- Email: [email protected]
[43] Basophils in allergic and inflammatory reactions
522
INFLAMMATION
[43]
and chemokinesis. The latter property reflects the tendency of a substance to increase random cell migration. This discrimination is best measured by a checkerboard design of chemotaxis. In this design, the substance to be evaluated is incorporated in multiple doses on either or both sides of the membrane. In this manner it is possible to distinguish pure chemotactic activity, pure chemokinetic activity, and mixed activity in the same preparation.S Many methods for assessing the results of the micropore chemotactic assay have been described. Generally, however, these fall into three basic types: (1) counting the number of cells that have arrived at a preselected distance, such as the distal surface of the filter; (2) measurement of the distance migrated by faster migrating cells (leading-front technique); and (3) measurement of the average distance migrated per cell (the locomotion or leukotactie index). The first method described is very sensitive for detecting differences in nonstimulated and chemotactic responses. It can be used effectively for measurement of chemotactic activity released from mast cells. The latter two methods are much more sensitive to defects in cell migration and are used for investigation of clinical defects in this process. 51 S. H. Zigmond and J. G. Hirsch, J. Exp. Med. 137, 387 (1973).
[ 4 3 ] B a s o p h i l s in A l l e r g i c a n d I n f l a m m a t o r y
Reactions
By FRANK M. GRAZIANO Introduction Basophils are granulocytic cells that differentiate in bone marrow, circulate in blood, and migrate into tissues in response to appropriate stimuli. They are the least common of the granulocytic cells and comprise 0.5- 1% of the total leukocyte population. Basophil morphology is dominated by intracytoplasmic granules, and the ultrastructure of these granules appears to differ from species to species. In humans, basophil granules vary in shape (round, oval, angular) and size ( 113- 260 A), are embedded in a less dense matrix, and are surrounded by a membrane) In guinea pigs, basophil granules are large oval structures that, depending on the plane of section, display a parallel array, hexagonal array, finely granular, or rectanA. Dvorak and H. Dvorak, Arch. Pathol. Lab. Med. 103, 551 (1979).
METHODS IN ENZYMOLOGY, VOL. 162
Copyright© 1988 by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
523
Flo. 1. Electron micrograph of a guinea pig basophil. The cytoplasm demonstrates typical large oval mediator-containing granules. × 14,080. (Insert a) Cytoplasmic granule. The dense granule particle appears in a less dense matrix surrounded by a perigranular membrane. X 27,500.
gular lattice o f granular material. A rim o f less dense finely granular or fibrillar material surrounds the matrix and separates it from the granule m e m b r a n e ~ (Fig. 1). A close relationship appears to exist between basophils and the granule-containing mononuclear mast cells, but in both
524
INFLAMMATION
[43]
functional and biochemical studies distinct differences between the two cell types have been reported. 2,3 In humans, basophils contain almost all of the blood histamine. 4 For many years, in vitro release ofbasophil histamine in allergic individuals has served as an assay of immediate hypersensitivity responsiveness? Knowledge of the actual participation of basophils in disease, however, has only come about with the appreciation that significant tissue infiltrates accompany some allergic diseases. 4,5 Attempts in the past to clarify the role of basophils have been greatly limited by the low number of these cells found in peripheral blood. 6,7 Recently, success in achieving high levels of purity and yield of basophils has been accomplished in humans with affinity chromatography,S- ~0 Percoll density gradient centrifugation,~ ~- ~4 centrifugal elutriation,15 cell panning procedures, 16,~7and flow microfluorometry.lS Normal circulating blood basophils have been identified with great difficulty in rodents, 19 appear to be abundant in turtles, 2° and have been isolated in substantial purity from guinea pigs. 2~,22 It is the purpose of this chapter to review basophil isolation procedures with special emphasis on human and guinea pig techniques.
2 D. MacGlashan, R. Schleimer, S. Peters, E. Schulman, G. K. Adams III, A. Sobotka, H. Newball, and L. Lichtenstein, Fed. Proc., Fed. Am. Soc. Exp. Biol. 142, 2504 (1983). 3 H. Dvorak and A. Dvorak, Hum. Pathol. 3, 454 (1972). 4 E. B. Mitchell and P. Askenase, Clin. Rev. Allergy 1,427 (1983). H. Dvorak, J. Allergy Clin. Immunol. 58, 229 (1976). 6 R. Day, Clin. Allergy 2, 205 (1972). 7 T. Ishizaka, R. DeBernardo, H. Tomioka, L. Lichtenstein, and K. Ishizaka, J. Immunol. 108, 1000 (1972). s D. MacGlashan and L. Lichtenstein, J. Immunol. 124, 2519 (1980). 9 j. Toll, J. Wikberg, and R. Andersson, Allergy 36, 411 (1981). 10j. Pruzansky and R. Patterson, J. Immunol. Methods 44, 183 (1981). 11 p. Raghuprasad, J. Immunol. 129, 2128 (1982). 12H. Kaufman, P. Levering, and K. DeVries, J. Immunol. Methods 57, 1 (1983). 13E. Leonard, R. Roberts, and A. Skeel, J. Leuk. Biol. 35, 169 (1984). 14E. Leonard and A. Skeel, J. Allergy Clin. Imrnunol. 76, 556 (1985). 13 M. DeBoer and D. Roos, J. Immunol. 136, 3447 (1986). ,6 F. Landry and S. Findlay, J. Immunol. Methods 63, 329 (1983). 17M. Lett-Brown, S. Robinson, H. Juneja, J. Farnam, and J. A. Grant, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 7438 (1985). 18G. Weil, W. Leiserson, and T. Chused, J. Immunol. Methods 58, 359 (1983). 19A. Dvorak, G. Nabel, K. Pyne, H. Cantor, H. Dvorak, and S. Galli, Blood59, 1279 (1982). 20 K. Mead, M. Borysenko, and S. Findlay, J. Immunol. 130, 334 (1983). 21 H. Dvorak, S. Selvaggio, A. Dvorak, R. Colvin, D. Lean, and J. Rypysc, J. Imrnunol. 113, 1694 (1974). 22 F. M. Graziano, W. Lipham, N. Swaminathan, J. Brendel, F. M. Graziano, Jr., and T. Warner, J. Immunol. Methods67, 157 (1984).
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
525
Methods for the Isolation of H u m a n Basophils Studies of human basophil function have progressed much more rapidly than studies of basophil biochemistry, pharmacology, and physiology. Recently, a number of methods have been developed to purify human basophils, and a more intensive study of this cell has proceeded. Since each method has its own advantages and disadvantages and no one method has proved to be clearly superior to all others, in this author's opinion, several important and productive methods for the purification of human basophils are discussed in detail below.
Procedures Using Affinity Chromatography There have been three major procedures developed which take advantage of a positive selection of basophils using affinity chromatography.8- i0 Two of the methods utilize antibody linked to an insoluble support, and the third utilizes the property of basophil adherence to glass beads. Procedure I. Methods utilized to purify human circulating basophils developed by MacGlashan and Lichtenstein8 were the first major step forward, allowing for the direct biochemical study of IgE-mediated phenomena in normal human basophils. The method of isolation takes advantage of unoccupied IgE receptors on basophils and the high affinity of these receptors for IgE antibody. In essence, the procedure entails passively sensitizing a mixed population of cells with antigen-specific IgE antibody (in this procedure antipenicillin IgE antibody) and passing this sensitized population of cells over a column of Sepharose to which the specific antigen [penicillin, i.e., benzylpenicilloyl (BPO)] 2z' has been coupled. IgEcoated basophils adhering to the column are then eluted with hapten antigen (penicillin hapten). The IgE-specific antipenicillin antibody (antibenzylpenicilloyl, prepared against the major penicillin determinant, BPO) critical to this procedure is isolated from penicillin-allergic individuals by passage of their serum over an affinity column of BPO linked to Sepharose CL-4B (Pharmacia, Piscataway, NJ). This column is prepared by first incubating the 22~Abbreviations: BPO, benzylpenicilloyl;CNBr, cyanogen bromide; PA, PIPES plus human serum albumin; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); BPO-fLys, benzylpenicilloyl-N-formyl-L-lysine; EDTA, ethylenediaminetetraacetic acid; REH, RPMI 1640 plus EDTA plus HEPES; Tris-ACM, Tris buffer plus human serum albumin, calcium, and magnesium; HBSS, Hanks' balanced salt solution; HEPES, N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid; PBS, phosphate-buffered saline; HLA, human leukocyte antigen; FCS, fetal calf serum; BSA, bovine serum albumin; FITC, fluorescein isothiocynate; FACS, fluorescence antibody ceil sorter; PBS-TC, PBS plus trisodium citrate; HG, HEPES plus gelatin; RBL, rat basophilic leukemia.
