WO1992021746A1 - Device and method for the analysis of rolling blood leukocytes and identifying inhibitors and promoters - Google Patents
Device and method for the analysis of rolling blood leukocytes and identifying inhibitors and promoters Download PDFInfo
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- WO1992021746A1 WO1992021746A1 PCT/US1992/004524 US9204524W WO9221746A1 WO 1992021746 A1 WO1992021746 A1 WO 1992021746A1 US 9204524 W US9204524 W US 9204524W WO 9221746 A1 WO9221746 A1 WO 9221746A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
- A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
- A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
- G01N33/56972—White blood cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
- A61M2202/0413—Blood
- A61M2202/0429—Red blood cells; Erythrocytes
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M2202/00—Special media to be introduced, removed or treated
- A61M2202/04—Liquids
- A61M2202/0413—Blood
- A61M2202/0439—White blood cells; Leucocytes
Definitions
- the present invention related to in vitro models of the in vivo rolling and arrest of leukocytes along the endothelial cell wall, an important part of the inflammatory response.
- the invention also relates to apparatuses and methods for collecting, purifying, and analyzing blood and blood components, and methods for identifying inhibitors or promoters of components of the inflammatory response.
- leukocytes white blood cells
- extravasation The migration of leukocytes (white blood cells) out of the blood and into tissues (extravasation) is the central event in the inflammatory response.
- Leukocyte emigration is responsible for the successful host response to tissue injury and infection, but is also potentially harmful and contributes to the pathology of many diseases and inflammatory disorders.
- HEV high endothelial venule
- Related receptors are expressed on other leukocyte subsets (id.) .
- the first step in leukocyte migration into tissues is margination, when leukocytes leave the central stream of flowing blood cells in a postcapillary venule and roll along the endothelial lining of the vessel (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)) .
- Leukocytic margination in postcapillary venules should be distinguished from the "marginating pool" of about 50% of leukocytes that may be in capillary beds in the lung or tissues and enter the circulation in response to exercise or epinephrine.
- Postcapillary venules are major sites of leukocyte emigration in inflammation, and there are few or no marginating leukocytes in these venules in the healthy state (Fiebig et al., 1991, Int. J. Microcirc. Clin. Exp. 10:127-144).
- leukocytes begin to interact with the vessel wall by rolling along the endotheliu within minutes after injury to adjacent tissue.
- the rolling response is seen throughout Vertebrata, in cold-blooded animals such as amphibians as well as in mammals (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)) .
- the number of rolling cells increases dramatically during the course of an inflammatory reaction (Atherton and Born, 1972, J. Physiol.
- a pseudopod is extended through the vessel at a junction between endothelial cells, and this often is accompanied by a flattening of the leukocyte against the vessel wall (Marchesi, 1961, Q. J. Exp. Physiol. 46:115-133). Transmigration continues as the pseudopod grows in ramifications and size until the entire cell body has emerged through a narrow gap between endothelial cells (Cohnheim, 1889, Lectures on General Pathology: A Handbook for Practitioners and Students (London: The New Sydenham Society)). Cells appear to reach the point at which they emigrate by rolling; no active migration along the vessel wall is evident by intravital microscopy. Both the rheology of blood and specific adhesive interactions may regulate the rolling response.
- Vascular permeability is increased, leading to plasma leakage and an increased hematocrit, and together with slower flow, leads to erythrocyte rouleaux formation.
- a combination of these factors causes leukocytes to be displaced to the marginal region of flow near the vessel wall (Chien, 1982, Adv. Shock Res. 8:71-80). This makes contact of a circulating leukocyte with the vessel wall more probable, but shear forces acting on the leukocyte at the vessel wall oppose adhesion to the endothelium.
- the velocity profile of a vessel shows no flow at the vessel wall and a parabolic increase toward the centerline. Because fluid velocity increases with distance from the wall, cells near the wall have torque exerted on them and will tumble even if not in contact with the wall.
- LFA-1 and not Mac-1 binds to ICAM-2 (de Fougerolles et al., 1991 J. Exp. Med. 174:253-267; Diamond et al., 1990, J. Cell Biol. 111:3129-3139), an endothelial cell molecule that is more closely related to ICAM-1 than these molecules are to other Ig superfamily members (Staunton et al., 1989, Nature 339:61-64).
- Stimulation of neutrophils with chemoattractants is required to activate binding of these integrins to ICAM-1 (Smith et al., 1989, J. Clin. Invest. 83:2008-2017; Diamond et al., 1990, J. Cell Biol. 111:3129-3139).
- Stimulation of neutrophil integrin avidity is a rapid response occurring in minutes, does not require increased integrin surface expression (Buyon et al., 1988, J. Immunol. 140:3156- 3160; Philips et al., 1988, J. Clin. Invest. 82:495- 501; Vedder and Harlan, 1988, J. Clin. Invest.
- ICAM-1 induction is a second mechanism for regulating inflammatory cell interactions that occurs on a time scale of hours and requires mRNA and protein synthesis (reviewed in Springer, 1990, Nature 346:425- 433) .
- ICAM-1 is expressed basally on endothelial cells but is greatly increased at inflammatory sites and by stimulation with lipopolysaccharide and cytokines such as IL-1 and TNF.
- ICAM-2 is expressed at higher surface density on resting endothelium but is not inducible (de Fougerolles et al., 1991, J. Exp. Med. 174:253-267).
- LFA-1 and Mac-1 together with pl50,95 comprise the leukocyte integrins, a subfamily of integrins that share a common ⁇ subunit (CD18) and have distinct ⁇ L, ⁇ M and ⁇ X (CDlla, b and c) a subunits (reviewed in Larson and Springer, 1990, Immunol. Rev. 114:181-217; Springer, 1990, Nature 346:425-433) .
- LFA-1 and Mac-1 ⁇ subunits are deficient in binding to endothelial cells in static adhesion assays (Buchanan et al., 1982, Blood 60:160-165; Harlan et al., 1985, Blood 66:167-178). However, when binding of neutrophils in shear flow is measured, the leukocyte integrin-dependent component of binding is lost at a shear stress below the physiologic range (Lawrence et al., 1990, Blood 75:227-237).
- VLA-4 that contains the ⁇ 4 (CD49d) subunit noncovalently associated with the ⁇ l (CD29) subunit, is expressed by lymphocytes, monocytes, and neural crest-derived cells, and can interact with vascular cell adhesion molecule-1 (VCAM-1) (Elices et al., 1990, Cell 60:577).
- VCAM-1 vascular cell adhesion molecule-1
- VCAM-1 is a member of the immunoglobulin (Ig) superfamily (Osborn et al., 1989, Cell 59:1203), but unlike the ICAMs, VCAM-1 is not expressed by lymphocytes (Wellicome et al., 1990, J. Immunol. 144:2558; Rice et al., 1990, J. Exp. Med. 171:1369). VCAM-1 expression is very low or absent on resting endothelial cells in culture but can be induced by cytokines such as TNF or IL-1 with kinetics of induction similar but not identical to that of
- ICAM-1 (Wellicome et al., 1990, J. Immunol. 144:2558; Carlos et al., 1990, Blood 76:965). Peak expression of VCAM-1 after continuous treatment of endothelial cells with TNF in culture occurs somewhat earlier than the peak expression of ICAM-1, but both persist at levels substantially higher than basal expression for at least 48 hr (Carlos et al., 1990, Blood 76:965).
- VLA-4 can also interact with fibronectin, binding to the alternatively spliced CS-1 region located C-terminal to the RGD site of fibronectin recognized by the integrin VLA-5 (Guan and Hynes, 1990, Cell 60:53; Wayner et al., 1989, J. Cell Biol. 109:1321; Hemler, 1990, Annu. Rev. Immunol. 8:365).
- VCAM-1 cDNA clones Two forms of VCAM-1 cDNA clones, which most likely represent alternatively spliced products, have been reported (Osborn et al., 1989, Cell 59:1203; Polte et al., 1990, Nucl. Acids Res.
- selectins are the most recently recognized class of leukocyte adhesion molecules (reviewed in Springer, 1990, Nature 346:425-433). They have an N-terminal lectin domain, one epidermal growth factor-like module, and from two to nine short consensus repeats. By contrast to integrins and immunoglobulin (Ig) family members, selectins have been found to date only on circulating cells and the endothelium, suggesting that they may be specialized for interactions within the vasculature.
- Ig immunoglobulin
- CD62 (PADGEM or GMP-140) is expressed in ⁇ granules of platelets and Weibel-Palade bodies of endothelial cells, and is mobilized to the plasma membranes of these cells after activation by mediators of inflammation and hemostasis, allowing these cells to bind neutrophils and monocytes at the site of tissue injury (Larsen et al., 1989, Cell 59:305-312; Geng et al., 1990, Nature 343:757-760). CD62 is rapidly unregulated on the endothelial cell surface, suggesting that it may be important early in inflammation (Hattori et al., 1989, J. Biol. Chem.
- ELAM-1 is synthesized by endothelial cells in response to inflammatory agents and promotes adhesion of neutrophils, monocytes, and a subpopulation of lymphocytes (Bevilacqua et al. , 1989, Science 243:1160-1165; Picker et al., 1991, Nature 349:796-798; Shimizu et al., 1991, Nature 349:799- 802) .
- the LAM-1 or LECAM-1 molecule is expressed on leukocytes and facilitates their binding to endothelium during lymphocyte recirculation through peripheral lymph nodes and neutrophil emigration at inflammatory sites (Jutila et al., 1989, J. Immunol. 143:3318-3324; Spertini et al. , 1991, Nature 349:691- 694; Watson et al., 1991, Nature 349:164-167).
- Carbohydrate ligands for selectins have recently been defined (reviewed in Springer and Lasky, 1991, Nature 349:196-197); that for CD62 has Lewis x as an important component (Larsen et al., 1990, Cell 63:467- 474) and also appears to be sialylated (Moore et al., 1991, J. Cell Biol. 112:491-499).
- Neutrophils bear Lewis x both on glycolipids and at the termini of N- and 0-linked oligosaccharides (Symington et al., 1985, J. Immunol. 134:2498-2506; Fukuda et al., 1984, J. Biol. Chem. 259:10925-10935).
- Antibodies to selectins and integrins additively inhibit neutrophil adhesion to endothelium, suggesting that they mediate distinct adhesion mechanisms (Luscinskas et al., 1989, J. Immunol. 142:2257-2263; Dobrina et al., 1989, Immunology 67:502-508; Smith et al., 1991, J. Clin. Invest. 87:608-618; Hallmann et al., 1991, Biochem. Biophys. Res. Commun. 174:236-243).
- the molecular basis of rolling does not appear to involve the leukocyte integrins, based on the inability of Mab to the leukocyte integrin common CD18 ⁇ subunit to inhibit rolling in vivo (Arfors et al., 1987, Blood 69:338-340) .
- Chemoattractants bind to serpentine family receptors on the surface of a leukocyte.
- a highly selective class of chemoattractants described in the last few years are small proteins of 70 to 80 amino acids that belong to a recently described protein family called the intercrines (Oppenheim et al., 1991, Ann. Rev. Immunol. 9:617-648).
- the chemoattractant receptors mediate pro-inflammatory and chemotactic actions, and transduce ligand-mediated signals through interactions with G proteins (GTP-binding proteins) .
- Actions mediated by chemoattractant receptors include stimulation of granule-enzyme release and superoxide anion production, upregulation of expression and activity of the cell adhesion molecule Mac-1 (CDIIb, CD18) , increased expression of CRl, a decrease in cell surface glycoprotein 100MEL-14 on neutrophils (Gerard and Gerard, 1991, Nature 349:6-14), and stimulation of neutrophil adherence to and emigration through activated endothelial cells (Huber et al., 1991,
- Interleukin can also act as an adhesion or migration inhibitor when added on the same side of activated endothelium as neutrophils (Huber et al., 1991, Science 254:99; Gimbrone et al., 1989, Science 246:1601). In vivo, these receptors may participate in anaphylactoid and septic shock (Gerard and Gerard, supra) .
- chemoattractant receptor is the one which binds formylpeptides.
- whole blood comprises red blood cells (RBC) , platelets, and white blood cells of various types known collectively as leukocytes.
- RBC red blood cells
- platelets and white blood cells of various types known collectively as leukocytes.
- Red blood cells and platelets can be separated from whole blood by centrifugation, leaving the blood product plasma.
