processes since the late 19th century Accumulation and trafficking of leukocytes in tissues under physiological and pathological conditions are orderly (typically neutrophils precede mononuclear cells) and selective because, in certain states, one or more leukocyte subsets are recruited preferentially (e.g., eosinophils in allergy) The current paradigm of recruitment is that of a multistep process involving the action of chemotactic signals (1,2) Classical chemoattractants include complement components, formyl peptides, and leukotriene B4 In addition, various cytokines are able to elicit directional migration of leukocytes Whereas molecules, such as monocyte colony-stimulating factor (M-CSF), tumor necrosis factor, and vascular endothelial growth factor (VEGF), exert chemotactic activity, the main chemotactic cytokines are a superfamily of molecules known as chemokines (for chemotactic cytokines) However, the in vivo role of M-CSF and VEGF as chemoattractants is well established Several independent lines of work lead to the identification of chemokines such as monocyte chemotactic protein-1 (MCP-1) and related molecules In the early 1970s it had already been noted that supernatants of activated blood mononuclear cells contained attractants active on monocytes and neutrophils (3) Subsequently, a chemotactic factor active on monocytes was identified in culture supernants of mouse (4) and human (5,6) tumor lines and was called tumor-derived chemotactic factor (TDCF) human (5–7) At the time, TDCF was rather unique in that it was active on monocytes but not on neutrophils (6) and From: Methods in Molecular Biology, vol 239: Cell Migration in Inflammation and Immunity Edited by: D D'Ambrosio and F Sinigaglia © Humana Press Inc., Totowa, NJ Sozzani et al had a low molecular weight (5,6) Moreover, correlative evidence suggested its involvement in the regulation of macrophage infiltration in murine and human tumors (5,6,8) A molecule with similar cellular specificity and physicochemical properties was independently identified in the culture supernatant of smooth muscle cells (SMDCF) (9) The JE gene had been identified as an immediate– early platelet-derived growth factor (PDGF)-inducible gene in fibroblasts (10,11) Thus, in the mid-1980s, a gene (JE) was in search of function and a monocytespecific attractant was waiting for molecular definition In 1989, MCP-1 was successfully purified from supernatants of a human glioma (12), a human monocytic leukemia (13) and a human sarcoma cell line (14–16): sequencing and molecular cloning revealed its relationship with the long-known JE gene (17–19) Here, we will focus on selected methods used to investigate chemoattractants at large, with emphasis on chemokines In particular, classic protocols used for studying cell movement including chemotaxis will be presented, along with methods for transendothelial migration and reverse transmigration In particular, sources of vascular endothelium and the generation of lymphatic endothelial cultures are discussed In vivo approaches to monitor leukocyte traffic are discussed elsewhere in this volume; here, we will describe the air-pouch model as a simple in vivo recruitment system Materials 2.1 Chemotaxis Micro 48-well Boyden chamber (Neuroprobe) Humidified 5% CO2 incubator Peripheral mononuclear cells (PBMCs) 5-µm Polycarbonate filters Glass slides RPMI 1640 medium (Biochrom KG) + 0.2% bovine serum albumin (BSA) Chemoattractants Diff-Quik (Harleco) 2.2 Polarization Assay Purified leukocytes (monocytes, neutrophils, lymphocytes) RPMI 1640 medium (Biochrom KG) + 1% fetal calf serum (FCS) (Hyclone) 10X Concentration chemoattractants 10% (v/v) Formaldehyde Humidified 5% CO2 incubator 2.3 In Vivo Air-Pouch Model Animals: CD1 mice, either sex Iota carrageenan, 1% in sterile, apirogen saline Chemotaxis and Vascular or Lymphatic Endothelium Syringes, mL, with a 25G needle Plastic Pasteur pipets, mL Hemocytometer Test