526
INFLAMMATION
[43]
cyanogen bromide (CNBr)-activated Sepharose beads with a solution of 0.1 M ethylenediamine (Sigma, St. Louis, MO) and 1 M NaCI, at pH 8.0 for 2 hr at room temperature. After washing, the beads are incubated for 90 min with a solution of 0.15 M sodium penicillin G (Sigma), 1 M NaC1, and 0.1 M NaHCO3 (maintained at pH 11). The beads are subsequently washed, and the penicillin adsorbent in PA buffer [piperazine-N,N'-bis(2ethanesulfonic acid) (PIPES) 25 mM, NaCI 150 mM, KC1 5 mM, adjusted to pH 7.3 with NaOH and containing 0.003% human serum albumin] is incubated batchwise with penicillin-allergic serum (1 volume of beads with 3 volumes of serum) for 5 hr at 4 °. After washing once again, bound IgE antibody is eluted with BPO-fLys (1 × 10-3 M benzylpenicilloyl-N-formyl-L-lysine), concentrated with no attempt to remove the BPO-fLys hapten, and used for passive sensitization of basophils. In the cell isolation procedure 50 ml of blood is obtained from nonallergic donors and mixed with saline containing ethylenediaminetetraacetic acid (EDTA) (0.8 ml of 0.1 M EDTA and 25 ml of saline/10 ml blood). The diluted blood is carefully layered over Hypaque (Winthrop, New York, NY)-Ficoll (Pharmacia) with a density of 1.086 g/ml (10 ml gradient solution/35 ml diluted blood) and subsequently centrifuged at room temperature for 30 min at 400 g. The lymphocyte-monocyte-basophil layer is removed, washed once with saline, twice with PA buffer, and then resuspended in a passive sensitization mix (total volume 0.125 ml) consisting of 1000-2000 ng/ml of purified IgE antibody (IgE-BPO-fLys, described above), 4 m M EDTA, 10/tg/ml heparin, and 1 X 10-4 M BPOfLys. Sensitization proceeds for 90 min at 37 °. Cells are then pelleted, resuspended in 0.2 ml of PA buffer, layered onto 1 ml ofautologous serum containing 4 m M EDTA, and centrifuged at 400 g for 8 min. After centrifugation, the cells are resuspended in l ml REH buffer [RPMI 1640 (Gibco) with L-glutamine, 25 m M HEPES, 5% fetal calf serum, 4 m M EDTA, 10 pg/ml heparin], pelleted once again, and resuspended in 0.1 ml of REH buffer. Sensitized cells are applied to a 1 ml packed column of BPO-Sepharose 6MB (Pharmacia, prepared with ethylenediamine as described above) in REH buffer and allowed to slowly sink into the column for 2 min. After this time, 15 ml of ice-cold REH buffer is washed over the column at a flow rate of 5 ml/min. Subsequent to this wash, cells bound to the column are desorbed at room temperature with 8 ml of REH buffer containing 2 × 10-3 MBPO-fLys. After the first 1 ml of this elution buffer is collected, the column flow is stopped for 5 min (to allow for full desorption of cells) then continued at 5 ml/min. Cells are collected, washed, stained with Wright stain for differential counts and with Alcian blue 23 for basophil counts in a Spier-Levy chamber. 23 H. Gilbert and L. Ornstein, Blood46, 279 (1975).
[43]
BASOPHILS IN I N F L A M M A T O R Y R E A C T I O N S
527
Basophil purities ranging from 65 to 80% and average yields of 60% have been reported with this procedure) The purity of the basophil preparation depends on the concentration ofbasophils after the Hypaque-Ficoll step (greater than 2% concentration yielded greater purity) and on the concentration of IgE-specific antibody used for passive sensitization (less than 1000 ng/ml oflgE anti-BPO antibody gave poor results). Morphologically, purified basophils appear intact and functionally the cells respond normally by releasing histamine to anti-IgE antibody. Histamine content (1 pg/cell) appears to be unaltered by the purification procedure. Disadvantages to the procedure include binding of the IgE antibody to the IgE Fc receptors (making passive sensitization with other IgE-specific antibody systems difficult to perform) and the low number of basophils that can be obtained. Procedure 2. The methods of Toll et al. 9 take advantage of IgE Fc receptors being occupied by IgE antibody on the surface of the basophil. In this procedure, basophils are passed over a column of Sepharose to which anti-IgE antibody is coupled. Basophils adhering to the column are eluted by differential buffer flow rate or mechanical agitation. Because of the nonspecific binding properties of CNBr-activated Sepharose 6MB (Pharmacia), a critical portion of this method is the preparation of the anti-IgE affinity column. In this procedure, 1 g of Sepharose 6MB is swollen in a beaker for 15 min with 1 m M HC1 solution. The beads are subsequently washed on a glass filter with 200 ml of the same HC1 solution, then resuspended in 5 ml of a 0.1 M carbonate-bicarbonate buffer containing 0.5 M NaC1, pH 8.3, and allowed to settle for 3 hr. At the end of this time, 3 mg of anti-IgE (Hoechst) in 6 ml of 0.1 M carbonatebicarbonate buffer (containing 0.5 M NaC1, pH 7.0) is added to the Sepharose 6MB and allowed to incubate at 4 ° in an end-over-end mixer overnight. After this coupling period, the beads are washed and resuspended in 1 M ethanolamine (pH 9.0) for a 2-hr incubation (ethanolamine blocks unreacted sites). Subsequently, 3 washing cycles are used to remove noncovalently adsorbed protein. Each cycle consists of a wash at pH 4 (0.1 M acetate buffer containing 0.5 MNaC1) followed by a wash at pH 8.3 (0.1 M carbonate-bicarbonate buffer containing 0.5 M NaC1). After the final wash cycle, the beads are resuspended in Tris-ACM buffer (containing Tris 25 mM, pH 7.5, NaC1 120 mM, KC1 5 mM, 0.3 mg/ml human serum albumin, 0.6 m M calcium, and 1 m M magnesium) and packed into a small plastic column (1 g of Sepharose 6MB yields 3.5 ml of column volume). Using this procedure, a 90% coupling efficiency for anti-IgE antibody has been reported) In the basophil isolation procedure, 1 part blood is mixed with 3 parts Tris buffer (without calcium and magnesium), and 6 ml of this diluted blood mixture is carefully layered onto 3 ml of a Hypaque-Ficoll gradient
528
INFLAMMATION
[43]
solution (10.5% w/v Hypaque, 6.4% w/v Ficoll, density 1.080 g/ml) and centrifuged for 30 min at 400 g (at 20°). The mononuclear band containing basophils (at the gradient-buffer interface) is washed in Tris buffer, and resuspended in Tffs-ACM buffer at 1 × 107 cells/ml. Five milliliters of this cell suspension is then loaded onto the anti-IgE affinity column at varying flow rates (at 0°). At a flow rate of 8 ml/hr basophils are tightly bound to the column and are eluted only by removing the beads from the column, suspending them in Tris-ACM buffer, stirring for 5 min, and recovering the bound cells in the supernatant fluid. At flow rates of 16 ml/ hr fractionation of basophils is observed, and preparations of high purity can be achieved. Using mechanical agitation, basophil purities of 40-60% may be obtained with a 30-45% recovery from blood. 9 Using selective elution, puffties of 80-95% can be obtained, but recovery is only 20-30% of the initial starting basophil concentration? Both basophil preparations are observed to be functional, releasing equivalent amounts of histamine to an anti-IgE stimulus at each step in the purification procedure. 9 Disadvantages to this procedure include the limited lifetime of the affinity column, variability in the coupling efficiency of anti-IgE antibody to Sepharose (leading to variability in basophil purification), and the low number of basophils that can be obtained. Procedure 3. The methods of Pruzansky and Patterson 1° employ three steps (Hypaque-Ficoll centrifugation, Hypaque centrifugation, glass bead chromatography) for basophil purification. Each step eliminates specific cell types: Hypaque-Ficoll gradients eliminate neutrophils and eosinophils; Hypaque gradient centfffugation, monocytes; glass beads, nonadherent lymphocytes. In the procedure, blood obtained from allergic donors (0.48 ml 10% EDTA added as anticoagulant) is layered directly onto a Hypaque-Ficoll gradient solution (Hypaque 10.6% w/v, Ficoll 6.6% w/v, density 1.0825 g/ ml; 15 ml blood per 10 ml Hypaque-Ficoll gradient) and centrifuged for 5 min at 1400 g. The mononuclear cell layer containing basophils (at the gradient-plasma interface) is washed with Tris buffer, pH 7.5 (see above, Procedure 2), and resuspended in a 1-ml volume of this same buffer. The cells are then carefully layered onto a gradient solution of Hypaque (1 ml cushion 12.7% w/v, 1 ml 10.6%, 1 ml 9.5%) and centrifuged at 500 g for 5 min (room temperature). Basophils appearing in the cell pellet at the bottom of the tube are washed twice with buffer and are ready for glass bead chromatography. Before use, washed (in water) glass beads (3M, St. Paul, MN) are saturated with whole autologous plasma (sedimented at high speed to remove platelets) supplemented with 4 ml of 0.1 M MgCI2 (required for cellular adhesive to beads; calcium is avoided here since
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
529
plasma clots are found in its presence) and 3 ml of 6% dextran. Saturated beads are added to a height of 8 cm in a glass column (1 × 12 cm), and washed cells (from the Hypaque gradient) resuspended in 1 ml of autologous plasma (supplemented as described above) are added dropwise to this column at room temperature. No incubation of the cell-loaded column is necessary, and nonadherent cells are eluted with autologous plasma (approximately 6 ml). The column is then filled with Tris buffer, pH 7.5, containing 0.1% EDTA, and the glass beads are agitated (gently stirred in the column with a glass rod). Desorbed cells are subsequently eluted with buffer, centrifuged, and differential and basophil counts, using Hansel stain (a modified rapid acting Wright stain, Lide Laboratories, St. Louis, MO), are performed on dried smears. 24 Basophils of 50% purity and 50% yield from whole blood have been reported with this procedure, and their functional nature is established by histamine release from cells of allergic donors known to be sensitive to mold) ° The greatest advantage to this procedure is that it can be performed in 1.5 hr. A disadvantage is that the purities obtained are not as great as with other methods.