- Removal of leukocytes to low levels in a blood sample is desirable to prevent diseases caused by transfusion of blood that contains donor leukocytes. These diseases include viral infections transmitted by leukocytes harboring viruses and infections in which the transfused leukocytes elicit allo-antibodies that cause transfusion reactions.
- enrichment of leukocytes is also desirable when different types of leukocytes are needed for a variety of clinical and research purposes.
- Centrifugal methods for separating blood do not entirely separate out the leukocytes; they may be present in substantial quantities in both the packed red blood cells and the platelet-concentrate fractions. Centrifugal methods are somewhat costly and the sterility of the product is such that it must be used within a short period of time.
- a number of other devices have been proposed in which fibers are packed into housings and whole blood allowed to pass through them in order to remove a portion of the white cell content. Typically, these devices are based on size separation but the various types of leukocytes are not widely divergent in size and most of these cells can deform so as to pass through much smaller openings than their normal size.
- the present invention provides in vitro models of the in vivo rolling and arrest of leukocytes along the endothelial cell wall, which are important steps in the migration of leukocytes out of the blood stream and into tissue, as part of the inflammatory response.
- the in vitro models of the invention provide a physiologically relevant model of such interactions, since the apparatuses of the invention can reproduce in vitro the sequential molecular interactions that are steps in leukocyte accumulation at an inflammatory site in vitro; and, furthermore, function under physiologic flow conditions resulting in physiologic shear stresses similar to those present in vivo at the sites of leukocyte transendothelial migration (extravasation) .
- Apparatuses embodying and methods relating to the in vitro models of the invention are also provided. Apparatuses embodying the models of the invention provide quick and effective methods for collecting, purifying, and analyzing leukocyte populations and subpopulations.
- the apparatus of the invention comprises a solid phase surface with rolling mediator molecules present
- the apparatus of the invention comprises a solid phase surface with both rolling mediators and integrin binding partners present thereon.
- the apparatuses of the invention can be used for collecting, concentrating, purifying, and analyzing blood and blood components, in particular, leukocytes and subsets thereof. Therapeutic and diagnostic methods based on the foregoing are also provided.
- the invention further relates to methods for identifying inhibitors or, alternatively, promoters (agonists, functional components) of the processes of leukocyte rolling and adhesion, important components of the inflammatory response.
- Pharmaceutical compositions and kits are also provided.
- FIGURES Figure 1. A Schematic Illustration of Leukocyte Rolling on a Substrate Containing a Selectin. Figure 2. A Schematic Cross-Section of an
- FIG. 3 A Schematic Perspective Illustration of an Embodiment of an Apparatus of the Invention.
- the apparatus shown is a parallel plate flow chamber, after Lawrence et al. (1987, Blood
- the chamber is shown upside down for illustration purposes; the glass slide with the artificial bilayer formed the lower parallel plate. It was mounted on the stage of an inverted microscope equipped with a video camera. The cell suspension was connected to the inlet manifold and a syringe pump to the outlet manifold.
- Figure 4 Schematic of parabolic flow profile in a blood vessel or parallel plate flow chamber.
- FIG. 5 Attachment of Neutrophils to Artificial Planar Bilayers during Flow. Resting or PMA-stimulated neutrophils were infused at varying wall shear stresses through the parallel-plate flow chamber. A planar membrane containing CD62, ICAM-1, or both at the density indicated below was formed on one side of the chamber. After 3 minutes of continuous flow to equilibrate adherence and deadherence to the bilayer, adherent neutrophils were quantitated. (A) and (B) represent two different sets of experiments: day-to-day variation throughout this work was slight as exemplified by binding of unactivated neutrophils to CD62 at 200 sites per ⁇ m 2 in both panels. Data are averaged from four to six (A) and two (B) experiments.
- Rolling neutrophils are visualized as round distinct cells by the video camera because their rolling velocity (5.2 ⁇ m/s here) is slow compared to the exposure time (30 frames per s) .
- nonadherent neutrophils tumbling in the shear flow appear as blurred streaks.
- the streaks of cells closest to the bilayer are short because laminar flow is slowest here.
- (B) Same as (A) , except on a bilayer containing 1,000 sites per ⁇ m 2 of ICAM-1. No rolling cells have accumulated. Nonadherent cells appear as streaks, and those closest to the bilayer have a velocity of about 500 ⁇ m/s.
- C-F Video images at 2 s intervals of neutrophils rolling on a bilayer containing 400 sites per ⁇ m 2 of CD62 at a shear stress of 7.3 dyn/cm 2 (40 x objective). Nuclei are resolved at this magnification, showing that the cells are rolling rather than sliding. Cells rolled at an average velocity of 8.2 ⁇ m/s; nonadherent cells tumbled too fast (>1700 ⁇ m/s) to be visualized at this flow rate and magnification.
- FIG. 7 Rolling Velocity as a Function of CD62 Density and Shear Stress.
- CD62 and ICAM-1 were used at the density indicated below. Error bars represent the SEM based on measurements from independent experiments. Experimental points at 14.6 dyn/cm 2 are not shown, but indicated by connecting lines. Closed circles: CD62 at 50 sites per ⁇ m 2 ; open circles: CD62 at 200 sites per ⁇ m 2 ; closed squares: CD62 at 200 sites per ⁇ m 2 plus ICAM-1 at 250 sites per ⁇ m 2 ; open triangles: CD62 at 400 sites per - ⁇ m 2 .
- FIG. 1 Neutrophils in Contact with Artificial Membranes under Static Conditions. Neutrophils, with or without stimulation with PMA, were allowed to adhere in the absence of flow to artificial bilayers containing 250 sites per ⁇ m 2 of ICAM-1 or 200 sites per ⁇ m 2 of CD62 for varying time periods, as indicated. (B)-(F) represent the same field of cells at one minute intervals. Time points prior to 3 min are not shown because it takes 2 min for all cells to settle onto the bilayer.
- FIG. 9 Detachment Assay following Static Incubation of Neutrophils on Artificial Membranes Containing either CD62, ICAM-1, or a mixture of CD62 and ICAM-1. Neutrophils were injected through a port in the side of the flow chamber and allowed to settle onto artificial bilayers containing 200 sites per ⁇ m 2 of CD62, 250 sites per ⁇ m 2 of ICAM-1, or both. For some experiments, PMA was added to the neutrophil suspension before it was injected into the flow chamber (broken line) . After 6 minutes of contact, shear stress was applied in staged increments. Neutrophils bound after 20 s at each shear stress point are expressed as the percentage of neutrophils that settled onto the bilayer in the initial contact period.
- Solid lines represent binding of unstimulated neutrophils to the membrane.
- Broken lines represent binding of PMA-stimulated neutrophils to the membrane.
- Error bars represent the SEM of three to five independent experiments (A) and two experiments (B) .
- Experimental points at 36 dyn/cm 2 are not shown but are indicated by connecting lines.
- FIG. 10 Stimulation with FMLP Arrests Neutrophil Rolling on Artificial Membranes Containing both CD62 and ICAM-1. Neutrophils were allowed to adhere to artificial bilayers containing CD62 (200 sites per ⁇ m 2 ) and ICAM-1 (250 sites per ⁇ m 2 ) during flow at a wall shear stress of 1.8 dyn/cm 2 (closed circles in (A)) or 0.73 dyn/cm 2 (open squares in (A)). Rolling velocity was constant for at least 5 min under these conditions. During the experiment, 10" 9 M FMLP was added to the medium being infused into the chamber and reached the cells rolling in the field of view 30 or 12 s later (marked 0 time) as verified in another experiment with a dye solution.
- B and C Distribution of rolling velocities of adherent neutrophils with time after FMLP exposure at 0.73 and 1.8 dyn/cm 2 , respectively.
- D Photomicrographs of neutrophils before the infusion of FMLP (0 time) and after exposure to 10 "9 M fMLP (5 min) on the same area of the planar membrane. fMLP addition induced arrest and spreading of the neutrophils. Flow was stopped for approximately 30 s to take the photographs in a separate experiment from the two experiments averaged for data in (A)-(C).
- E Shear resistance of neutrophil binding. Neutrophils were allowed to attach at 0.73 dyn/cm 2 and shear resistance was determined 5 min after fMLP exposure.
- the present invention provides in vitro models of leukocyte rolling and adhesion, which are functional at physiologic shear stresses.
- the present invention is based in part on the discovery that under physiologic flow conditions, leukocyte rolling mediated by interactions between selectins and their leukocyte binding partners is a prerequisite for chemoattractant-stimulated interaction of integrins on leukocytes with integrin binding partners, that results in arrest of the leukocytes.
- leukocyte extravasation appears to involve the following sequential steps: (1) reversible adhesion of flowing leukocytes to the blood vessel wall and subsequent rolling, mediated by interactions between selectins and their carbohydrate ligands; (2) leukocyte activation mediated by binding of chemoattractants, presumably diffusing out of the inflammatory site or expressed on the surface of the endothelium, to their receptors on the leukocyte cell surface; and (3) arrest and stable attachment of the rolling leukocyte mediated by binding of leukocyte integrins to immunoglobulin (Ig) family member molecules on the endothelium.
- Ig immunoglobulin
- the apparatus of the invention comprises a solid phase surface with rolling mediator molecules present thereon.
- rolling mediators include but are not limited to selectins and selectin ligands, expressed on endothelium in vivo, which have binding partners expressed on leukocytes.
- the apparatus of the invention comprises a solid phase surface with both rolling mediators and integrin binding partners present thereon.
- the apparatuses of the invention can be used for collecting, concentrating, purifying, and analyzing blood and blood components, in particular, leukocytes and subsets thereof.
- the invention further relates to methods for identifying inhibitors or, alternatively, promoters (agonists, functional components) of the processes of leukocyte rolling and adhesion, important components of the inflammatory response.
- Pharmaceutical compositions and kits are also provided.
- a selectin is a rolling receptor.
- neutrophils bind to and roll on CD62 in artificial bilayers.
- the rolling interaction through CD62 is a prerequisite for chemoattractant-stimulated interaction of integrins on neutrophils with ICAM-l that arrests rolling and dramatically strengthens adhesion. This essentially reproduces in vitro the steps of leukocyte accumulation at an inflammatory site in vivo.
- the present invention provides two general types of apparatuses: one which provides an n vitro model of leukocyte reversible adhesion and subsequent rolling along an endothelial vessel wall .in vivo (hereinafter “the rolling model”); and one which provides an .in vitro model of leukocyte arrest and stable attachment to the endothelium subsequent to rolling and activation by chemoattractant binding (hereinafter “the arrest model”) ; both of which models are functional at physiologic shear stresses.
- the rolling model comprises a solid phase surface with a rolling mediator present thereon. Interaction of the rolling mediator (preferably, a selectin or selectin ligand) with its binding partner on a leukocyte cell surface, at physiologic shear stresses, mediates reversible attachment and rolling of the leukocyte on the solid phase surface of the rolling model.
- the rolling model apparatus comprises, on a solid phase, planar lipid bilayers containing the selectin CD62.
- rolling refers to the literal rolling of leukocytes along a surface containing a rolling mediator, which rolling is induced by the interaction of the rolling mediator with the leukocyte in the presence of fluid drag forces arising from relative movement between the surface containing the rolling mediator and a medium containing the leukocytes.
- This rolling by leukocytes is seen throughout the Vertebrata, in cold blooded animals such as amphibians as well as in mammals.
- the number of rolling cells in vivo increases dramatically during the course of an inflammatory reaction and is important in the accumulation of cells at the site of injury.
- the arrest model comprises a solid phase surface with both a rolling mediator and an integrin binding partner present thereon, both with the same specificity for the leukocyte cell subset of interest (see infra) .
- a rolling mediator In the presence of a chemoattractant with matching cellular specificity, and under approximate physiologic shear conditions, interaction of the rolling mediator with its binding partner on the leukocyte leads to rolling of the leukocyte along the solid phase surface, followed by activation due to chemoattractant binding, and stable attachment and arrest of the leukocyte upon interaction of the leukocyte integrin with its binding partner on the solid phase surface.
- the arrest model apparatus comprises, on a solid phase surface, planar lipid bilayers containing the selectin CD62 and the integrin binding partner ICAM-l; and is preferably used with a formyl peptide such as N- formyl-methionyl leucyl phenylalanine (fMLP) as the activating chemoattractant.
- a formyl peptide such as N- formyl-methionyl leucyl phenylalanine (fMLP) as the activating chemoattractant.