tubes Centrifuge Sterile apirogen saline 2.4 Adhesion to Endothelium Endothelial cells (ECs) obtained with a well-established methodology (20) Tissue culture medium, M199 with 20% FBS +50 µg/mL endothelial cell growth supplement (ECGS) (Collaborative Research) + 100 µg/mL heparin (Sigma) This is referred to as a complete medium Freshly isolated eukocytes 96-Well plates (Falcon, Becton Dickinson) Cotton fiocs (Johnson and Johnson) 51Cr (Amersham, 37 MBq, µCi) Humidified 5% CO2 incubator Gamma counter windowed for 51Cr Phosphate-buffered saline (PBS) (Biochrom KG) 2.5 Transendothelial Migration ECs obtained with a well-established methodology (20) Tissue culture medium, M199 with 20% FBS +50 µg/mL ECGS (Collaborative Research) + 100 µg/mL heparin (Sigma) This is referred to as complete medium Freshly isolated leukocytes Single-well Boyden chambers (Neuroprobe) Nitrocellulose filter (12-mm diameter , 5-µm pore, Sartorius) Polyvinyl-pirrolidonet (PVP)-free polycarbonate filter (12-mm diameter, 5-µm pore, Sartorius) Fibronectin (Sigma) 24-Well plates (Falcon, Becton Dickinson) Cotton fiocs (Johnson and Johnson) 10 51Cr (Amersham, 37 MBq, µCi) 11 Humidified 5% CO2 incubator 12 Gamma counter windowed for 51Cr 13 PBS (Biochrom KG) 2.6 Reverse Transmigration In Vitro ECs obtained with a well-established methodology (20) Tissue culture medium, M199 with 20% FBS +50 µg/mL ECGS (Collaborative Research) + 100 µg/mL heparin (Sigma) This is referred to as a complete medium Freshly isolated leukocytes Single-well Boyden chambers (Neuroprobe) Sozzani et al 10 11 12 13 14 Nitrocellulose filter (12-mm diameter, 5-µm pore, Sartorius) PVP-free polycarbonate filter (12-mm diameter, 5-µm pore, Sartorius) Fibronectin (Sigma) 24-Well plates (Falcon, Becton Dickinson) Cotton fiocs (Johnson and Johnson) 51 Cr (Amersham, 37 MBq, µCi) Humidified 5% CO2 incubator Gamma counter windowed for 51Cr PBS (Biochrom KG) Stripping buffer: 20 mM NH4OH, 0.5% (v/v) Triton X-100 2.7 Endothelial Cells 2.7.1 Generation of Endothelioma Cell Lines Note: All culture reagents are from Gibco unless otherwise specified 15 Days’ gestation fetuses 0.05% Trypsin + 0.02% EDTA Dulbecco’s modified Eagle’s medium (DMEM) medium + 20% FCS 6- and 12-Well tissue culture plates Retrovirus vector N-TKmT G418 Ca2+- and Mg2+-free PBS or saline 2.7.2 Generation of Lymphatic Endothelial Cell Lines Note: All culture reagents are from Gibco unless otherwise specified 10 11 12 Incomplete Freund adjuvant (Sigma) 0.05% Trypsin + 0.02% ethylenediamine tetraacetic acid (EDTA) DMEM medium + 10% FCS (HyClone) Nonessential amino acids (NEAA) Na Pyruvate (NaPyr) 6-Well tissue culture plates and T25 flasks (Falcon) Ca2+- and Mg2+-free PBS or saline Supernatant of Sarcoma 180 ECGS (Sigma) Heparin (Sigma) Gelatin (Sigma) solution (1% in PBS) Collagenase CLS type I (Worthington Biochem) Methods 3.1 Chemotaxis Chemotaxis is defined as the directional locomotion of cells sensing a gradient of the stimulus Chemotaxis has been extensively studied with leukocytes Chemotaxis and Vascular or Lymphatic Endothelium that are “professional migrants,” but a variety of cell types, including fibroblasts, melanoma cells, keratinocytes, and vascular endothelial cells, exhibit directional locomotion in vitro Two main techniques have been used to measure migration in vitro: migration under agarose and chemotaxis across porous membranes Although the former approach may more closely resemble the in vivo conditions, the latter is easier to quantitate and allows analysis of directional versus random locomotion We will therefore focus on the description of migration through a porous membrane using the modified Boyden microchamber method (21,22) Aliquot 25 µL chemoattractant in each lower well The 25-µL volume may have some variations (2–3 µL more or less), depending on the microchamber used It is worth calibrating the lower wells in advance, so that having seeded the chemoattractant, the