Procedures Utilizing Percoll Density Gradients The techniques described above have been successful in purifying basophils. Despite this and the fact that histamine release from these cells has been demonstrated, there is a lingering concern that the multiple manipulations of and positive selection for basophils may cause changes in cell metabolic function which are not readily apparent. Recently, procedures utilizing Percoll density gradient centrifugation have been developed.tZ-~5 These methods do not involve multiple manipulations or positive selection of basophils and generally are rapidly performed. Each of these reports observed basophils at Percoll densities between 1.072 and 1.082 g/ml) ~-~4 The most specific density for basophil banding was observed to be 1.076 g/ml. H While each study had slight nuances in forming Percoll gradients, and handling of cells, a general procedure is described below. Percoll stock solution is prepared by mixing 90 ml of a commercial Percoll solution with 8.96 ml of 10 × H a n k s ' balanced salt solution (HBSS) (without calcium, magnesium, or bicarbonate), 0.45 ml of 1 N HC1, and 1 ml of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 7.3 (final density 1.123 g/ml). Specific densities of Percoll are prepared by mixing Percoll stock and HBSS without calcium or magnesium. Final adjustment of the density with either stock Percoll or 24 j. Pruzansky and R. Patterson, Int. Arch. Allergy Appl. Immunol. 38, 522 (1970).
530
INFLAMMATION
[43]
HBSS is facilitated by density measurement with either a pycnometer or refractometer (Fischer Scientific, Silver Spring, MD) (see also this series, Vol. 108 [9]). The density of Percoll is altered by temperature; therefore, if gradient centrifugation is to be performed at room temperature, all solutions should be made and stored at this temperature. Discontinuous gradients of Percoll are formed by layering (approximately 3 ml) each Percoll solution (greatest density first) in a 15-ml polystyrene conical-bottom centrifuge tube (No. 2095, Falcon, Oxnard, CA) using a peristaltic pump (Miniplus II, Gibson) at a rate of 30 ml/hr. In most reports, blood anticoagulated with EDTA (1 ml 0.1 M EDTA/10 ml blood) is layered directly onto the Percoll gradient and centrifuged in a swinging bucket rotor at 600 g for 20 min at room temperature. Total basophil recovery is assessed by absolute counts using Alcian blue23 and differential counts on cytocentrifuged smears using Wright stain. Basophil purity obtained with Percoll density gradient centrifugation depends on various factors including the following: (1) Number of circulating basophils. A greater number of basophils in donor blood facilitates greater purity and yield. Leukocytosis, on the other hand, dramatically decreases basophil purity without affecting basophil recovery. (2) Technical problems. If an abrupt disturbance of the Percoll gradient is made when layering blood (either by pipet tip or blood dropping into the Percoll) a dramatic decrease in basophil purity can be observed. Similar decreases in basophil purity can also be observed if disturbance of the cell band occurs while withdrawing it from the gradient or if a small quantity of the cell layer is left behind. The results of reports utilizing the general procedure described above are summarized in the following discussion. In the report of Raghuprasad, H a discontinuous Percoll gradient of densities 1.072, 1.074, 1.076, and 1.078 g/ml is used to separate whole anticoagulated blood. Basophil purity is highest at 1.076 and 1.078 g/ml densities, with average purities of approximately 25% obtained (range 6-50% and 6-60%, respectively). Basophils are also found at 1.072 g/ml (average 5% purity) and 1.074 g/ml densities (average 15% purity). Basophil recovery and purity are highest in donors having high basophil counts (approximately 2% of total leukocytes; does not necessarily correspond to an atopic state), and lymphocytes and neutrophils are the most frequent contaminants present in basophil preparations. Centrifugation of cells pooled from the 1.076 and 1.078 g/ml densities and application to a second discontinuous Percoll gradient consisting of two densities (1.072 and 1.080 g/ml) lead to an enhancement of purity (average 40%, range 15- 83%) but a decrease in yield (20% of total basophils). The functional nature of the isolated basophils may be verified by histamine release using several secretagogues (anti-IgE, complement component 5a, and alternaria antigen in sensitive individuals).
[43]
BASOPHILS IN I N F L A M M A T O R Y REACTIONS
531
Kaufman et al. 12 also used discontinuous Percoll gradients (densities 1.072, 1.082, 1.100 g/ml) and observed basophils to band between the 1.072 and 1.082 g/ml densities. The range of purity is between 8 and 20% with a basophil recovery of 70% of those applied to the gradient. This method differs from that previously described by the fact that a leukocyte preparation (obtained by unit gravity sedimentation for 30-40 min at 37 ° in 6% dextran) instead of whole blood is layered onto the Percoll gradient. In another method, ~3whole blood (EDTA anticoagulated) is layered onto a discontinuous Percoll gradient (densities 1.070, 1.079, 1.090 g/ml), and, as in the previous method, basophils are observed to band between densities of 1.070 and 1.079 g/ml. The mean purity of the isolated basophils is 19% (range 5 - 53%), and lymphocytes are the main contaminating cells) a However, on closer scrutiny, basophils from some donors consistently band in two different layers of the discontinuous Percoll gradient (the plasma- 1.07 g/ml Percoll interface and the 1.070- 1.079 g/ml Percoll interface).~4 While the greatest number of basophils is still found to be in 1.070- 1.079 g/ml interface, in some donors the majority of basophils are found in the plasma-1.070 g/ml Percoll interface. From this and other observations, the existence of two populations of basophils has been suggested. These data alone do not prove the existence of multiple populations of basophils, but they do raise the possibility that subpopulations of basophils exist and perhaps have distinct chemical composition and function.