- attached cells can be dissociated from the solid phase surface within the apparatus by exposing them to divalent cation chelating agents such as citrate, EDTA, EGTA, etc. , and washed away from the solid phase surface; the surface can then be re-used.
- the apparatus of the arrest model can also be used as in vitro model of leukocyte rolling without subsequent arrest, by use of such device in the absence of the activating chemoattractant required for subsequent arrest.
- a rolling mediator and an integrin binding partner must be present on the surface of the solid phase of the arrest model, and a chemoattractant must be used, that have respective cognate binding partners present on such leukocyte cell subset.
- the binding partner for the rolling mediator on the surface of the solid phase of the apparatus must be present on such leukocyte cell subset.
- Leukocyte cell subsets include but are not limited to neutrophils, eosinophils, basophils, mast cells (collectively known as granulocytes or polymorphonuclear leukocytes) , monocytes, macrophages, and lymphocytes (both T lymphocytes and B lymphocytes) .
- platelets shall also be deemed included within leukocytes, unless clearly excluded by context or otherwise.
- Exemplary leukocyte cell subset specificities of interactions with various rolling mediators, chemoattractants, and integrin binding partners are described infra.
- rolling mediator molecules may be present on the solid phase surface(s) of the apparatuses of the invention.
- Binding partner of a molecule as used herein refers to a receptor or ligand interacting with such molecule, e.g., by binding or other type of noncovalent association.
- Rolling of leukocytes on a solid surface which contains one or more affixed rolling mediators, and arrest of leukocytes on such a surface which also contains one or more integrin binding partners, can occur at physiologic flow rates, and the methods of the invention are designed to produce physiologic flow rates and induce physiologic shear stresses. It is well known that in Newtonian fluids at Reynolds numbers less than 2000, flow is laminar. Furthermore, fluid velocity is zero at the vessel wall and increases parabolically toward the center of the vessel. The change in velocity per change in radial displacement away from the vessel wall is called the shear rate and is highest at the wall. Shear stress scales linearly with the fluid forces acting on a cell under laminar flow conditions and is the product of shear rate and the fluid viscosity.
- Preferred shear stresses induced by methods of the invention bracket the range estimated to exist in vivo in post-capillary venules, namely, 0.5-30 dyn/cm 2 .
- the viscosity of water at 37°C (0.007 poise) can often be used as an approximation of the viscosity of the flow medium.
- the wall shear rate is given by T/ ⁇ .
- Physiologic flow rates are those flow rates sufficient to induce shear stresses in the apparatus of the invention of between about 0.5 to about 30 dynes per square centimeter. Particularly preferred flow rates are sufficient to induce shear stresses of between about 0.5 and about 4.0 dynes per square centimeter, with those in the range of about 0.5-2.0 dyn/cm 2 most preferred.
- Any rolling mediator known in the art, with a binding partner present on a leukocyte, can be used in the practice of the invention, by providing it on the solid phases of the apparatuses of the invention.
- rolling mediator shall mean any molecule capable of interacting with a leukocyte so as to mediate rolling of the leukocyte on the surface containing the rolling mediator.
- the rolling mediator is a selectin or a binding partner (ligand) of a selectin.
- Such rolling mediators include but are not limited to those described in Table 1, which also discloses the presently known cell subset specificity of binding partners for such rolling mediators (see also
- MECCA-79 (lymph node All leukocytes, in addressin) particular L, N, M
- N neutrophils
- M monocytes
- L lymphocytes
- sMT a subset of T lymphocytes and memory T lymphocytes
- the binding partner for MECCA-79 (lymph node addressin) (Berg et al., 1991, J. Cell Biol. 114:343) is the homing receptor selectin, also called LAM-1, LECAM-l, or L-selectin, which is expressed on all leukocytes and facilitates lymphocyte binding to endothelium during blood circulation through peripheral lymph nodes and lymphocyte and neutrophil binding to endothelium at inflammatory sites.
- LAM-1 homing receptor selectin
- LECAM-l L-selectin
- the granule membrane protein (CD62) also called PADGEM and GMP- 140, is a granule-associated glycoprotein of platelets and endothelial cells that is brought to the cell surface after stimulation by thrombogenic agents, allowing platelets and endothelial cells to bind neutrophils and monocytes at the site of tissue injury.
- All known selectins have an N-terminal domain that is homologous to a variety of Ca +2 - dependent animal lectins (thus the name selectin) , one epidermal growth factor (EGF)-like module, and from two to nine short consensus repeats.
- Molecules composed of short consensus repeats of the type found in selectins have random configurations as revealed by electron microscopy, suggesting a high degree of segmental flexibility.
- Location of the ligand for selectin is at the termini of long carbohydrate structures on the leukocyte that also may confer flexibility.
- rolling mediators includes molecules that competitively block binding of neutrophils or other leukocytes to rolling mediators such as selectins.
- Selectin counter-structure ligands have been identified (see Springer and Lasky, 1991, Nature 349:196-197) and certain molecules are now known to compete with selectins, especially the LAM-1 glycoprotein, for binding sites and thus inhibit binding of selectins to neutrophils. These molecules may thus contain binding sites that are identical, or closely related to, the neutrophil (or other leukocyte) binding site(s) on the selectin glycoprotein. These molecules include fucoidin, sulfatides, polyphosphomannose-ester (PPME) and sulfated glucans and sulfated polysaccharides (e.g. dextran sulfate, xylan sulfate) (see Skinner et al. , 1991, J. Biol. Chem. 206:5371-74).
- PPME polyphosphomannose-ester
- sulfated glucans e.g. dextran sulfate, xylan sulfate
- Rolling mediators can be obtained from any source known in the art, and are preferably purified for use in the apparatuses of the invention. Purification can be carried out by standard methods commonly known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, and sizing column chromatography) , centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
- chromatography e.g., ion exchange, affinity, and sizing column chromatography
- centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
- blood components containing selectins can be isolated from fresh blood using gel filtration (Larsen et al., 1989, Cell 59:305-312) or dextran-sedimentation and density separation over
- Selectins can be purified from blood components by immunoaffinity chromatography, e.g., by binding to its binding partner or antibody) . Briefly, selectins are extracted from cell materials by addition of detergent and, after centrifugation to remove organelles, the crude preparation can be applied to a chromatographic column containing anti- selectin antibody (see Larsen et al., supra) .
- CD62 expression on platelets provides a convenient source for biochemical isolation (id.) .
- ELAM-1 can be purified as described (Lobb et al., 1991, J. Immunol. 147:124).
- MECCA-79 can be purified as described (Berg et al., 1991, J. Cell Biol. 114:343).
- Recombinant DNA methods using well-known techniques can also be used to prepare purified selectins.
- Selectins on endothelium have been cloned and sequenced (see Springer, 1990, Nature 346:425) .
- Chemical synthesis may also be used, which can be automated, e.g., by use of peptide synthesizers.
- Use of the arrest model apparatuses of the invention to achieve leukocyte arrest after rolling comprises providing a chemoattractant to the rolling cell.
- a chemoattractant having its receptor present on a leukocyte or leukocyte cell subset of interest, which functions in the arrest model to allow arrest of the leukocyte or the subset thereof, can be used in the present invention.
- Such chemoattractants can include but are not limited to those presented in Table 2:
- LLB4 Leukotriene B4
- Mon Platelet activating factor Neut Mon, Eo, Baso, (PAF)
- any chemoattractants known in the art can be assayed for functional activity in and then used with the arrest model apparatuses of the invention.
- chemoattractants include but are not limited to the following: lymphokines (e.g., interleukin (IL)-l, IL- 2, IL-4, etc.), collagen, fibrin fragments, oxidized lipid components from cell membranes, histamine (active on eosinophils) (Clark et al., 1975, J. Exp. Med. 142:1462-1476), eosinophilotactic peptides (ECF-A) (active on eosinophils) (Goetzl and Austen, 1976, J.
- lymphocyte chemoattractants for use should promote chemotaxis, rather than chemokinesis.
- the chemoattractants can be obtained from any source known in the art, and are preferably purified for use with the arrest model apparatuses of the invention. Purification can be by standard methods known in the art, including but not limited to chromatography (e.g., ion exchange, affinity, and sizing column chromatography) , centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Chemoattractants can also be prepared by chemical synthetic techniques (e.g., by use of a peptide synthesizer) , or by recombinant DNA methods by expression of a cloned chemoattractant nucleotide sequence.
- Such molecules are integrin binding partners, which include but are not limited to known Ig family members, normally present on endothelium, such as those listed in Table 3 (see Butcher, 1991, Cell 67:1033-1036): TABLE 3
- ICAM intercellular adhesion molecule
- LFA-1 lymphocyte function-associated antigen-1
- VCAM vascular cell adhesion molecule
- VLA very late activation antigen
- N neutrophils
- M monocytes
- L (B and T) lymphocytes
- sT subset of T lymphocytes
- mL memory T lymphocytes
- vL virgin T lymphocytes
- aL activated (B and T) lymphocytes
- aT activated T lymphocytes
- sL subset of (B and T) lymphocytes
- Integrin binding partners are preferably purified prior to use in the arrest model apparatuses, and can be obtained and purified by use of any method known in the art, e.g., by methods as described supra for the rolling mediators.
- ICAM-l can be purified as described infra in Section 6.3.2 hereof. 5
- cloned ICAM-l (Staunton et al., 1988, Cell 52:925-933) can be expressed by recombinant methods known in the art.
- a cloned nucleic acid encoding ICAM-2 or a functional derivative thereof can be expressed to obtain ICAM-2.
- VCAM-1 cDNA clones (Osborn et al., 1989, Cell 59:1203; Polte et al. , 1990, Nucl. Acids Res. 18:5901) are expressed.
- Preferred solid phases are translucent materials such as glass, plastic, quartz, etc.
- Well-known solid phases include beads formed from glass, polystyrene, polypropylene, dextran, and other materials, tubes formed from or coated with such materials, or a __ fibrous support matrix made from one or more of these materials.
- the solid phase includes planar shapes (e.g., plates, sheets) such as glass or plastic slides or coverslips.
- the solid phase 0 includes one or more solid or hollow glass fibers or tubes. These solid fibers or tubes can be packed within a housing or cartridge which preferably contains an inlet and an outlet so that blood containing leukocytes can flow through the cartridge 5 in a unidirectional manner.
- the solid phase is plastic.
- One or more surfaces of the solid phase(s) can contain the rolling mediators, or rolling mediators and integrin binding partners, as the case may be.
- the rolling mediator proteins and integrin binding partner proteins can be placed on the surface of the solid phase in any manner, e.g., by affixing or adsorbing them directly thereto, coating a solution or suspension containing the proteins on the surfaces, etc. , as long as the proteins are accessible to leukocytes flowing by the surfaces.
- one or more of the desired rolling mediator and/or integrin binding proteins can be either covalently or non-covalently affixed directly to the solid phase by techniques such as covalent bonding via an amide or ester linkage or adsorption.
- the rolling mediator and/or integrin binding partner is immobilized by incorporation into lipid bilayers.
- Lipid bilayers can be prepared by any method known in the art, e.g., from liposomes. Preparation of liposomes can be achieved by any of several well known procedures (See, for example, Mimms et al., 1981, Biochemistry 20:833-840) .
- one or more surfaces of the solid phase is coated with a liposome suspension.
- the liposome suspension is spread on glass substrates to form planar lipid bilayers containing the incorporated rolling mediators (see also Sections 6.3.3 and 6.3.4 infra) , or rolling mediators and integrin binding partners.
- phosphatidylcholine is used in the preparation of liposomes.
- Other phospholipids can also be used, including but not limited to phosphatidylserine, phosphatidylinositol, and phosphatidylethanolamine.
- the lipid vesicles can also contain other lipid-soluble molecules such as cholesterol.
- a 7:2 ratio mixture of phosphatidylcholine to cholesterol can be used to make lipid vesicles incorporating the rolling mediators and/or integrin binding partners of the invention (see e.g., Dustin and Springer, 1988, J. Cell Biol. 107:321) .
- Lipid vesicles incorporating a rolling mediator, alone or in combination with an integrin binding partner can also be made as described in Smith et al. (1989, J. Clin. Invest. 83:2008-2017).
- the rolling mediators and/or integrin binding partners are affixed to a solid phase such as glass or plastic by direct coating.