liquid in the lower well forms a small convex surface that guarantees a perfect adhesion of the filter, avoiding air-bubble formation Put the filter (25 × 85 mm) on the lower compartment To avoid confusion in filter orientation, cut an angle of the filter Mount the silicon trimming and cover piece Press the cover piece tightly to avoid air bubbles Seed 50 àL cell suspension (1.5 ì 106 cells) in the upper well by leaning the pipet tip on the border of the well and quickly ejecting the cell suspension Incubate the chamber at 37°C in 5% CO2 for 1.5 h Unscrew and turn over the chamber Hold the upper compartment tightly and remove the lower compartment, keeping the silicon trimming and the filter adhered to the upper compartment of the chamber At this point, the migrated cells are on the upper surface of the filter Lift the filter and hold it with a clamp on each end (clamps purchased from the manufacturer of the chamber—Neuroprobe, Maryland, USA) Wash the opaque side of the filter, where the nonmigrated cells remain, by passing this side over PBS Do not immerse the entire filter in PBS or the migrated cells will be lost Remove all nonmigrating cells by scraping the opaque surface of the filter against the special rubber policeman (purchased from the manufacturer) 10 Stain the filter with Diff-Quik 11 Place the filter on glass slides and cout the migrated cells present on the bright surface of the filter Count 5–10 microscopic fields at ×1000 final magnification 3.2 Polarization Assay The early phase of leukocyte response to chemotactic factors is characterized by shape change Chemoattract stimulation results in the formation of a frontal lamellipodia that contains all of the machinery for cell movement and a rear uropode (23) This front-tail polarization is rapid, being detectable within minutes, and can easily be observed at the microscope without the need of any special equipment Leukocyte polarization is not chemotaxis However, for Sozzani et al the measurement of cell polarization, it is a predictive, and inexpensive, way to investigate chemotactic factors (23) Prepare cells (106/mL) in RPMI 1640 with 1% FCS and prepare 200 µL samples (use at least duplicate tubes) Prepare agonists (e.g., fMLP, C5a; Sigma) at a 10X concentration Prewarm cells at 37°C for Add the agonists in a volume of 20 µL Stimulate the cells at 37°C for 10 Stop the stimulation by the addition of an equal volume (200 µL) of ice-cold 10% (v/v) formaldhyde The readout of the experiment is the evaluation of the percentage of polarized cells (head/tail) at the microscope (×400) At least 200 total cells per sample need to be counteted 3.3 In Vivo Air-Pouch Model In vivo leukocyte recruitment can be easily investigated by the use of the air-pouch model This technique consists in the creation of a pouch in the back of the mice The pouch needs to be prepared some days in advance by the the injection of sterile air, to allow the internal formation of an epithelial layer (24) The advantage of this technique is that a chemotactic factor or a pleyotropic inflammatory agent can be injected locally and cell recruitment can be evaluated by the collection of the local essudate Cytokines (e.g., chemokines), lipid mediators, and other components of the inflammatory reaction can also be tested in the inflammatory essadute In our experience, this technique works at the best when pro-inflammatory mediators are inoculated (e.g., endotoxin, interleukin-1, carrageenan) In these conditions, infiltration of mononuclear cells (monocytes and lymphocytes) as well granulocytes is easily detected Interleukin (IL)-8 and C5a also represent active in vivo chemotactic signals that provide clear results On the contrary, recruitment of monocytes by chemokines may represent a more difficult task Inject mice subcutaneously on their back with mL of sterile air (syringes are prepared under a laminar-flow hood) After d, inject the pouches again