Procedures Utilizing Percoll Density Gradient Centrifugation and Cell Panning Techniques Landry and Findlay ~6 have recently described a basophil purification procedure which combines both Percoll density gradient centrifugation and cell panning techniques, using monoclonal antilymphocyte antibodies. Cell panning techniques negatively select for basophils by the selective removal of unwanted or contaminating cells. In this procedure, stock Percoll (Pharmacia) is prepared by mixing 9 parts commercial Percoll, and 1 part PIPES (10 × buffer) (PIPES 250 mM, NaC1 1180 mM, KC1 50 mM, NaOH 430 mM, and 0.1% 0.1 M EDTA). Leukocyte-rich plasma is prepared by mixing whole blood (anticoagulated with 0.1 M EDTA) with 20% v/v dextran and allowing the mixture to settle at unit gravity for 90 min. The resultant leukocyte preparation is then carefully layered over a discontinuous Percoll gradient prepared by layering 10 ml of a 55% PercoU solution (5.5 ml stock Percoll plus 4.5 ml PIPES 1 × buffer) over 10 ml of a 65% Percoll solution (6.5 ml stock Percoll plus 3.5 ml PIPES 1 × buffer). This gradient is centrifuged at 400 g for 20 min at room temperature. After centrifugation three bands of cells can be seen. The second band of cells found between the 55 and 65% Percoll layers
532
INFLAMMATION
[43]
contains an enriched population of basophils plus contaminating mononuclear cells and neutrophils. This layer is carefully withdrawn, washed twice (250 g for l0 rain at 4 °) with 30 ml of PIPES buffer containing 0.03% human serum albumin (PA), and further purification of basophils is obtained with cell panning techniques. For the preparation of panning plates (see also this series, Vol. 108 [l l ]), plastic petri dishes (Fisher 8-757-12) are coated with 100/d (0.1 mg) of goat anti-mouse IgG (Tago No. 4150) in l0 ml of phosphate-buffered saline (PBS) by incubating at 4 ° for 12 hr. After incubation, the antibody solution is decanted. The plates washed 3 times with l0 ml of PBS after which they are ready for use. Hates may be stored (wrapped in Parafilm) with PBS containing 2% fetal calf serum (FCS) at - 2 0 °. In the panning procedure, cells from band 2 of the Percoll gradient (2-4 × l05) are resuspended in 1 ml of PA buffer. Monoclonal antibodies (anti-Leu-1 and anti-HLA-DR, Becton Dickinson, Sunnyvale, CA) are added to the cells at a final concentration of 1/~g/106 cells, and the mixture is incubated for l hr at 4 °. After incubation, the cells are centrifuged (240 g for 5 min at 4°), resuspended in 3 ml of PBS containing 2% FCS (PBS-FCS, at 4°), added to panning plates, and incubated at 4 ° for 60 min (swirling of plate is performed every 20 min). After this incubation, the unabsorbed cells are decanted, combined with cells obtained by 3 gentle washings of the plate (with 10 ml of PBS-FCS), centrifuged at 240 g for l0 min at 4 °, and resuspended in PA buffer. Counting of basophils is performed using the Alcian blue method. 23 The second cell band in the Percoll gradient obtained from fractionated leukocyte-rich plasma contains basophils of highest purity (mean 14.6 _+ 7.9%)/16 Further purification of these basophils using cell panning techniques increases the purity to a mean of 34 _4- 15% (range 19-54%), with 55% yield of basophils.16 This is approximately a 30-fold purification. The functional capability of the basophils is verified by histamine release using anti-IgE as a secretagogue. Lett-Brown et al. 17 have recently reported a basophil purification technique similar to that described above. The primary difference in their procedure is the Percoll density gradient centrifugation step. In their method, the procedure described by Leonard et al. 13,14 (outlined above) is utilized. Basophil-enriched fractions withdrawn from Percoll are subsequently panned as described above (an OKT 11 monoclonal antibody is used in place of the anti-Leu-1 monoclonal antibody). In selected donors, functional basophils with purities of greater than 90% and yields from blood of approximately 50% may be achieved. 17 The major drawback is the small number of highly purified basophils (5-10 × 105 cells) that can be obtained. While mediator release experiments are feasible with small numbers ofbasophils, biochemical studies usually require larger
[43]
BASOPHILS IN I N F L A M M A T O R Y R E A C T I O N S
533
numbers of pure cells. The use of leukopheresis products may enhance the productivity of these techniques.
Procedures Utilizing Flow Microfluorometric Techniques (see also this series, VoL 108 [19]) Flow microfluorometry allows for the isolation of viable and highly purified basophils from blood. This method is the simplest to perform, requiring only commercially available anti-IgE antibody and access to a fluoroescence cell sorter. The technique as described by Weil et al. is consists of two steps. In the first step, blood collected in EDTA (final concentration 5 mM) and diluted with an equal volume of HBSS containing 0.1% bovine serum albumin (BSA) (HBSS-BSA) is layered over a HypaqueFicoll solution (specific gravity 1.077 g/ml) and centrifuged at 800 g for 15 min, and the mononuclear cell layer containing basophils washed 3 times with HBSS-BSA. Washed cells (5 × 107/ml) are incubated with a 1 : 50 dilution of fluorescein isothiocyanate (FITC)-conjugated goat antihuman IgE (epsilon-specific IgG fraction, Cappel Laboratories, Cochranville, PA) at 5 ° for 30 min. Following this incubation, the cells are washed twice with cold HBSS-BSA, and labeled and unlabeled cells are sorted with a FACS II cell sorter (Becton Dickinson) using two sorting rates. The first enrichment sort is performed at a rate of 12 × 103/sec without coincidence constraints. These enriched cells are then sorted at a rate of 4 × 103/sec with coincidence constraints (i.e., only positive cells without cells in adjacent droplets sorted). Basophil purities of 50-65% could be obtained with the first sorting and purities of 97-99% with the second sort. is These cells are viable, and a histamine content of 1.7 pg/cell agrees with other reports published for human basophils. Limitations to this procedure include the following: (1) Sorted cells having been exposed to anti-IgE under conditions preventing histamine release are inactivated and cannot be used to study release using antigen-dependent mechanisms. (2) The number of cells able to be prepared by this technique is small and would not lend itself to biochemical studies.
Procedures Utilizing Centrifugal Elutriation (see also this series, Vol. 108 [20]) All of the methods described above have been successful to some extent in isolating and purifying human basophils. The number of basophils isolated using these procedures is usually low (of the order of 105- 106 cells), and generally the techniques are not suitable or capable of being modified for the isolation and purification of large numbers of basophils. The recent report of DeBoer and Roos ~5 utilizing Percoll density gradient
534
INFLAMMATION
[43 ]
centrifugation, centrifugal elutriation, and increased quantities of donor blood has, however, demonstrated much greater success in isolating and purifying large numbers of human basophils. In this method a stock Percoll solution is prepared by mixing 93 ml of Percoll with 7 ml of a solution containing 1.54 M NaC1 and 0.1 M NaH2PO4 (all solutions are kept at room temperature). Specific Percoll densities are prepared by mixing stock Percoll with PBS (140 m M NaCI, 9.2 m M Na2HPO4, 1.3 m M NaH2PO4, pH 7.4). The final Percoll solution also contains human serum albumin (5 /tg/ml) and trisodium citrate (13 m3/). In the isolation procedure, 1 unit of normal donor blood (500 ml) is collected in a plastic bag containing 75 ml of an ACD solution (containing a hydrous disodium citrate, 100 mM, and anhydrous glucose, 128 mM, pH 5.0). Within 1 hr after collection, the blood is centrifuged in the plastic bag (4000 g, 5 min, room temperature), and the plasma is separated from cells. The upper 50- 100 ml of the packed cells (buffy coat) contains 70% of the leukocytes. This buffy coat is collected ~5and diluted to 200 ml with PBS containing 13 m M trisodium citrate (PBS-TC). Mononuclear cells and basophils are separated from neutrophils, eosinophils, and red blood cells by centrifugation (1000 g, 20 min, room temperature) of the buffy coat over a Percoll solution of 1.077 g/ml density (8 tubes each containing 12 ml of Percoll solution). The supernatant autologous plasma (SP) from this centrifugation is saved, and the cells at the plasma-Percoll interface are diluted to 200 ml with PBS-TC and centrifuged at 400 g for 5 min at room temperature. To remove platelets, the resultant cell pellets are resuspended first in 100 ml then in 50 ml of the SP solution. After each resuspension of cells in SP, the cells are centrifuged at 400 g for 5 min at room temperature. The final cell pellet is resuspended in 10-20 ml of the SP solution and injected into a JE-6 elutriation rotor powered by a J-2 I C centrifuge (Beckman). The elutriation medium is PBS-TC plus 5 mg/ml human serum albumin. To separate the various cell populations, the flow rate is maintained at 20 ml/min while the rotor speed is diminished from 4000 to 0 rpm. Fractions of 100-200 ml of the outflow are collected at rotor speeds of 3700, 2600, 2500, and 0 rpm. The last fraction (0 rpm) contains 80% of the basophils present in the starting preparation. This fraction is centrifuged (400 g, 5 min, room temperature), resuspended in 4 ml of Percoll 1.067 g/ml), and layered over 2 ml of Percoll of 1.075 g/ml density. A top layer of 0.2 ml of PBS-TC containing 5 mg/ml of human serum albumin is added, and the gradient is centrifuged at 1000 g for 10 min at room temperature. After centrifugation, two interfaces containing cells are observed. The lower Percoll interface (1.067-1.075 g/ml densities) contains the basophils. Basophil purity of 70% (range 45- 87%, as measured by Alcian blue)
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
535
and yield of 60% from the buffy coat have been reported. ~5 More importantly, however, 4 - 19 X 106 basophils can be obtained using this procedure. Histamine content (1-2 pg/cells) and releasability (30-50% to antiIgE) have been shown in these preparations. ~5The success and consistency of this procedure has been predicated on the following: (1) the capabilities of centi'ifugal elutriation; (2) suspension of the cells in Percoll (1.067 g/ml density) before applying to the Percoll solution of higher density (this prevents carryover of cells into a layer with a higher specific gravity than the cells); and (3) prevention of partial degranulation of basophils in the procedure (this is avoided by preventing platelet aggregation), accomplished by platelet removal with a low-speed centrifugation in autologous plasma. Methods for the Isolation of Basophils from Other Species Although present, basophils in rodents are much less abundant and, therefore, are less well studied. ~9In dogs, basophils make up less than 1% of the leukocyte population, and their concentration can be enhanced to 1-2% using double-density Hypaque-Ficoll centrifugation.25 Naturally abundant basophils have been observed in the snapping turtle Chelydra serpentina. 2° Normal circulating basophil levels of 50-63% have been reported, and these cells have been shown to release histamine in response to a rabbit anti-turtle immunoglobulin. Studies aimed at purifying and further characterizing these cells have not as yet been reported. To date, basophil function and techniques for isolation from blood have been most thoroughly studied in humans and guinea pigs .21'22Procedures for isolation of guinea pig basophils are discussed in detail below. Procedure
Techniques for purification of guinea pig basophils utilizing Ficoll density gradient centrifugation2! and negative selection using a rabbit antileukocyte antibody and Percoll density gradient centrifugation have been reported. 22 However, the most consistent and reliable technique utilized for guinea pig basophil purification involves both centrifugal elutriation and Percoll density gradient centrifugation.26 This procedure is described below.