- a rolling mediator or integrin binding partner in solution at a concentration in the range of 20-100 ⁇ g/ml in 1% octylglucoside detergent can be diluted 1:10 in Tris-saline (pH 8.0), 2 mM MgCl 2 at the time of addition of the solution to a plastic or glass surface, followed by incubation for 16 h at 4°C to allow the protein to adsorb to the surface, followed by washing of the coated surface with 1% bovine serum albumin in Tris-saline (pH 8.0), 2 mM MgCl 2 buffer (see, Dustin and Springer, 1989, Nature 341:619).
- rolling mediators and/or integrin binding partner proteins present in lipid-vesicle-coated solid phases are preferred over plastic surfaces coated directly with such proteins, which are in turn preferred over glass surfaces coated directly with such proteins, since there is a higher background binding of leukocytes to the solid phase, when the solid phase is glass with directly adsorbed proteins, relative to when the solid phase is plastic with directly absorbed proteins, relative to when the solid phase is a surface coated with lipid vesicles containing such proteins.
- FIG. 1 shows a solid phase 10 upon which is affixed one or more rolling mediators 12.
- a sample of blood including components 14 (e.g., leukocytes or a subset thereof) which can bind with the rolling mediator and other components 16 (e.g., erythrocytes (red blood cells)) which cannot bind with the rolling mediator, are introduced under conditions providing relative movement between the solid phase 10 and the sample, so that blood components 14 and 16 come into contact with the solid phase 10.
- Those components 14 that bind to the selectin will contact the solid phase and reversibly adhere thereto.
- these components 14 that bind with the rolling mediator will also begin to roll along solid phase 10 under physiologic flow conditions.
- the rate of rolling is very slow, on the order of microns per second.
- Components 16 that are not responsive to the rolling mediator will continue to move rapidly across the solid phase without adhering to it, or tumble along the solid phase, as illustrated by the arrows shown associated with component 16.
- the rolling velocity of the adhering components 14 is several orders of magnitude slower than the velocity of the freely moving components 16 and this serves as one basis for the separation and purification methods of the invention, as described infra .
- Components 16 that do not adhere to the solid phase 10 flow out and are removed from the system.
- solid phase 10 also contains an integrin binding partner 17, in addition to the rolling mediator 12.
- the components 14 that (i) bind (or otherwise interact) with the rolling mediator 12 and roll along solid phase 10, and (ii) express a receptor that binds the chemoattractant, and (iii) express an integrin that binds (or otherwise interacts with) the integrin binding partner 17, will become arrested on the solid phase.
- ah apparatus of the invention comprises (a) a solid phase having a plurality of rolling mediator molecules on a surface of the solid phase; (b) inlet means for receiving a fluid sample and for permitting the sample to enter onto the surface of the solid phase; and (c) outlet means for permitting the fluid sample after it has flowed across at least a portion of the surface of the solid phase to exit said surface.
- the solid phase further comprises a plurality of integrin binding partners on its surface.
- the inlet and outlet means can be entrance and exit slots or holes, channels, tubes, pipes, etc.
- the apparatuses can further comprise a means associated with the inlet means, for introducing the fluid sample into the inlet means, e.g., ports, injection systems, etc.
- the apparatuses can further comprise a means associated with the outlet means, for removing a fluid sample from the outlet means, e.g., a pump. Other examples are described infra.
- FIG. 2 shows a schematic diagram of one embodiment directed to an apparatus of the rolling model of the invention.
- a housing 20 contains a plurality of solid glass fibers 22 having affixed on their peripheral surfaces 24 one or more rolling mediators 26.
- Solid glass fibers 22 can be formed by using well-known procedures. The thickness of the fibers should be as small as possible to maximize the surface in contact with the sample, although the smallest possible fibers are necessarily constrained by mechanical and manufacturing procedures.
- Blood 28 is introduced via inlet 30 into the housing 20 containing bundled fibers.
- the fibers may optionally be separated from each other by a plurality of dividers or panels (not shown) .
- the housing can be of any inert material including glass, plastic or other polymer such as polytetrafluoroethylene (PTFE- TefIon®) .
- Blood or other medium containing or suspected of containing blood cells e.g., whole blood, artificial blood, plasma, heparinized blood, or combinations or components of the foregoing, or other suspending vehicles for blood cells including buffers and the like
- Blood can be fed under slight pressure so that the flow will exit the housing through outlet port 34.
- blood can be introduced into the housing at atmospheric or subambient pressures in which the outlet port 34 is attached to a slight vacuum, the term "vacuum” meaning a pressure lower than the atmospheric pressure.
- Blood components that are capable of binding to the selectin(s) affixed to the bundled fibers will reversibly adhere to them and roll. Blood components that do not adhere to rolling mediator(s) 26 on the bundled fibers are pulled across the exteriors of the fibers and exit the housing at port 34.
- the peripheral surfaces 24 in Figure 2 also have affixed thereon one or more integrin binding partners, such that, in the presence of the appropriate chemoattractant, leukocytes which (i) express binding partners for the rolling mediator 26, (ii) express binding partners for the chemoattractant, and (iii) express the integrin recognizing the integrin binding partner, will stop and become arrested on the surfaces 24.
- leukocytes which (i) express binding partners for the rolling mediator 26, (ii) express binding partners for the chemoattractant, and (iii) express the integrin recognizing the integrin binding partner, will stop and become arrested on the surfaces 24.
- Relative movement of the solid phase and the sample containing or suspected of containing blood cells is accomplished using a variety of methods.
- bulk flow i.e., flow of both fluid and particles in the fluid
- a peristaltic pump or syringe pump is preferred for this purpose.
- Relative movement between sample and solid phase can also be provided by capillary action which will draw the sample across one or more surfaces of a solid phase.
- Other physical methods that do not rely on bulk flow can include electrophoretic methods in which certain blood cell subpopulations (e.g. , T and B cells, neutrophils and monocytes) which differ in their electrophoretic mobility can be exposed to an electric field. The leukocytes will be charged accordingly and will be induced to move within the fluid across an appropriate solid phase.
- blood cell subpopulations e.g. , T and B cells, neutrophils and monocytes
- the apparatuses of the invention can be used in various methods relating to the collection. purification, concentration, and analysis of blood and blood cells.
- the apparatuses of the invention are used to effect the collection of leukocytes or cell subset(s) thereof.
- the collection of the leukocytes is accomplished based on the cells' differential mobility (slower speed) in passage across the solid phase of the invention, due to the cells' rolling arising from interaction with the rolling mediator molecules. That is, leukocytes which express a cell-surface binding partner for a rolling mediator (e.g., selectin) present on a solid phase of a rolling model apparatus can be collected from a medium flowing at physiologic flow rates through the rolling model apparatus, by removing the solid phase while such leukocytes are still reversibly adhering and rolling thereon.
- a rolling mediator e.g., selectin
- such cells can be collected by collecting medium after passage over the solid phase at a time subsequent to passage of the non-rolling blood components; however, this method is not preferred, due to the long time period required for passage of the rolling leukocytes over the solid phase (see infra) .
- the type(s) of leukocyte that are collected depends on the cellular specificity of the binding partners for the rolling mediator(s) present on the solid phase (see Table 1, supra) .
- a chemoattractant (or a plurality of chemoattractants) is introduced, preferably by addition to the medium flowing through the apparatus, without stopping flow.
- Rolling leukocytes that are activated by the chemoattractant. and that express an integrin on their cell surface which interacts with its binding partner on the solid phase of the arrest model apparatus, will become arrested on and strongly adherent to the solid phase.
- the solid phase can then be removed, and the bound cells eluted for subsequent use (e.g., by exposure to chelating agents such as citrate, EDTA, EGTA, etc.).
- the type of leukocyte that is collected depends on the cellular specificity of the binding partners for the rolling mediator(s) , chemoattractant(s) , and integrin binding partner(s) that are used (i.e., what type of leukocyte expresses binding partners for these molecules) .
- a blood sample containing leukocytes and a solid phase containing at least one affixed rolling mediator protein are moved relative to each other under physiologic flow conditions.
- the rolling mediator is the selectin CD62.
- This selectin is specific for neutrophils and monocytes and will not bind to lymphocytes or red blood cells.
- a different rolling mediator or more than one rolling mediator can be affixed to the solid phase in order to bind with, and cause adherence of, a different type or a plurality of leukocyte subsets, depending on the cellular specificity of the rolling mediator's binding partner (see Table 1).
- a plurality of rolling mediator analogs can also be affixed to the solid phase, such as sulfated glycans, fucoidin, or PPME (polyphosphomannose-ester) .
- the medium (e.g., blood sample) containing leukocytes is allowed to flow over the substrate at flow rates sufficient to induce a shear stress preferably of between about 0.5 and about 2.0 dynes per square centimeter.
- a shear stress preferably of between about 0.5 and about 2.0 dynes per square centimeter.
- rolling will be induced in the leukocytes that come into contact with the rolling mediator affixed to the solid phase.
- In vitro rolling velocities of leukocytes on CD62 are comparable to in vivo rolling velocities, as described below in the examples sections. Velocities generally range from under 2 to over 30 microns per second, depending on the site density of selectin and the shear stress. These velocities are several orders of magnitude slow than the fluid velocities necessary to induce the physiologic shear stress.
- the rolling leukocytes will not traverse the solid phase of the apparatus under most conditions. For example, leukocytes rolling between 2 and 30 microns per second will traverse a 10 centimeter long substrate in about 1 to 15 hours, for longer than most convenient collection procedures.
- a particularly preferred washing solution includes saline that contains calcium ions. Calcium ions are preferred because selectins have a N-terminal domain which is homologous to a variety of Ca 2+ -dependent animal lectins and, therefore, selectin binding is calcium-dependent.
- leukocytes can be eluted from the solid phase by using a chelating agent such as citrate or ethylenediaminetetraacetic acid (EDTA) to bind calcium ions, thus enabling leukocytes to be released from the solid phase.
- a chelating agent such as citrate or ethylenediaminetetraacetic acid (EDTA) to bind calcium ions, thus enabling leukocytes to be released from the solid phase.
- EDTA ethylenediaminetetraacetic acid
- neutrophils and monocytes can be thus collected.
- the above-described embodiment can also be adapted to collect cells using an arrest model apparatus, preferably containing CD62 as the rolling mediator and ICAM-l as the integrin binding partner and with the use of fMLP as the chemoattractant, for the collection of neutrophils and monocytes.
- the above collection methods can also result in and be used for the concentration, purification, and/or quantification of the rolling or arrested leukocytes. Quantification can be by various methods known in the art, including visualization of rolling cells, or staining methods, as described infra.
- the collection methods of the invention can be used to extract substantially all of the leukocytes or a cell subset thereof from a given volume of medium.
- the total number of leukocytes remaining in the bulk sample is less than 1 x 10 6 .
- the distance between the fibers 22 can be adjusted by altering the dimensions and spacing of the fibers. Blood components are then forced to pass through narrower gaps between fibers, which gaps preferably are in the range of from about 10 to about 100 ⁇ m (somewhat larger than the width of a typical leukocyte, i.e., 6-7 ⁇ m) .
- the flow rate can be controlled so that the shear stress is sufficient to allow all the leukocytes to come into contact with the rolling mediator on the solid phase.
- a preferred shear stress is in the range of from 0.5-2 dynes/cm 2 .
- the site density or concentration of selectin on the solid phase can also be increased to accomplish the same ends. A site density of up to about 1,000 sites/ ⁇ m 2 can be used, with 100 sites/ ⁇ m 2 the preferred site density.
- the volume and numbers of leukocytes per volume of blood can be determined by quantitation of the rolling or arrested leukocytes and by measuring the volume of flowing blood.
- the leukocytes can be eluted from a known blood volume using the apparatus of the invention and their total numbers separately determined using well known methods.
- the inlet and outlet manifolds 48, 50 are also in communication with respective entrance and exit slots 54, 56 in the deck.
- Base 46 contains a recessed lip 58 on an upper surface 59 of the base into which is placed a compressible gasket 60 having an aperture 62, which aperture being sized and shaped to be congruent with the deck 52.
- Glass slide 42 or other similar planar surface with an artificial bilayer containing one or more rolling mediators is placed on the gasket in communication with the gasket aperture.
- a vacuum source 64 is connected to the base 46 and this vacuum source applies enough force compress the gasket 60 and slide 42 together and prevent their disengagement from the rest of the base assembly.