with mL of sterile air On d 6, inject mL of 1% carrageenan into the pouches Controls are injected with mL of saline On d (24 h later), sacrifice animals Incise the skin on the back and gently detach it to expose the surface of the air sac Carefully inject the pouch with mL saline; then, make a small incision in the upper part, recover the liquid with a plastic Pasteur pipet, and immediately put it in a test tube in ice Record the total amount of liquid collected Take an aliquot (usually 100 µL) for counting the cells; if differential counting is needed, spin the cells in a cytocentrifuge and stain with Diff-Quik Chemotaxis and Vascular or Lymphatic Endothelium If measurement of soluble factors has to be performed, centrifuge the remaining fluid at 500g for 10 at 4°C, and collect and store the supernatants at –20°C until use The method described here uses carrageenan as a local stimulus for leukocyte recruitment; other stimuli may be used as well, basically following the same protocol It must be noted, however, that water-soluble stimuli (e.g., proteins) must be prepared in 0.5% sterile (endotoxin-free) carboxymethylcellulose to avoid the fast absorption of the agent from the local site of injection The carrageenan injection cause a recruitment that lasts a long time, for d, whereas other stimuli (e.g., IL-1β, recruit cells for shorter times For IL-1β or IL-8, peak cell recruitment is observed at h Conditions here reported refer to CD1 mice If other strains of mice are used, experimental conditions need to be validated 3.4 Adhesion to Endothelium The emigration of leukocytes from blood to tissues is essential for mediating immune surveillence and mounting inflammatory responses The interaction of leukocytes with endothelial cells (ECs) can be divided into four sequential steps: tethering, triggering, strong adhesion, and migration The selectin family of adhesion molecules mediates tethering; strong adhesion is mediated by the integrin family, which need to be activated (triggering), and, finally, migration is induced by local promigratory factors, including some cytokines and chemokines (25,26) We have studied the adhesive properties and transendothelial migration of leukocytes, but this method may also apply for investigation of other cell types (e.g., tumor cells) Protocols and describe radioisotopic assays for monitoring adhesion and transendothelial migration, based on an assay described in ref 27 Some leukocytes (e.g., dendritic cells, lymphocytes) have a peculiar trafficking pattern from tissues into the lumen of blood or lymphatic vessels To mimick this basal-to-apical process of migration in vitro, we established a transmigration assay, described in ref 20—the reverse transmigration assay Various leukocyte subsets (neutrophils, monocytes, natural killer [NK] cells, or lymphocytes) are separated from buffy coats of normal blood donors, as described (22,28) Resuspend cells at 107 cells/mL in RPMI 1640 medium +10% FBS (complete medium) and label by incubation with 100 µCi 51Cr for h at 37°C After labeling, wash extensively and resuspend in complete medium Culture ECs in 96-well plates (1 × 104/well) in order to reach a confluent monolayer in 36–48 h Stimulate designed wells with IL-1 (10 ng/mL) during the last 18 h of culture Incubate ECs with 100 µL 51Cr-labeled cells resuspended at 107 cells/mL and incubate at 37°C for 30 Sozzani et al Carefully remove the supernantant and wash the cells twice to remove nonadherent cells Incubate the adherent cells with 100 µL of NaOH M + 1% sodium dodecyl sulfate (SDS) for and count radioactivity using a gamma counter Express cell adhesion as percentage of input cells The spontaneous adhesion of resting leukocytes to unstimulated ECs varies for different subsets For instance, the adhesion of NK cells is usually 5–15%, a value intermediate between that of monocytes (20–40%) and the very low value of T-cells and polymorphonuclear cells (PMNs) (