25 D. Austin, S. Chan, A. Malley, J. Hanifin, and C. Hirshman, J. Lab. Clin. Med. 17, 53 (1985). 26 B. Undem, J. Brendel, T. Hirth, C. Buckner, and F. M. Graziano, Am. Rev. Respir. Dis. 133, 763 (1986).
536
INFLAMMATION
[43]
Basophils comprise only a small percentage of the total nucleated cells in guinea pig blood. However, the animals can be treated to increase the number of basophils which, in turn, facilitates purification. A striking basophilia can be obtained in guinea pigs sensitized with whole sheep blood for 12 days (daily i.p. injections of 2 ml whole blood diluted 1 : 2 with PBS). 21,22On day 13, blood obtained from six animals and containing 0.1 M EDTA is thoroughly mixed with 3% gelatin (2 parts blood to 1 part gelatin) in a test tube and placed at an angle of 30 ° in a 37 ° water bath. After incubation for 45 min, the leukocyte-rich plasma is withdrawn and centrifuged at 400 g (room temperature) for 30 min. Pelleted cells are washed once in HG (HEPES 10 mM, NaC1 137 mM, KC1 5 mM, 0.5 mg/ml gelatin, 5.5 m M glucose, pH adjusted to 7.4 with NaOH) containing 4 m M EDTA, and residual red cells are removed using hypotonic lysis (see this series, Vol. 108 [6]). Leukocyte preparations (containing an average of 6% basophils) 22'26 are then fractionated using a JE-6 elutriator rotor driven by a J-21B Centrifuge (Beckman Instruments, Palo Alto, CA). Rotor speed is controlled with a 10-turn potentiometer which allows for accurate rpm selection (within 10 rpm). Before each run, the system is washed sequentially with ethanol (200 ml), distilled water (1000 ml), and PBS (200 ml). Blood leukocyte suspensions containing basophils are loaded into the elutriator with a rotor speed of 4150 rpm, and the flow rate is adjusted to and maintained at 30 ml/min. Leukocytes are fractionated by incrementally decreasing the rotor speed to 3600, 2930, 2760, 2100, and 0 rpm. After each adjustment in rotor speed, two to four 45-ml fractions are collected. An additional 90-ml collection is obtained at the end of each run with the centrifuge turned off. Elutriated fractions containing the highest percentage of basophils (fractions collected after reduction in rotor speed to 2760 and 2100 rpm) are pooled, centrifuged, resuspended in l ml of HG (10-50 × 106 cells/ml), and layered onto a discontinuous Percoll gradient (1.06, 1.07, and 1.08 g/ml). 26 Centrifugation is carried out at 400 g for 20 min at room temperature, and cell bands are carefully removed and washed with HG. Total basophil counts are performed at each step during the purification procedure using the Alcian blue method. 23 Differential counts are obtained on cytocentrifuged (Cytospin, Shandon Elliot Corp.) smears stained with Wright stain. Cell viability (90-98%) is monitored at each step during the isolation procedure by Trypan blue dye exclusion. Guinea pig basophils (5 - 10 × l06 basophils) of greater than 90% purity and 40-50% yield from whole blood are consistently observed at the 1,070- 1.080 g/ml Percoll interface using this procedure. 26 Use of centrifugal elutriation alone will consistently give basophil purity of 40-50%. 26 Morphologically, the cells appear to be intact (see Fig. 1), and, functionally, histamine release from these cells can be demonstrated with antigen
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
537
(sheep ~,-globulin), concanavalin A, rabbit anti-IgG~ antibody, and 12-0tetradecanoylphorbol 13-acetate (TPA). Rat Basophilic Leukemia Cells The problems inherent in studying normal basophils (obtaining large quantities of pure cells for biochemical studies, possible heterogeneity of basophil populations) discussed above have focused attention on the study of a rat basophilic leukemia tumor cell line. In 1973 Eccleston et al. 27 first described the development of basophilic leukemia in a Wistar rat fed fl-chlorethylamine (a potent inducer of myeloid or lymphatic leukemia). Leukemic cells obtained from such animals (RBL cells) can be serially transplanted by intraperitoneal injection into rats, resulting in the development of a solid t u m o r . 27 A number of investigators have shown these tumor cells to contain histamine and serotonin and have used them for study of the structure and binding characteristics of IgE surface receptors present on these cells. 27-31 RBL cells have been adapted to suspension cell culture and have been maintained for prolonged periods of time with little morphologic or functional change in cell characteristics compared to original minced tumor cells. 2s The original cell lines, while useful in studying the IgE receptor, failed to be triggered for IgE-mediated histamine release and, therefore, were not useful in studying the mechanisms of this process.27,2s Subsequently, several cloned histamine-releasing cell lines and nonhistamine-releasing cloned variant sublines defective at different steps in the histaminereleasing process have been reported) 2-~ The defects at different sites of the release pathway have allowed for a dissection of the biochemical events involved in histamine release. 33-3s RBL cell lines have been and will 27 E. Eccleston, J. Leonard, S. Lowe, and H. Welford, Nature (New Biol.} 244, 73 (1973). 2s A. Kulczycki, C. Isersky, and H. Metzger, J. Exp. Med. 139, 600 (1974). 29 A. Froese, R. Helm, D. Conrad, C. Isersky, T. Ishizaka, and A. Kulczycki, Immunology 46, 107 (1981). 30 H. Metzger, Immunol. Rev. 41, 186 (1979). 31 H. Metzger, A. Goetze, J. Kanellopoulos, D. Holowka, and C. Fewtrell, Fed. Proc., Fed. Am. Soc. Exp. Biol. 41, 8 (1982). 32 E. Barsumian, C. Isersky, M. Petrino, and R. Siraganian, Eur. J. Irnmunol. 11, 317 (1981). 33A. McGivney, F. Crews, F. Hiraha, J. Axelrod, and R. Siraganian, Proc. Natl. Acad. Sci. U.S.A. 78, 6176 (1981). R. Siraganian, A. McGivney, E. Barsumian, F. Crews, F. Hiraha, and J. Axelrod, Fed. Proc., Fed. Am. Soc. Exp. Biol. 41, 30 (1982). 35 C. Meyer, L. Wahl, B. Shader, and R. Siraganian, J. Immunol. 131, 911 (1983). 36 j. Rivera, J. M. Mullins, K. Furuichi, and C. Isersky, J. Immunol. 136, 623 (1986). 37 M. Garcia-Gil and R. Siraganian, J. Immunol. 136, 259 (1986). 3s M. Gareia-Gil and R. Siraganian, J. Imrnunol. 136, 3825 (1986).