- a cell suspension is connected to the inlet manifold and preferably a syringe pump (not shown) is connected to the outlet manifold.
- Another embodiment of an analysis -method uses a solid substrate affixed with a plurality of different rolling mediators. Each rolling mediator can be confined to a distinct area or zone of the solid substrate. The rolling mediators are chosen so that each zone will allow adhesion of a different leukocyte cell subset or subsets (e.g., monocyte, neutrophil, eosinophil, basophil, and the like) .
- a blood sample is obtained from a patient and a small amount of blood or other medium containing blood cells is allowed to contact the solid substrate. Leukocytes of various types will bind to the respective rolling mediators.
- the flow can be generated by a syringe or syringe pump, as described above and allowed to flow across the parallel plate chamber of Figure 3.
- the substrate which can be of small size, can be moved through the blood sample to provide relative movement. This is most conveniently done by attaching the solid phase to an elongated member (e.g., a "dipstick") and moving the substrate back and forth within the sample at controlled velocities. Different leukocytes flowing across the substrate will roll and reversibly adhere to their respective rolling mediators and will be physically separated on the substrate.
- the substrate can be washed, as above, to remove unbound material and the adhering leukocytes stained using fluorescent or other labels well known in the art (e.g., Wright's Crimson) .
- a further embodiment of an analysis method that utilizes a solid substrate affixed with a plurality of different rolling mediators can include, as described above, a solid phase in which rolling mediators are confined to distinct areas or zones.
- the solid phase can include one or more rolling mediators chosen so that substantially all of the leukocytes are concentrated in a particular zone of the solid phase. This initial concentration of leukocytes can take place within a so-called "starting zone.” After cells are concentrated onto the starting zone, they can be further separated and analyzed using, for example, subsequent electrophoresis onto a solid phase containing a plurality of different rolling mediators.
- the different leukocyte populations will then be separated on this so-called "separation zone.”
- Cells can then be counted and analyzed on the separation zone using a variety of conventional methods. For example, the separation zone can be scanned and the absorbance determined, which absorbance is a function of the leukocyte population density. Further, the solid substrate can be viewed under a microscope and numbers of leukocytes, separated by leukocyte subset into distinct “zones", can be counted. The slide can be preserved and cells further examined by microscopy if desired.
- the foregoing methods of analysis can also be adapted for use with an arrest model apparatus, with provision of the appropriate chemoattractant(s) and integrin binding partner(s) .
- the apparatuses and methods of the invention can also be adapted to provide for visual analysis of leukocyte rolling velocities.
- the parallel flow apparatus of Figure 3 is attached to a microscope stage, and the blood components flowing across the surfaces are viewed through the microscope objective by way of a video cassette recorder (VCR) . Images are recorded on a time-lapse VCR at real time and then played back at slower speed. The location of cells can be marked at any given time and the location of the individual cell is determined at some finite time afterwards.
- VCR video cassette recorder
- the kinds and numbers of leukocytes that rollingly adhere to a substrate can be determined and their individual velocities can be recorded.
- the analysis is completed by constructing a frequency histogram of the numbers of leukocytes (or a cell subset thereof) rolling at a particular velocity. This analysis also results in a determination of the rolling rate of a particular kind (subset) of leukocyte. Rolling leukocytes are visualized as round, distinct cells by the video camera because their rolling velocity is on the order of microns per second, which rolling velocity is extremely slow compared to the exposure time of the VCR camera (typically 30 frames per second) .
- non-adherent blood components flowing and tumbling through the apparatus tumble in the shear flow and appear as blurred streaks.
- non ⁇ adherent cells and those tumbling closest to the solid phase have a velocity of about 500 microns per second.
- Confirmation of rolling can be determined by visualizing the nuclei of the leukocytes under magnification. Determination of the rotating position of the nuclei distinguishes between leukocytes that merely slide along the surface of the substrate and those that rollingly adhere.
- Leukocytes in a given medium can optionally be differentiated from erythrocytes prior to introducing the sample into the flow chamber by labelling the leukocytes, e.g., with acridine orange or quinicrine dihydrochloride (see Nobis et al. , 1985, Microvasc. Res. 29:295-304).
- erythrocytes can be lysed prior to introduction of the labelled leukocytes into a flow chamber of the invention (e.g., by NH 4 C1 lysis; see e.g., 1991, Current Protocols in Immunology, ch. 3.1, J.E.
- the leukocytes are irradiated under ultraviolet (UV) light.
- UV ultraviolet
- the acridine orange or quinicrine dihydrochloride stain will absorb this radiation and emit light at visible wavelengths (510-540 nm) .
- the positions of leukocytes on the substrate are compared under UV irradiation in successive frames of the VCR recording to determine rolling velocities, and a histogram of velocities is constructed. Each peak in the histogram is associated with a specific cell population that rolls at a particular rate.
- Other methods of labelling leukocytes or cell subsets thereof can also be used, e.g. , by use of an antibody directed to a leukocyte cell surface antigen, which antibody is labeled with a detectable marker.
- a quartz cell with a hollow bore having an internal diameter between about 50 and about 500 microns is provided.
- the hollow bore is brought into contact with rolling mediators so that one or more rolling mediators are affixed to the inner wall of the hollow bore.
- Leukocytes and erythrocytes can be physically separated by lysing the erythrocytes as described above, or leukocytes can be stained with a fluorescent stain, thus differentially tagging them.
- a blood sample containing leukocytes to be differentiated is allowed to flow through the chamber affixed with one or more rolling mediators, and light of sufficient wavelength to excite the fluorescent label of the leukocytes is directed at the chamber. Emission of light from the rolling cells is determined.
- the total time interval over which fluorescent energy is received from the excited label on the leukocyte will be a function of its rolling velocity. This is because the light beam width can be made constant (preferably about 50-100 microns 2 ) and the amount of time required for any individual leukocyte to traverse the beam is the beam width divided by the cell velocity.
- laser light is used.
- Non-coherent light is also suitable.
- one embodiment of a laser-based method comprises providing fluorescently tagged leukocytes and flowing the leukocytes through a tube, the inner surfaces of which contain one or more rolling mediators.
- the sample is irradiated with laser light and the duration of fluorescent light emitted by each cell of the sample is measured as the cell flows in a direction perpendicular to the optical axis of the laser beam.
- Leukocytes can be discriminated on the basis of the distribution of signals that are indicative of the intensities of the sensed fluorescent light.
- the value of emitted light data can be stored using A/D converters in a computer system, or directly visualized with a video camera, such procedures being well known to those of ordinary skill in the art.
- cytometric methods can be calibrated by comparing the velocity of a given, but unknown type of leukocyte, to the velocity of a known type of leukocyte.
- the methods can also be used to produce a frequency histogram of leukocyte rolling velocities, which can be used in a diagnostic context, as described infra.
- an apparatus can comprise a solid phase, optionally contained within a housing.
- This solid phase contains one or more rolling mediators affixed to it, as described above (and, in an arrest model, one or more integrin binding partners) .
- the housing can include a means for withdrawing the sample from an outlet end and a means for introducing sample into an inlet end.
- the apparatus can contain a plurality of solid fibers upon whose cylindrical surfaces are immobilized one or more rolling mediators (and, in an arrest model, one or more integrin binding partners) .
- the ibers can be contained within a cartridge or housing with a means for introducing a fluid sample (e.g., of blood) into the housing and a means for withdrawing the sample from the housing so that the sample flows over the external surfaces of the fibers.
- a fluid sample e.g., of blood
- the size of fibers, their number, and the site density of rolling mediator can be adjusted to provide for a cartridge having different capacities for accumulating leukocytes, or cell subsets thereof.
- Apparatuses for use in diagnostic contexts can also be arranged into convenient kits. For example, a solid phase, such as a microscope slide or coverslip, can be provided that contains a plurality of rolling mediators arranged in spatially distinct zones.
- the solid phase can be attached to a dipstick or other arrangement such as the parallel flow chamber described above, to provide for relative movement between the solid phase and the sample.
- the solid phase can contain both rolling mediators and integrin binding partners in spatially distinct zones, for use with the addition of a chemoattractant, to promote affixing of the cells, in an arrest model.
- Another embodiment of a kit of the invention includes, in one or more containers, the following components of an apparatus of the invention: (a) liposomes or other artificial lipid bilayer components or precursors (e.g., phospolipids such as phosphatidylcholine) ; and (b) rolling mediator molecules.
- such a kit comprises, in one or more containers, liposomes and CD62.
- a kit of the invention comprises, in one or more containers or precursors: (a) liposomes or other artificial lipid bilayer components or precursors; (b) rolling mediator molecules; and (c) integrin binding partners.
- Such a kit can further comprise a chemoattractant in a container.
- such a kit comprises, in one or more containers: liposomes, CD62, ICAM-l, and the chemoattractant fMLP.
- the methods of collection, purification, and analysis of blood and blood components provided by the present invention have diagnostic and therapeutic utilities.
- the collection methods can be used to determine the number of leukocytes or cell subset thereof in a sample of blood or other fluid derived from a patient, or the number of leukocytes or cell subset thereof which roll at particular velocities in such a sample.
- Such number can be compared to the amount present in an equivalent sample from a normal or healthy subject, or a subject in remission from a disease or disorder, or the same patient at an earlier time period, whereby increases or decreases relative to such amount indicate the presence or progression of a disease or disorder, the stage of the disease or disorder, or the response to therapy in such patient, thus providing methods of detection, diagnosis, staging, and monitoring of treatment.
- the methods of the invention can be used to determine a percentage, consisting of the number of leukocytes or cell subset thereof per number of one or more other cell types in the sample, or consisting of the percentage of leukocytes rolling at a particular velocity, which percentage can be similarly compared to the percentage in an equivalent sample from subjects as described above, to detect, diagnosis, stage, and monitor treatment of diseases and disorders.
- the numbers of total leukocytes per volume of blood can be used diagnostically.
- the numbers of leukocytes from a patient with an unknown disease can be compared to the numbers of leukocytes in a blood sample that is characteristic of a disease. Comparison of the two samples can determine the presence or absence of the disease in the patient from whom the original sample is derived.
- the diseases or disorders suitable to diagnosis, staging, and/or monitoring in the foregoing methods include those in which there is a disturbance in the normal amount of a leukocyte or cell subset thereof, so that the changed amount is characteristic of a diseased condition.
- the number of circulating white blood cells (leukocytes) may be markedly decreased or increased in a variety of clinical disorders.
- disorders involving an increase in leukocytes or a subset thereof include but are not limited to hematologic malignancies such as leukemias, lymphomas, e.g., acute and chronic myeloid and lymphatic leukemias, including chronic myelogenous leukemia, adult T cell leukemia, non-Hodgkins lymphoma, chronic lymphatic leukemia, plasma cell myeloma, etc.
- hematologic malignancies such as leukemias, lymphomas, e.g., acute and chronic myeloid and lymphatic leukemias, including chronic myelogenous leukemia, adult T cell leukemia, non-Hodgkins lymphoma, chronic lymphatic leukemia, plasma cell myeloma, etc.
- eosinophils may occur in asthma or parasitic infections.
- An increase in the number of white blood cells in circulating blood is due mainly to granulocytosis. The most extreme and important increases are, however, encountered in the various
- leukopenias such as neutropenia, Hodgkin's disease, etc.
- An abnormally low white blood cell count may occur because of decreased numbers of any one of the specific types of leukocytes within the circulating blood, but most often leukopenia involves the neutrophils.
- Low lymphocyte counts are much more common and are associated with specific clinical syndromes (e.g., Hodgkin's disease, non-lymphocidic leukemias) .
- T lymphocytes Other diseases or disorders involving decreased levels of T lymphocytes include the immunodeficiency disorders congenital thymic aplasia (DiGeorge syndrome) and severe combined immunodeficiency disease (in which both T and B cells may be completely absent) , as well as AIDS (Acquired Immune Deficiency Syndrome; involving a decrease in CD4 + T cells) . Additionally, the histograms described in
- Section 5.7.1, supra. can be used in a diagnostic, staging, or monitoring context.
- blood samples taken from subjects known to have one or more of the foregoing diseases can be analyzed using the methods of this invention to produce a velocity histogram, and the resulting histogram provides a rolling velocity "fingerprint" that is characteristic of the disease.
- a blood sample from a patient having an unknown disease is then analyzed using the methods of this invention, and the velocity histogram is compared to this standard "fingerprint" to determine the presence or absence of the disease in the unknown blood sample.