538
INFLAMMATION
[44]
continue to be a convenient model for study of the mechanisms of mediator release. A rat basophilic leukemia cell line (CRL 1378-RBL-l) is available from the American Type Culture Collection, Rockville, MD. M e d i a t o r R e l e a s e b y Basophils Mediator release (e.g., histamine, arachidonic acid metabolites) from isolated and purified basophils has been documented in humans s- 15,39and other species. 19-21,25,26In the general procedure for mediator release, purified cells (generally 5 - 10 × 104 basophils per assay tube) in 0 . 5 - 1 ml of isotonic buffer (e.g., HEPES or PIPES buffer) containing 1 m M calcium and magnesium are reacted with varying concentrations of secretagogue, e.g., anti-IgE (0.01- 1 gg/ml) s (Calbiochem-Behring Corp.), antigen (e.g., sheep ),-globulin, 0.1 - 10 gg/ml), 22,26 or concanavalin A (0.1 - 3 #g/ml). 4° Mediators released into the supernatant fluid can be measured with one of the available methods (e.g., this volume [42]). 39R. Schleimer, C. Fox, R. Naclerio, M. Plaut, P. Creticos, A. Togias, J. Warner, A KageySobotka, and L. Lichtenstein, J. Allergy Clin. Immunol. 76, 369 (1985). 4op. Siraganian and R. Siraganian, J. Immunol. 112, 2117 (1974).
[44] H u m a n
Neutrophil
Degranulation
By DANIEL G. WRIGHT Introduction Neutrophils are wandering, phagocytic white blood cells that are critical to host defenses against bacterial and fungal infections. They are also the first circulating leukocytes to arrive at a site of acute inflammation. Indeed, the extravascular accumulation of these cells in tissues is a fundamental histologic hallmark of the acute inflammatory response. Inflammatory neutrophils are recognized to play a key role in mediating the dissolution and remodeling of connective tissues at sites of inflammation. Neutrophils injure or destroy connective tissue cells and digest or alter extracellular matrix proteins by at least two mechanisms, generation of toxic oxygen metabolites (see this series, Vols. 105 and 132) and secretion of proteolytic enzymes. Unlike the monocyte/macrophage family of phagocytes, neutrophils retain a very limited capacity for d e n o v o protein synthesis once they are METHODS IN ENZYMOLOGY, VOL. 162
Copyright © 1988 by Academic Press, Inc. All fights of reproduction in any form r-~-~rved.
INFLAMMATION
[43]
and chemokinesis. The latter property reflects the tendency of a substance to increase random cell migration. This discrimination is best measured by a checkerboard design of chemotaxis. In this design, the substance to be evaluated is incorporated in multiple doses on either or both sides of the membrane. In this manner it is possible to distinguish pure chemotactic activity, pure chemokinetic activity, and mixed activity in the same preparation.S Many methods for assessing the results of the micropore chemotactic assay have been described. Generally, however, these fall into three basic types: (1) counting the number of cells that have arrived at a preselected distance, such as the distal surface of the filter; (2) measurement of the distance migrated by faster migrating cells (leading-front technique); and (3) measurement of the average distance migrated per cell (the locomotion or leukotactie index). The first method described is very sensitive for detecting differences in nonstimulated and chemotactic responses. It can be used effectively for measurement of chemotactic activity released from mast cells. The latter two methods are much more sensitive to defects in cell migration and are used for investigation of clinical defects in this process. 51 S. H. Zigmond and J. G. Hirsch, J. Exp. Med. 137, 387 (1973).
[ 4 3 ] B a s o p h i l s in A l l e r g i c a n d I n f l a m m a t o r y
Reactions
By FRANK M. GRAZIANO Introduction Basophils are granulocytic cells that differentiate in bone marrow, circulate in blood, and migrate into tissues in response to appropriate stimuli. They are the least common of the granulocytic cells and comprise 0.5- 1% of the total leukocyte population. Basophil morphology is dominated by intracytoplasmic granules, and the ultrastructure of these granules appears to differ from species to species. In humans, basophil granules vary in shape (round, oval, angular) and size ( 113- 260 A), are embedded in a less dense matrix, and are surrounded by a membrane) In guinea pigs, basophil granules are large oval structures that, depending on the plane of section, display a parallel array, hexagonal array, finely granular, or rectanA. Dvorak and H. Dvorak, Arch. Pathol. Lab. Med. 103, 551 (1979).
METHODS IN ENZYMOLOGY, VOL. 162
Copyright© 1988 by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
523
Flo. 1. Electron micrograph of a guinea pig basophil. The cytoplasm demonstrates typical large oval mediator-containing granules. × 14,080. (Insert a) Cytoplasmic granule. The dense granule particle appears in a less dense matrix surrounded by a perigranular membrane. X 27,500.
gular lattice o f granular material. A rim o f less dense finely granular or fibrillar material surrounds the matrix and separates it from the granule m e m b r a n e ~ (Fig. 1). A close relationship appears to exist between basophils and the granule-containing mononuclear mast cells, but in both
524
INFLAMMATION
[43]
functional and biochemical studies distinct differences between the two cell types have been reported. 2,3 In humans, basophils contain almost all of the blood histamine. 4 For many years, in vitro release ofbasophil histamine in allergic individuals has served as an assay of immediate hypersensitivity responsiveness? Knowledge of the actual participation of basophils in disease, however, has only come about with the appreciation that significant tissue infiltrates accompany some allergic diseases. 4,5 Attempts in the past to clarify the role of basophils have been greatly limited by the low number of these cells found in peripheral blood. 6,7 Recently, success in achieving high levels of purity and yield of basophils has been accomplished in humans with affinity chromatography,S- ~0 Percoll density gradient centrifugation,~ ~- ~4 centrifugal elutriation,15 cell panning procedures, 16,~7and flow microfluorometry.lS Normal circulating blood basophils have been identified with great difficulty in rodents, 19 appear to be abundant in turtles, 2° and have been isolated in substantial purity from guinea pigs. 2~,22 It is the purpose of this chapter to review basophil isolation procedures with special emphasis on human and guinea pig techniques.
2 D. MacGlashan, R. Schleimer, S. Peters, E. Schulman, G. K. Adams III, A. Sobotka, H. Newball, and L. Lichtenstein, Fed. Proc., Fed. Am. Soc. Exp. Biol. 142, 2504 (1983). 3 H. Dvorak and A. Dvorak, Hum. Pathol. 3, 454 (1972). 4 E. B. Mitchell and P. Askenase, Clin. Rev. Allergy 1,427 (1983). H. Dvorak, J. Allergy Clin. Immunol. 58, 229 (1976). 6 R. Day, Clin. Allergy 2, 205 (1972). 7 T. Ishizaka, R. DeBernardo, H. Tomioka, L. Lichtenstein, and K. Ishizaka, J. Immunol. 108, 1000 (1972). s D. MacGlashan and L. Lichtenstein, J. Immunol. 124, 2519 (1980). 9 j. Toll, J. Wikberg, and R. Andersson, Allergy 36, 411 (1981). 10j. Pruzansky and R. Patterson, J. Immunol. Methods 44, 183 (1981). 11 p. Raghuprasad, J. Immunol. 129, 2128 (1982). 12H. Kaufman, P. Levering, and K. DeVries, J. Immunol. Methods 57, 1 (1983). 13E. Leonard, R. Roberts, and A. Skeel, J. Leuk. Biol. 35, 169 (1984). 14E. Leonard and A. Skeel, J. Allergy Clin. Imrnunol. 76, 556 (1985). 13 M. DeBoer and D. Roos, J. Immunol. 136, 3447 (1986). ,6 F. Landry and S. Findlay, J. Immunol. Methods 63, 329 (1983). 17M. Lett-Brown, S. Robinson, H. Juneja, J. Farnam, and J. A. Grant, Fed. Proc., Fed. Am. Soc. Exp. Biol. 44, 7438 (1985). 18G. Weil, W. Leiserson, and T. Chused, J. Immunol. Methods 58, 359 (1983). 19A. Dvorak, G. Nabel, K. Pyne, H. Cantor, H. Dvorak, and S. Galli, Blood59, 1279 (1982). 20 K. Mead, M. Borysenko, and S. Findlay, J. Immunol. 130, 334 (1983). 21 H. Dvorak, S. Selvaggio, A. Dvorak, R. Colvin, D. Lean, and J. Rypysc, J. Imrnunol. 113, 1694 (1974). 22 F. M. Graziano, W. Lipham, N. Swaminathan, J. Brendel, F. M. Graziano, Jr., and T. Warner, J. Immunol. Methods67, 157 (1984).
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
525
Methods for the Isolation of H u m a n Basophils Studies of human basophil function have progressed much more rapidly than studies of basophil biochemistry, pharmacology, and physiology. Recently, a number of methods have been developed to purify human basophils, and a more intensive study of this cell has proceeded. Since each method has its own advantages and disadvantages and no one method has proved to be clearly superior to all others, in this author's opinion, several important and productive methods for the purification of human basophils are discussed in detail below.
Procedures Using Affinity Chromatography There have been three major procedures developed which take advantage of a positive selection of basophils using affinity chromatography.8- i0 Two of the methods utilize antibody linked to an insoluble support, and the third utilizes the property of basophil adherence to glass beads. Procedure I. Methods utilized to purify human circulating basophils developed by MacGlashan and Lichtenstein8 were the first major step forward, allowing for the direct biochemical study of IgE-mediated phenomena in normal human basophils. The method of isolation takes advantage of unoccupied IgE receptors on basophils and the high affinity of these receptors for IgE antibody. In essence, the procedure entails passively sensitizing a mixed population of cells with antigen-specific IgE antibody (in this procedure antipenicillin IgE antibody) and passing this sensitized population of cells over a column of Sepharose to which the specific antigen [penicillin, i.e., benzylpenicilloyl (BPO)] 2z' has been coupled. IgEcoated basophils adhering to the column are then eluted with hapten antigen (penicillin hapten). The IgE-specific antipenicillin antibody (antibenzylpenicilloyl, prepared against the major penicillin determinant, BPO) critical to this procedure is isolated from penicillin-allergic individuals by passage of their serum over an affinity column of BPO linked to Sepharose CL-4B (Pharmacia, Piscataway, NJ). This column is prepared by first incubating the 22~Abbreviations: BPO, benzylpenicilloyl;CNBr, cyanogen bromide; PA, PIPES plus human serum albumin; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); BPO-fLys, benzylpenicilloyl-N-formyl-L-lysine; EDTA, ethylenediaminetetraacetic acid; REH, RPMI 1640 plus EDTA plus HEPES; Tris-ACM, Tris buffer plus human serum albumin, calcium, and magnesium; HBSS, Hanks' balanced salt solution; HEPES, N-2-hydroxyethylpiperazineN'-2-ethanesulfonic acid; PBS, phosphate-buffered saline; HLA, human leukocyte antigen; FCS, fetal calf serum; BSA, bovine serum albumin; FITC, fluorescein isothiocynate; FACS, fluorescence antibody ceil sorter; PBS-TC, PBS plus trisodium citrate; HG, HEPES plus gelatin; RBL, rat basophilic leukemia.
526
INFLAMMATION
[43]
cyanogen bromide (CNBr)-activated Sepharose beads with a solution of 0.1 M ethylenediamine (Sigma, St. Louis, MO) and 1 M NaCI, at pH 8.0 for 2 hr at room temperature. After washing, the beads are incubated for 90 min with a solution of 0.15 M sodium penicillin G (Sigma), 1 M NaC1, and 0.1 M NaHCO3 (maintained at pH 11). The beads are subsequently washed, and the penicillin adsorbent in PA buffer [piperazine-N,N'-bis(2ethanesulfonic acid) (PIPES) 25 mM, NaCI 150 mM, KC1 5 mM, adjusted to pH 7.3 with NaOH and containing 0.003% human serum albumin] is incubated batchwise with penicillin-allergic serum (1 volume of beads with 3 volumes of serum) for 5 hr at 4 °. After washing once again, bound IgE antibody is eluted with BPO-fLys (1 × 10-3 M benzylpenicilloyl-N-formyl-L-lysine), concentrated with no attempt to remove the BPO-fLys hapten, and used for passive sensitization of basophils. In the cell isolation procedure 50 ml of blood is obtained from nonallergic donors and mixed with saline containing ethylenediaminetetraacetic acid (EDTA) (0.8 ml of 0.1 M EDTA and 25 ml of saline/10 ml blood). The diluted blood is carefully layered over Hypaque (Winthrop, New York, NY)-Ficoll (Pharmacia) with a density of 1.086 g/ml (10 ml gradient solution/35 ml diluted blood) and subsequently centrifuged at room temperature for 30 min at 400 g. The lymphocyte-monocyte-basophil layer is removed, washed once with saline, twice with PA buffer, and then resuspended in a passive sensitization mix (total volume 0.125 ml) consisting of 1000-2000 ng/ml of purified IgE antibody (IgE-BPO-fLys, described above), 4 m M EDTA, 10/tg/ml heparin, and 1 X 10-4 M BPOfLys. Sensitization proceeds for 90 min at 37 °. Cells are then pelleted, resuspended in 0.2 ml of PA buffer, layered onto 1 ml ofautologous serum containing 4 m M EDTA, and centrifuged at 400 g for 8 min. After centrifugation, the cells are resuspended in l ml REH buffer [RPMI 1640 (Gibco) with L-glutamine, 25 m M HEPES, 5% fetal calf serum, 4 m M EDTA, 10 pg/ml heparin], pelleted once again, and resuspended in 0.1 ml of REH buffer. Sensitized cells are applied to a 1 ml packed column of BPO-Sepharose 6MB (Pharmacia, prepared with ethylenediamine as described above) in REH buffer and allowed to slowly sink into the column for 2 min. After this time, 15 ml of ice-cold REH buffer is washed over the column at a flow rate of 5 ml/min. Subsequent to this wash, cells bound to the column are desorbed at room temperature with 8 ml of REH buffer containing 2 × 10-3 MBPO-fLys. After the first 1 ml of this elution buffer is collected, the column flow is stopped for 5 min (to allow for full desorption of cells) then continued at 5 ml/min. Cells are collected, washed, stained with Wright stain for differential counts and with Alcian blue 23 for basophil counts in a Spier-Levy chamber. 23 H. Gilbert and L. Ornstein, Blood46, 279 (1975).
[43]
BASOPHILS IN I N F L A M M A T O R Y R E A C T I O N S
527
Basophil purities ranging from 65 to 80% and average yields of 60% have been reported with this procedure) The purity of the basophil preparation depends on the concentration ofbasophils after the Hypaque-Ficoll step (greater than 2% concentration yielded greater purity) and on the concentration of IgE-specific antibody used for passive sensitization (less than 1000 ng/ml oflgE anti-BPO antibody gave poor results). Morphologically, purified basophils appear intact and functionally the cells respond normally by releasing histamine to anti-IgE antibody. Histamine content (1 pg/cell) appears to be unaltered by the purification procedure. Disadvantages to the procedure include binding of the IgE antibody to the IgE Fc receptors (making passive sensitization with other IgE-specific antibody systems difficult to perform) and the low number of basophils that can be obtained. Procedure 2. The methods of Toll et al. 9 take advantage of IgE Fc receptors being occupied by IgE antibody on the surface of the basophil. In this procedure, basophils are passed over a column of Sepharose to which anti-IgE antibody is coupled. Basophils adhering to the column are eluted by differential buffer flow rate or mechanical agitation. Because of the nonspecific binding properties of CNBr-activated Sepharose 6MB (Pharmacia), a critical portion of this method is the preparation of the anti-IgE affinity column. In this procedure, 1 g of Sepharose 6MB is swollen in a beaker for 15 min with 1 m M HC1 solution. The beads are subsequently washed on a glass filter with 200 ml of the same HC1 solution, then resuspended in 5 ml of a 0.1 M carbonate-bicarbonate buffer containing 0.5 M NaC1, pH 8.3, and allowed to settle for 3 hr. At the end of this time, 3 mg of anti-IgE (Hoechst) in 6 ml of 0.1 M carbonatebicarbonate buffer (containing 0.5 M NaC1, pH 7.0) is added to the Sepharose 6MB and allowed to incubate at 4 ° in an end-over-end mixer overnight. After this coupling period, the beads are washed and resuspended in 1 M ethanolamine (pH 9.0) for a 2-hr incubation (ethanolamine blocks unreacted sites). Subsequently, 3 washing cycles are used to remove noncovalently adsorbed protein. Each cycle consists of a wash at pH 4 (0.1 M acetate buffer containing 0.5 MNaC1) followed by a wash at pH 8.3 (0.1 M carbonate-bicarbonate buffer containing 0.5 M NaC1). After the final wash cycle, the beads are resuspended in Tris-ACM buffer (containing Tris 25 mM, pH 7.5, NaC1 120 mM, KC1 5 mM, 0.3 mg/ml human serum albumin, 0.6 m M calcium, and 1 m M magnesium) and packed into a small plastic column (1 g of Sepharose 6MB yields 3.5 ml of column volume). Using this procedure, a 90% coupling efficiency for anti-IgE antibody has been reported) In the basophil isolation procedure, 1 part blood is mixed with 3 parts Tris buffer (without calcium and magnesium), and 6 ml of this diluted blood mixture is carefully layered onto 3 ml of a Hypaque-Ficoll gradient
528
INFLAMMATION
[43]