- Methods designed to differentiate leukocytes based on rolling induced by interactions with different types of rolling mediators also provide a convenient method of determining numbers of different leukocytes.
- apparatuses and methods of the invention can be used in a therapeutic context to monitor the progression of a disease or the progression of therapy by taking frequency spectra or leukocyte rolling velocities from blood samples over a period of time.
- the methods of collection, purification, and analysis of the invention have utility for the diagnosis of diseases and disorders involving a defect in leukocyte rolling, chemoattractant activation, and/or arrest at the endothelial cell wall, by detecting a decrease in the percentage of leukocytes or a cell subset thereof from a patient which are able to roll, or roll and arrest, in the apparatuses of the invention, relative to such percentage of leukocytes from a healthy patient.
- leukocyte adhesion deficiency (Anderson and Springer, 1987, Ann. Rev. Med.
- leukocytes or a subset thereof from a subject, or blood components obtained after passage through an apparatus of the invention and thus deleted of such cells can be analyzed in vitro by carrying out diagnostic tests known in the art, e.g., analysis for expression of certain cell-surface antigens (e.g., associated with a malignancy or with infection by a pathogenic microorganism) , karotype analysis, etc.
- diagnostic tests known in the art, e.g., analysis for expression of certain cell-surface antigens (e.g., associated with a malignancy or with infection by a pathogenic microorganism) , karotype analysis, etc.
- the invention can be used ex vivo in a manner similar to a blood phoresis device. Blood is taken directly from a patient's blood vessel and is allowed to flow into the inlet port of the apparatus of Figure 2.
- Various valving arrangements that are well-known to those of ordinary skill in the art may be attached to one or another end of the apparatus to provide for shunting of blood flow away from the apparatus during washing and elution of leukocytes.
- citrate anticoagulated whole blood derived from a patient in a clinical setting can be introduced into the apparatuses of the invention and analyzed as described above.
- the collection and purification methods of the invention also have therapeutic utility, by providing collected, purified, and/or concentrated blood components which can be administered to a patient, in a transfusion procedure.
- leukocytes or a subset thereof isolated by the methods of the invention can be administered to a patient suffering from decreased levels of such leukocytes.
- fluid passed through an apparatus of the invention and thereby substantially deleted or decreased in leukocytes or a subset thereof can be administered to a patient in need of blood or blood components but for whom administration of such leukocytes is not desirable.
- the therapeutic and diagnostic methods described herein have application to subjects that are preferably mammals, including cows, dogs, pigs, and most preferably, humans.
- the apparatuses and methods of the present invention can be used for the identification of inhibitors (e.g., antagonists) or promoters (agonists/functional components or enhancers) of the adhesion receptor-mediated migration of leukocytes through the endothelium (extravasation) .
- inhibitors and promoters respectively inhibit and promote the inflammatory response, and thus have therapeutic utilities.
- the inhibitors and promoters are identified by detecting their abilities to respectively inhibit or promote the rolling of leukocytes or a cell subset thereof in a rolling model apparatus of the invention, or to respectively inhibit or promote the rolling and arrest of leukocytes or a cell subset thereof in an arrest model apparatus of the invention.
- the models of the invention can provide for the identification of inhibitors and promoters of the inflammatory response that are therapeutically useful in vivo.
- the methods of the invention can be used to identify an inhibitor of the inflammatory response which acts by inhibiting one or more of the steps involved in leukocyte rolling (by use of a rolling model apparatus) or leukocyte rolling and arrest (by use of an arrest model apparatus) in an apparatus of the invention.
- inhibitors thus identified can inhibit a rolling mediator-binding partner interaction, a chemoattractant-chemoattractant receptor interaction, and/or an integrin-integrin binding partner interaction.
- the inhibition may be competitive or non-competitive.
- an inhibitor of the inflammatory response is thus identified as follows.
- the rolling mediator(s) incorporated onto the solid phase surfac (s) , and the flow medium containing leukocytes are selected so as to provide for rolling of the leukocytes or one or more cell subsets thereof on the solid phase(s) .
- the molecule to be tested is introduced into the flow medium prior to passage though the apparatus, or preferably, during passage, when leukocytes have begun rolling.
- a decrease in rolling e.g., as measured by a decrease in the percentage of leukocytes that are rolling, or a decrease in their velocity, or a decrease in the number of rolling leukocytes per volume
- the molecule is an inhibitor of leukocyte extravasation, a component of the inflammatory response.
- the rolling mediator(s) and integrin binding partner(s) incorporated onto the solid phase surface(s) , the chemoattractant(s) introduced into the flow medium, and the flow medium containing leukocytes are selected so as to provide for rolling and arrest of the leukocytes or one or more cell subsets thereof on the solid phase(s) .
- the molecule to be tested is introduced into the flow medium prior to passage through the apparatus, or during passage, when leukocytes have begun rolling.
- a decrease in arrest of leukocytes indicates that the molecule is an inhibitor of leukocyte rolling, chemoattractant activation, and/or arrest, and thus an inhibitor of leukocyte extravasation, a component of the inflammatory response.
- Molecules to be tested for inhibitory activity can be any of interest, including but not limited to antibodies (preferably monoclonal, most preferably human or humanized monoclonal, or antigen- binding domains thereof) to any member of the following receptor-ligand pairs: rolling mediator- binding partner; chemoattractant-chemoattractant receptor; integrin-integrin binding partner, or a neutralizing epitope thereof, e.g., sialylated Lewis x or GDP-fucose; peptide antagonists and peptidomimetics, etc.
- antibodies preferably monoclonal, most preferably human or humanized monoclonal, or antigen- binding domains thereof
- a preferred specific embodiment for testing numerous compounds for inhibitory activity is as follows: As cells are continuously rolling on E-selectin, P-selectin, or the ligand of L-selectin in a parallel plate flow chamber of a rolling model apparatus of the invention, a test compound is injected for some duration of time just upstream of the observation point. The flow brings the compound, with some dilution factor (approximately 2-fold) , into the area of observation. Its effect on the percentage of rollingly adherent cells, or their number per volume flow medium, or their velocity, is then measured.
- Injection is stopped for a sufficient length of time to allow the compound to flow downstream, and for cells to accumulate from upstream and for rolling to reach equilibrium again, then a new compound is injected and the process is continuously repeated.
- cycle time 2 minutes, it is possible to screen 720 compounds per flow chamber per day.
- Multiple ports for simultaneous injection of compounds, with injection of buffer in intervening ports to keep the compounds separate in distinctive streams, may be used to increase throughput.
- the ports are placed perpendicular to the direction of flow and thus the streams are parallel. Cells rolling in each stream may be visualized using multiple microscope objectives or by moving the same objective from one area to another.
- the promoters of the inflammatory response detected according to the present invention can act by increasing the efficiency of the adhesion process in an apparatus of the invention, or by acting as a functional component thereof (e.g., a rolling mediator, chemoattractant, or integrin binding partner) .
- a functional component e.g., a rolling mediator, chemoattractant, or integrin binding partner
- Such a functional component is detected by its ability to promote rolling or arrest in a model where this was previously lacking (e.g., due to lack of appropriate cellular specificity of a rolling mediator or integrin binding partner previously present in the apparatus, or lack of any rolling mediator or integrin binding partner) .
- an arrest model apparatus can be used to identify a compound which is a chemoattractant or to identify an integrin binding partner, functional in leukocyte extravasation.
- a rolling model apparatus or an arrest model apparatus can be used to identify an endothelial cell rolling mediator functional in leukocyte extravasation.
- an arrest model apparatus in which the rolling mediator and integrin binding partner present on a solid phase thereof have binding partners situated on the leukocytes present in the flow medium to be passed through the apparatus.
- a sample containing the molecule to be tested for chemoattractant activity is introduced into the flow medium passing through the apparatus, and it is determined whether any leukocytes are arrested on the solid phase.
- CD62 and ICAM-l are used as the rolling mediator and integrin binding partner, respectively, to assay for chemoattractants with receptors on neutrophils and/or monocytes.
- chemoattractants can be used as either inhibitors or promoters of the inflammatory response depending on how they are administered.
- a chemoattractant gradient directing leukocytes toward a specific tissue is expected to be pro-inflammatory at such tissue
- general systemic administration of a chemoattractant is expected to be inhibitory to leukocyte extravasation, since the systemically administered chemoattractant would competitively inhibit leukocyte recognition of chemoattractant gradients directing its migration toward tissues.
- an arrest model apparatus is used, in which the rolling mediators on a solid phase thereof have binding partners situated on the leukocytes present in the flow medium to be passed through the apparatus.
- One or more chemoattractants are introduced into the flow medium.
- a chemoattractant is used which has a receptor on the same subset(s) of leukocytes that express the rolling mediator binding partner.
- a compound or molecule to be tested for integrin binding partner function is affixed onto the surface of the solid phase. After the flow medium is passed through the apparatus, it is determined whether any leukocytes have arrested on the solid phase. Arrest of leukocytes indicates that the molecule is an integrin binding partner which recognizes an integrin present on the same leukocyte subset(s) that express the rolling mediator binding partner and the chemoattractant receptor.
- the molecule to be tested for rolling mediator activity is incorporated onto the solid phase surface(s) of the rolling model apparatus, and flow medium containing leukocytes is passed through. Rolling of the leukocytes along the solid phase indicates that the molecule has rolling mediator activity and that the leukocytes express a binding partner for the rolling mediator.
- the molecule to be tested for rolling mediator activity is incorporated onto the solid phase surfac (s) of the arrest model apparatus. Also incorporated onto the solid phase surface(s) is an integrin binding partner.
- a chemoattractant is introduced into the flow medium passing through the apparatus.
- the flow medium contains leukocytes which express the integrin recognizing the integrin binding partner and express a receptor for the chemoattractant. After passage of the flow medium through the apparatus, it is determined whether any leukocytes have arrested on the solid phase. Arrest of leukocytes indicates that the test molecule has rolling mediator activity and that the leukocytes which express the integrin and the chemoattractant receptor also express a binding partner for the test molecule.
- a molecule can also be identified as a functional component in the processes of leukocyte rolling, or rolling and arrest, or as an enhancer thereof, by the methods described supra in which an increase in number or percentage of cells rolling or arrested, is detected relative to the number or percentage of such cells in the absence of the test molecule.
- the inhibitors and promoters of the invention have use therapeutically in diseases or disorders involving inflammation, and which involve extravasation of leukocytes.
- the invention provides methods of reducing inflammation, and of treating or preventing disorders associated therewith, by administration to a subject of an effective amount of the inhibitory compounds of the invention.
- the invention provides methods of stimulating the inflammatory response, and treating or preventing disorders associated with a deficit in the desired inflammatory response, by administration to a subject of an effective amount of the pro- inflammatory compounds (promoters) of the invention.
- the subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
- Disease and disorders which can be treated by administration of a therapeutically effective amount of the inhibitory compounds of the invention include but are not limited to the following:
- Inflammatory arthritis e.g., rheumatoid arthritis, seronegative spondeloarthritites (Behcets disease, Reiter's syndrome, etc.), juvenile rheumatoid arthritis, vasculitis, psoriatic arthritis, polydermatomyositis.
- SLE Systemic lupus erythematosus
- Inflammatory dermatoses e.g., psoriasis, dermatitis herpetiformis, eczema, necrotizing and cutaneous vasculitis, bullous diseases. Reperfusion injury. Septic shock (Sepsis) .
- ARDS Adult respiratory distress syndrome
- autoimmune disorders In addition to the autoimmune disorders SLE and rheumatoid arthritis, disorders such as glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic conditions, autoimmune thyroiditis, allograft rejection (e.g., rejection of transplanted kidney, heart, or liver) , Crohn's disease, and graft-versus-host disease can be treated.
- SLE and rheumatoid arthritis disorders such as glomerulonephritis, juvenile onset diabetes, multiple sclerosis, allergic conditions, autoimmune thyroiditis, allograft rejection (e.g., rejection of transplanted kidney, heart, or liver) , Crohn's disease, and graft-versus-host disease can be treated.
- Thermal injury burn
- the main complications due to burn are inflammatory in nature, including shock, and pulmonary edema.
- Cardiopulmonarv bypass Systemic inflammation has been associated with the use of pump- oxygenator systems in cardiopulmonary bypass and hemodialysis, which can lead to organ dysfunction, termed the post-pump syndrome or post-perfusion syndrome.
- the inhibitors of the invention can be treated with the inhibitors of the invention, including but not limited to those associated with hemolytic anemia, hemodialysis, blood transfusion, certain hematologic malignancies, pneumonia, post- ischemic myocardial inflammation and necrosis, barotrauma (decompression sickness) , ulcerative colitis, inflammatory bowel disease, atherosclerosis, cytokine-induced toxicity, necrotizig enterocolitis, granulocyte-transfusion-associated syndromes, Reynaud' ⁇ syndrome, multiple organ injury syndromes secondary to septicemia or trauma, and acute purulent meningitis or other central nervous system inflammatory disorders.
- the inhibitors of the inflammatory response which bind to ICAM-l can be used to treat or prevent viral infections such as rhinoviral infection, since the rhinovirus binds to ICAM-l on human cells and thereby initiates infection of the cells.
- Diseases or disorders that can be treated by the pro-inflammatory compounds of the invention include but are not limited to immunosuppression (e.g., due to AIDS, cancer chemotherapy, radiation therapy, corticosteroid therapy, or other therapy for autoimmune disease) , and congenital immunodeficiencies.
- immunosuppression e.g., due to AIDS, cancer chemotherapy, radiation therapy, corticosteroid therapy, or other therapy for autoimmune disease
- congenital immunodeficiencies e.g., due to AIDS, cancer chemotherapy, radiation therapy, corticosteroid therapy, or other therapy for autoimmune disease
- any animal model system known in the art may be used prior to administration to humans.
- animal models are available to demonstrate the efficacy of anti- inflammatory compounds of the invention in the treatment of adult respiratory distress syndrome (ARDS) .
- ARDS adult respiratory distress syndrome
- animal models are available to demonstrate the efficacy of anti- inflammatory compounds of the invention in the treatment of adult respiratory distress syndrome (ARDS) .
- ARDS adult respiratory distress syndrome
- New Zealand white rabbits infused with activated complement Nuytinck et al., 1986, Brit. J. Exp. Pathol. 67:537-548
- cerulean- induced acute pancreatitis in rats (Guice et al.,
- Two animal models of sepsis which can be used are a rat cecal ligation and puncture model (von Allmen et al., 1990, J. Surg. Res. 48:476-480) and a sheep common bile duct contamination model (Barke et al., 1990, Arch. Surg. 125:437-440).
- An animal model system for rheumatoid arthritis is that consisting of animals of the autoimmune MRL/1 mouse strain (Murphy, E.D. and Roths, J.B., 1978, in Genetic Control of Autoimmune Disease. Rose, N.R., et al. , eds., Elsevier/North-Holland, New York, pp. 207-219) , that develop a spontaneous rheumatoid arthritis-like disease (Hang et al., 1982, J. Exp. Med. 155:1690-1701).
- THERAPEUTIC ADMINISTRATION AND COMPOSITIONS Various delivery systems are known and can be used to administer the compounds of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, expression by recombinant cells, etc.
- Other methods of introduction include but are not limited to intradermal, intramuscular, intraperitonea1, intravenous, subcutaneous, intranasal, and oral routes.
- the compounds may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
- Administration can be systemic or local, e.g., direct injection at the inflamed joint of someone suffering from rheumatoid arthritis.
- compositions comprise a therapeutically effective amount of a compound of the invention, and a pharmaceutically acceptable carrier or excipient.
- a pharmaceutically acceptable carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
- the formulation should suit the mode of administration.
- the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
- the composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
- the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
- Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
- the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
- compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
- the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
- the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
- composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
- an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
- the compounds of the invention can be formulated as neutral or salt forms.
- Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc. , and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.
- the amount of the compound of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
- the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
- Associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
- PHYSIOLOGIC FLOW RATES DISTINCTION FROM AND PREREQUISITE FOR ADHESION THROUGH INTEGRINS
- neutrophil binding to planar bilayers containing CD62 and ICAM-l was found to be highly specific. Neutrophils did not bind to planar membranes containing phosphatidylcholine alone. Neutrophil binding to planar membranes containing CD62 was 98% reversible by incubation with EDTA for 10 min. Binding of N-formyl methionyl leucyl phenylalanine (fMLP)-stimulated neutrophils to planar membranes containing ICAM-l was inhibited 97% by a combination of anti-LFA-1 (TS1/22) and anti-Mac-1 (LPM19c) ⁇ subunit antibodies, in agreement with previous reports (Smith et al., 1989, J. Clin.
- TS1/22 anti-LFA-1
- LPM19c anti-Mac-1
- Shear stress The change in velocity per change in radial displacement is called shear and is highest at the wall.
- Shear stress the product of shear and viscosity, better correlates with the forces acting on a cell under flow.
- Shear stresses of 1-10 dyn/cm 2 have been measured for postcapillary venules (Heisig, 1968, Adv. Microcirc. 1:89-94), and in the classic studies of Atherton and Born (1972, J. Physiol. 222:447-474), leukocytic rolling was observed at shear stresses that we calculate to be 1.5-4.0 dyn/cm 2 .
- Flowing neutrophils readily bound to artificial bilayers containing CD62 at wall shear stresses within the physiologic range (Fig. 5A) .
- Lymphocytes also fail to bind through CD2 to LFA-3 (1,000 sites per ⁇ m 2 ) under flow conditions (not shown), but do so at stasis (Chan et al., 1991, J. Cell Biol. 115:245-255).
- the failure of several integrin-lg superfamily interactions and Ig-Ig superfamily (CD2-LFA-3) interactions to occur under flow conditions is distinct from the efficiency of interaction through the selectin CD62.
- Rolling velocity increased with increasing shear force (Fig. 7) .
- the rolling velocity was proportional to the shear force at low shear stresses, and then began to plateau. This may reflect the effect of torque acting on a rolling but deformable object, leading to a greater contact area and less fluid drag.
- Rolling velocity was dependent on the site density of CD62 (Fig. 7) .
- the higher the CD62 density the more slowly the neutrophils rolled at a given shear stress. This is as predicted, because a higher number of receptor-ligand interactions will lead to a greater resistance to the fluid drag force.
- the slower rolling velocities at higher site density correlated with the increased effectiveness of attachment.
- Inclusion of ICAM-l in artificial bilayers containing CD62 did not alter rolling velocity (Fig. 7) , correlating with its lack of effect on attachment.
- Rolling did not appear to result in any alteration in the cells or the substrate.
- the rolling velocity of groups of cells followed along the substrate did not appear to change with time.
- the rolling velocity of cells on CD62 substrates was unaltered for at least 15 min at a single observation condition, and the same substrate could be used for observations at many different shear stresses, without any alteration in rolling velocity measurements for the same shear stress replicated at the beginning and end of the set of observations.
- the direction of flow was reversed, the direction of rolling was reversed and the cells rolled back "over their tracks" with the same velocity as in the forward direction.
- Measured rolling velocities are from 2 or 4 s measurement periods on the indicated substrates (CD62 at 200 sites per ⁇ m 2 ; ICAM-l at 1000 sites per ⁇ m 2 ) ; the percentage of cells that rolled in this time period on CD62 is shown in parentheses. Velocities of resting neutrophils on ICAM-l at shear stresses of 0.73 dyn/cm 2 and above were measured from the leading edge of the streak on four consecutive frames. Asterisks indicate that cells moved too fast for velocity measurements. Measurements for PMA-stimulated neutrophils were made after cells were allowed to adhere to the substrate under static conditions for 6 min.
- Hydrodynamic calculations are for a 7 ⁇ m diameter hard sphere in a shear flow with no interaction with the wall, at the indicated distance from the wall.
- the measurements on the ICAM-l substrate are for the cells flowing closest to the substrate, as determined by the focal plane.
- the unstimulated neutrophils flowing over the ICAM-l substrate moved at a velocity 100-fold greater than the cells rolling on the CD62 substrate. Because of the torque exerted by shear flow, a spherical object will rotate at an angular velocity of one-half the shear rate, so its motion, particularly if close to the wall, is not qualitatively different from rolling along a flat surface.
- Predicted velocities of a sphere 7 ⁇ m in diameter having no interaction with a smooth wall Goldman et al., 1967, Chem. Engineer Sci.
- Neutrophils on CD62 bilayers remained round even after prolonged contact of up to 12 min (Fig. 8G) , in agreement with observations on rolling cells under flow conditions.
- Neutrophil activation with PMA induces a bipolar shape change even when cells are held in suspension; PMA-stimulated neutrophils had a bipolar appearance on CD62 bilayers but no spreading was observed (Fig. 8H) .
- To examine adherence under static conditions neutrophils were allowed to settle onto the planar membrane, and after 6 min of contact, controlled flow was used to create a detachment force. Binding to CD62 and ICAM-l was compared, with and without PMA present. This assay highlighted significant differences in patterns of adhesion strengthening between the CD18 and the CD62 pathways.
- leukocyte rolling a hallmark of the early stages of an inflammatory response, can be reconstituted in vitro on artificial lipid membranes containing an endothelial cell selectin, CD62.
- Neutrophils formed reversible rolling attachments to CD62 at physiologically relevant shear stresses.
- Another endothelial cell adhesion molecule, ICAM-l did not support rolling adhesions, and was found to be significantly less effective than CD62 as a ligand for neutrophils under flow conditions. However, ICAM-l was required for subsequent spreading and the development of a shear resistant attachment.
- the selectin CD62 was distinctive from integrin and Ig family members both in ability to mediate rolling and to mediate adhesion during flow at physiologic shear stresses.
- the avidity of neutrophil integrins for ICAM-l was stimulated with PMA or fMLP, flowing neutrophils did not bind to ICAM-l significantly over a range of physiologic shear stresses at which CD62 was highly effective. This was despite the ability of activated neutrophils to form attachments in static assays to 4-fold lower densities of ICAM-l. These attachments to ICAM-l were greater than 100-fold more shear resistant than attachments to CD62.
- Integrins on activated T cells also failed to bind to ICAM-l and fibronectin at physiologic shear stresses. Furthermore, an Ig family-Ig family interaction between CD2 on T lymphocytes and LFA-3 in artificial bilayers failed to occur at physiologic shear stresses. Even when adhesion of neutrophils or lymphocytes to ICAM-l or lymphocytes to LFA-3 was initiated under static conditions, firm adhesion was unable to be converted to a rolling adhesion when shear flow was applied; cells either remained adherent or were completely dislodged. The shear stress required to dislodge 50% of cells binding through CD2 to the transmembrane isoform of LFA-3 is 1.5 dyn/cm 2 (Chan et al.
- CD62 is stored in Weibel-Palade bodies of endothelial cells (Bonfanti et al., 1989, Blood 73:1109-1112; McEver et al., 1989, J. Clin. Invest. 84:92-99), which in response to stimuli such as thrombin and histamine fuse with the plasma membrane and thereby upregulate expression of CD62.
- An increase from 20 to 50 sites per ⁇ m 2 within 5 min after stimulation and a decline to baseline levels by 30 min is seen for endothelium cultured in vitro (Hattori et al., 1989, J. Biol. Chem. 264:7768-7771).
- endothelial cells in vitro there is a decline in CD62 and Weibel-Palade body content and therefore densities of CD62 could be higher in vivo.
- CD62 is a receptor that mediates rolling
- Thrombin stimulation mediates a transient binding of neutrophils that is CD18 independent and likely due to CD62 expression (Zimmerman and Mclntyre, 1988, J. Clin. Invest. 81:531-537; Geng et al., 1990, Nature 343:757-760).
- Neutrophil binding to primary culture endothelial cells at 2.0 dyn/cm 2 wall shear stress is stimulated by thrombin and is characterized by the initiation of rolling detectable within less than a 1 min of thrombin exposure, suggesting the involvement of CD62 (M.B. Lawrence and L.V. Mclntire, unpublished data) .
- CD62 is an excellent candidate for the initiation of neutrophil rolling during the early stages of an inflammatory response.
- Rolling velocities on CD62 were comparable to in vivo rolling velocities. On bilayers containing CD62, velocities ranged from under 2 to over 30 ⁇ m/s, depending on site density and the shear stress. Mean rolling velocities in vivo were 10 ⁇ m/s in mouse mesentery venules and 20 ⁇ m/s in hamster cheek pouch venules (Atherton and Born, 1973, J. Physiol. 233:157-165). Rolling velocities on CD62 were proportional to the flow rate at low shear stresses, but increases were not proportional at higher shear stresses. Atherton and Born (1973, J. Physiol.
- Wall shear stresses at which rolling was observed in the measurements of Atherton and Born (1972, J. Physiol. 222:447-474; 1973, J. Physiol. 233:157-165) ranged from 1.5 to 4.0 dyn/cm 2 .
- These values are similar to the shear stresses at which neutrophils form rolling attachments to bilayers containing CD62, and . also to monolayers of endothelial cells stimulated with cytokines (Lawrence et al. , 1990, Blood 75(1) :227-237) or thrombin (M.B. Lawrence and L.V. Mclntire, unpublished data) .
- selectins have not been shown to mediate rolling; however, this family of molecules may be specialized to mediate rolling adhesions, and rolling observed in vivo may involve contributions from all three.
- LECAM-1 contributes to interaction of unactivated neutrophils with cytokine stimulated endothelial cells at physiologic shear stresses (Smith et al., 1991, J. Clin. invest. 87:609-618) .
- the selectins so far studied appear to be structures that are capable of mediating the initial attachment of neutrophils to the vessel wall.
- the structure of selectins and their carbohydrate ligands appears ideally suited for their function in rolling and adhesion at high shear stresses.
- Diffusion of the extracellular ligand binding domain of an adhesion receptor has two components, one due to segmental flexibility of the tether by which it is attached to the membrane and the other due to lateral diffusion in the membrane bilayer of the membrane anchor. With an appropriately high number of segments and high segmental flexibility, the former type of diffusion can occur more rapidly than the latter, since proteins of adhesion receptor size have diffusion coefficients on the order of 10" 7 cm 2 /s (Tanford, 1961, Physical Chemistry of
- the (sialylated) Lewis x ligand of CD62 is found at the termini of long carbohydrate structures that are predicted to confer flexibility (Fukuda et al., 1984, J. Biol Chem. 259:10925-10935).
- the location of the lectin binding domain and the Lewis x determinant at the termini of their respective structures maximizes diffusiveness due to segmental flexibility.
- the diffusion coefficient is inversely related to size; therefore, sialylated Lewis x, by virtue of its small size (molecular weight 779) relative to protein adhesion ligands, allows a faster rate for diffusion-limited processes.
- Adhesiveness through integrins and the selectin CD62 differ drastically in the time scale required for their development. Binding of flowing cells to a substrate places stringent time constraints on adhesive bond formation that appear to prevent leukocyte integrin interactions with ICAM-l, even when neutrophils are activated. CD62 mediates adhesion on a time scale at which the leukocyte integrin interactions are ineffective. Bond formation through CD62 appears so rapid that little adhesion strengthening is apparent when binding under flow and static conditions are compared. Adhesion strengthening through integrins may take time to develop both because the globular putative ligand binding regions are quite large and are attached by two stalks to the membrane (Nermut et al., 1988, EMBO J.
- CD62 binds at saturation to 20,000 sites per cell with a similar affinity or avidity of 10 9 M" 1 to both resting and PMA-stimulated neutrophils (Moore et al., 1991, J. Cell Biol. 112:491-499).
- PMA-stimulated and resting neutrophils When we bound PMA-stimulated and resting neutrophils to artificial bilayers containing CD62 at stasis and subjected them to detachment with shear, PMA-stimulated cells bound less efficiently, but the decrease in efficiency compared to untreated cells was much less marked than for binding under conditions of shear.
- Activated neutrophils with their bipolar, elongated shapes would experience higher transient torques than unstimulated, round cells in contact with a substrate. The shape change may therefore be an important factor that impedes adhesion in shear flow, and may help prevent activated leukocytes, if they fail to emigrate at an inflammatory site, from attaching and emigrating at an uninvolved site downstream.
- the rolling interaction may promote the integrin-ICAM-1 interaction both because it facilitates close physical interaction between integrins and ICAM-l, and because the neutrophil is rolling approximately two orders of magnitude more slowly than a tumbling neutrophil near the wall, which would raise the chance of enough bonds being formed for the rolling adhesion to be converted to a stationary one.
- Our findings suggest that activated integrins contribute to leukocyte arrest at sites of endothelial cell junctions and contribute to the mechanism for transendothelial migration.
- the neutrophil interaction with CD62 is highly reversible, and should not impede subsequent migration mediated by leukocyte integrins, since interactions through the leukocyte integrins are much stronger.
- the slowed rolling allows even more efficient exposure to chemostimulants and thereby sets in motion a positive feedback loop that results in arrest of the rolling leukocyte and finally spreading, integrin-mediated adhesion strengthening, and transendothelial migration.
- neutrophil rolling and subsequent arrest and adhesion strengthening at an inflammatory site in vivo can be accurately reproduced in vitro with a small number of purified components: a lipid bilayer, the selectin CD62, the Ig family member ICAM-l, and the chemostimulant fMLP.
- Virus-transformed B lymphoblastoid JY cell clone 33 (Hollander et al., 1988, J. Immunol. 141:4283-4290), as previously described (Marlin and Springer, 1987, Cell 51:813-819). Briefly, a Triton X-100 lysate was passed over a RRl/1 Sepharose column, and the column was eluted with a buffer containing 1% octyl- ⁇ -D-glucopyranoside (0G) so that ICAM-l could be incorporated into liposomes.
- CD62 was a generous gift of Drs. S. Sajer and B. Furie, and was purified as previously described (Larsen et al. , 1989, Cell 59:305-312) .
- the lipid film was redissolved at 0.4 ⁇ M in 250 ⁇ l of 25 ⁇ M Tris-HCl (pH 8.0)/150 ⁇ M NaCl (TS) , 2% (w/v) OG, and was mixed with 250 ⁇ l of detergent solution containing approximately 1-18 ⁇ g/ml CD62, 6-30 ⁇ g/ml ICAM-l, or both in TS, 1% OG, followed by three changes of dialysis against TS at 4°C over 36 hr. After the removal of OG by dialysis, the liposome suspension was stored at 4°C under argon to minimize oxidation of lipids.
- PLANAR BILAYERS Planar bilayers were formed by incubating drops of liposome suspension on glass coverslips or slides at 22°C for 30 min. Prior to use, all glass surfaces were boiled in detergent (Linbro 7 x solution, Flow Lab, McLean, VA) for 15 min, rinsed extensively in deionized distilled water for at least 24 hr, and then stored in ethanol. For site number determinations, glass coverslips (5 mm diameter, no.
- Liposomes were reconstituted with different quantities of either immunoaffinity-purified ICAM-l or CD62, and planar membranes were formed as described above.
- Monoclonal antibodies R6.5 to ICAM-l (Smith et al., 1988, J. Clin. Invest. 82:1746-1756) and AC1.2 to CD62 (Larsen et al., 1989, Cell 59:305-312) were iodinated to a known specific activity of about 70 ⁇ Ci/ ⁇ g, and site densities of ICAM-l and CD62 were determined by saturation binding, as previously described (Dustin and Springer, 1988, J. Cell Biol. 107:321-331).
- Site numbers assume binding of one IgG molecule per antigen molecule because saturation binding favors monomeric binding and because transmembrane proteins are immobile on glass-supported bilayers (McConnell et al., 1986, Biochim. Biophys. Acta 864:95-106) and at the highest density were on average too far apart (32 nm) for bivalent binding. After initial measurements of bilayer incorporation, protein concentrations were adjusted to give round numbers of sites per ⁇ m 2 . The actual site densities were determined twice for each liposome preparation at each density, in triplicate.
- the neutrophils were washed into HBSS supplemented with 10 mM HEPES, 1.0 mM Mg 2+ , and 1.2 mM Ca 2+ at pH 7.3, since CD18 interactions with ICAM-l require divalent cations (Marlin and Springer, 1987, Cell 51:813-819), as does the CD62 interaction with its counterstructure (Geng et al., 1990, Nature 343:757-760).
- a glass slide containing a planar bilayer was assembled in a parallel-plate laminar flow chamber (260 ⁇ m gap thickness) in which a uniform wall shear stress is generated.
- the flow chamber was mounted on the stage of an inverted phase-contrast microscope (Diaphot-TMD, Nikon Inc., Garden City, NY) .
- inverted phase-contrast microscope Diaphot-TMD, Nikon Inc., Garden City, NY
- neutrophils were resuspended at a concentration of 10 6 /ml in HBSS supplemented with 10 mM HEPES, 1.2 mM Ca 2+ , and 1.0 mM Mg 2+ ) and drawn through the chamber at controlled flow rates with a syringe pump attached to the outlet.
- the wall shear stress was calculated from a momentum balance on a Newtonian fluid, assuming a viscosity of 1.0 centipoise.
- the flow rate was stepped down to allow measurements of cell binding at different shear stresses. Three minutes was allowed for equilibration before the number of cells per unit area was measured. Measurements on different areas of the bilayer were averaged, and no evidence for nonuniformity was found. Attached neutrophils and their motion were observed with phase-contrast objectives and quantitated by analysis of videotaped images. For activation studies, neutrophils were treated with PMA (30 ng/ml, final concentration) for 5 min before perfusing the cell suspension through the flow chamber.
- neutrophils (4 x 10 6 /ml) were injected into the chamber through a port and allowed to settle.
- PMA was added to the neutrophils 1 minute before injection into the flow chamber. All cells came in contact with the bilayer within 120 s, as indicated by their entry into the same focal plane. Controlled flow was applied following a 6 min incubation period. The initial shear force was 0.5 dyn/cm 2 ; this force was increased every 20 s to a maximum of 36 dyn/cm 2 . All experiments were recorded on videotapes, and multiple fields of view were examined for each data point.
Abstract
Description
Claims
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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AU21772/92A AU663324B2 (en) | 1991-05-30 | 1992-05-29 | Device and method for the analysis of rolling blood leukocytes |
JP5500579A JPH06508265A (en) | 1991-05-30 | 1992-05-29 | Apparatus and method for analysis of rolling blood leukocytes and identification of inhibitors and promoters |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US70784191A | 1991-05-30 | 1991-05-30 | |
US707,841 | 1991-05-30 | ||
US07/887,444 US5460945A (en) | 1991-05-30 | 1992-05-20 | Device and method for analysis of blood components and identifying inhibitors and promoters of the inflammatory response |
US887,444 | 1992-05-20 |
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WO1992021746A1 true WO1992021746A1 (en) | 1992-12-10 |
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PCT/US1992/004524 WO1992021746A1 (en) | 1991-05-30 | 1992-05-29 | Device and method for the analysis of rolling blood leukocytes and identifying inhibitors and promoters |
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Country | Link |
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EP (1) | EP0586596A4 (en) |
JP (1) | JPH06508265A (en) |
AU (1) | AU663324B2 (en) |
CA (1) | CA2110292A1 (en) |
IE (1) | IE921698A1 (en) |
IL (1) | IL102030A0 (en) |
NO (1) | NO934321D0 (en) |
NZ (1) | NZ242896A (en) |
WO (1) | WO1992021746A1 (en) |
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1992
- 1992-05-26 NZ NZ242896A patent/NZ242896A/en unknown
- 1992-05-27 IL IL102030A patent/IL102030A0/en unknown
- 1992-05-29 JP JP5500579A patent/JPH06508265A/en active Pending
- 1992-05-29 CA CA002110292A patent/CA2110292A1/en not_active Abandoned
- 1992-05-29 WO PCT/US1992/004524 patent/WO1992021746A1/en not_active Application Discontinuation
- 1992-05-29 AU AU21772/92A patent/AU663324B2/en not_active Ceased
- 1992-05-29 EP EP92913842A patent/EP0586596A4/en not_active Withdrawn
- 1992-07-01 IE IE169892A patent/IE921698A1/en unknown
-
1993
- 1993-11-29 NO NO934321A patent/NO934321D0/en unknown
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Also Published As
Publication number | Publication date |
---|---|
CA2110292A1 (en) | 1992-12-10 |
AU663324B2 (en) | 1995-10-05 |
IL102030A0 (en) | 1992-12-30 |
AU2177292A (en) | 1993-01-08 |
NZ242896A (en) | 1996-05-28 |
JPH06508265A (en) | 1994-09-22 |
NO934321D0 (en) | 1993-11-29 |
EP0586596A4 (en) | 1996-06-05 |
EP0586596A1 (en) | 1994-03-16 |
IE921698A1 (en) | 1992-12-02 |
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