solution (10.5% w/v Hypaque, 6.4% w/v Ficoll, density 1.080 g/ml) and centrifuged for 30 min at 400 g (at 20°). The mononuclear band containing basophils (at the gradient-buffer interface) is washed in Tris buffer, and resuspended in Tffs-ACM buffer at 1 × 107 cells/ml. Five milliliters of this cell suspension is then loaded onto the anti-IgE affinity column at varying flow rates (at 0°). At a flow rate of 8 ml/hr basophils are tightly bound to the column and are eluted only by removing the beads from the column, suspending them in Tris-ACM buffer, stirring for 5 min, and recovering the bound cells in the supernatant fluid. At flow rates of 16 ml/ hr fractionation of basophils is observed, and preparations of high purity can be achieved. Using mechanical agitation, basophil purities of 40-60% may be obtained with a 30-45% recovery from blood. 9 Using selective elution, puffties of 80-95% can be obtained, but recovery is only 20-30% of the initial starting basophil concentration? Both basophil preparations are observed to be functional, releasing equivalent amounts of histamine to an anti-IgE stimulus at each step in the purification procedure. 9 Disadvantages to this procedure include the limited lifetime of the affinity column, variability in the coupling efficiency of anti-IgE antibody to Sepharose (leading to variability in basophil purification), and the low number of basophils that can be obtained. Procedure 3. The methods of Pruzansky and Patterson 1° employ three steps (Hypaque-Ficoll centrifugation, Hypaque centrifugation, glass bead chromatography) for basophil purification. Each step eliminates specific cell types: Hypaque-Ficoll gradients eliminate neutrophils and eosinophils; Hypaque gradient centfffugation, monocytes; glass beads, nonadherent lymphocytes. In the procedure, blood obtained from allergic donors (0.48 ml 10% EDTA added as anticoagulant) is layered directly onto a Hypaque-Ficoll gradient solution (Hypaque 10.6% w/v, Ficoll 6.6% w/v, density 1.0825 g/ ml; 15 ml blood per 10 ml Hypaque-Ficoll gradient) and centrifuged for 5 min at 1400 g. The mononuclear cell layer containing basophils (at the gradient-plasma interface) is washed with Tris buffer, pH 7.5 (see above, Procedure 2), and resuspended in a 1-ml volume of this same buffer. The cells are then carefully layered onto a gradient solution of Hypaque (1 ml cushion 12.7% w/v, 1 ml 10.6%, 1 ml 9.5%) and centrifuged at 500 g for 5 min (room temperature). Basophils appearing in the cell pellet at the bottom of the tube are washed twice with buffer and are ready for glass bead chromatography. Before use, washed (in water) glass beads (3M, St. Paul, MN) are saturated with whole autologous plasma (sedimented at high speed to remove platelets) supplemented with 4 ml of 0.1 M MgCI2 (required for cellular adhesive to beads; calcium is avoided here since
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
529
plasma clots are found in its presence) and 3 ml of 6% dextran. Saturated beads are added to a height of 8 cm in a glass column (1 × 12 cm), and washed cells (from the Hypaque gradient) resuspended in 1 ml of autologous plasma (supplemented as described above) are added dropwise to this column at room temperature. No incubation of the cell-loaded column is necessary, and nonadherent cells are eluted with autologous plasma (approximately 6 ml). The column is then filled with Tris buffer, pH 7.5, containing 0.1% EDTA, and the glass beads are agitated (gently stirred in the column with a glass rod). Desorbed cells are subsequently eluted with buffer, centrifuged, and differential and basophil counts, using Hansel stain (a modified rapid acting Wright stain, Lide Laboratories, St. Louis, MO), are performed on dried smears. 24 Basophils of 50% purity and 50% yield from whole blood have been reported with this procedure, and their functional nature is established by histamine release from cells of allergic donors known to be sensitive to mold) ° The greatest advantage to this procedure is that it can be performed in 1.5 hr. A disadvantage is that the purities obtained are not as great as with other methods.
Procedures Utilizing Percoll Density Gradients The techniques described above have been successful in purifying basophils. Despite this and the fact that histamine release from these cells has been demonstrated, there is a lingering concern that the multiple manipulations of and positive selection for basophils may cause changes in cell metabolic function which are not readily apparent. Recently, procedures utilizing Percoll density gradient centrifugation have been developed.tZ-~5 These methods do not involve multiple manipulations or positive selection of basophils and generally are rapidly performed. Each of these reports observed basophils at Percoll densities between 1.072 and 1.082 g/ml) ~-~4 The most specific density for basophil banding was observed to be 1.076 g/ml. H While each study had slight nuances in forming Percoll gradients, and handling of cells, a general procedure is described below. Percoll stock solution is prepared by mixing 90 ml of a commercial Percoll solution with 8.96 ml of 10 × H a n k s ' balanced salt solution (HBSS) (without calcium, magnesium, or bicarbonate), 0.45 ml of 1 N HC1, and 1 ml of N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer, pH 7.3 (final density 1.123 g/ml). Specific densities of Percoll are prepared by mixing Percoll stock and HBSS without calcium or magnesium. Final adjustment of the density with either stock Percoll or 24 j. Pruzansky and R. Patterson, Int. Arch. Allergy Appl. Immunol. 38, 522 (1970).
530
INFLAMMATION
[43]
HBSS is facilitated by density measurement with either a pycnometer or refractometer (Fischer Scientific, Silver Spring, MD) (see also this series, Vol. 108 [9]). The density of Percoll is altered by temperature; therefore, if gradient centrifugation is to be performed at room temperature, all solutions should be made and stored at this temperature. Discontinuous gradients of Percoll are formed by layering (approximately 3 ml) each Percoll solution (greatest density first) in a 15-ml polystyrene conical-bottom centrifuge tube (No. 2095, Falcon, Oxnard, CA) using a peristaltic pump (Miniplus II, Gibson) at a rate of 30 ml/hr. In most reports, blood anticoagulated with EDTA (1 ml 0.1 M EDTA/10 ml blood) is layered directly onto the Percoll gradient and centrifuged in a swinging bucket rotor at 600 g for 20 min at room temperature. Total basophil recovery is assessed by absolute counts using Alcian blue23 and differential counts on cytocentrifuged smears using Wright stain. Basophil purity obtained with Percoll density gradient centrifugation depends on various factors including the following: (1) Number of circulating basophils. A greater number of basophils in donor blood facilitates greater purity and yield. Leukocytosis, on the other hand, dramatically decreases basophil purity without affecting basophil recovery. (2) Technical problems. If an abrupt disturbance of the Percoll gradient is made when layering blood (either by pipet tip or blood dropping into the Percoll) a dramatic decrease in basophil purity can be observed. Similar decreases in basophil purity can also be observed if disturbance of the cell band occurs while withdrawing it from the gradient or if a small quantity of the cell layer is left behind. The results of reports utilizing the general procedure described above are summarized in the following discussion. In the report of Raghuprasad, H a discontinuous Percoll gradient of densities 1.072, 1.074, 1.076, and 1.078 g/ml is used to separate whole anticoagulated blood. Basophil purity is highest at 1.076 and 1.078 g/ml densities, with average purities of approximately 25% obtained (range 6-50% and 6-60%, respectively). Basophils are also found at 1.072 g/ml (average 5% purity) and 1.074 g/ml densities (average 15% purity). Basophil recovery and purity are highest in donors having high basophil counts (approximately 2% of total leukocytes; does not necessarily correspond to an atopic state), and lymphocytes and neutrophils are the most frequent contaminants present in basophil preparations. Centrifugation of cells pooled from the 1.076 and 1.078 g/ml densities and application to a second discontinuous Percoll gradient consisting of two densities (1.072 and 1.080 g/ml) lead to an enhancement of purity (average 40%, range 15- 83%) but a decrease in yield (20% of total basophils). The functional nature of the isolated basophils may be verified by histamine release using several secretagogues (anti-IgE, complement component 5a, and alternaria antigen in sensitive individuals).
[43]
BASOPHILS IN I N F L A M M A T O R Y REACTIONS
531
Kaufman et al. 12 also used discontinuous Percoll gradients (densities 1.072, 1.082, 1.100 g/ml) and observed basophils to band between the 1.072 and 1.082 g/ml densities. The range of purity is between 8 and 20% with a basophil recovery of 70% of those applied to the gradient. This method differs from that previously described by the fact that a leukocyte preparation (obtained by unit gravity sedimentation for 30-40 min at 37 ° in 6% dextran) instead of whole blood is layered onto the Percoll gradient. In another method, ~3whole blood (EDTA anticoagulated) is layered onto a discontinuous Percoll gradient (densities 1.070, 1.079, 1.090 g/ml), and, as in the previous method, basophils are observed to band between densities of 1.070 and 1.079 g/ml. The mean purity of the isolated basophils is 19% (range 5 - 53%), and lymphocytes are the main contaminating cells) a However, on closer scrutiny, basophils from some donors consistently band in two different layers of the discontinuous Percoll gradient (the plasma- 1.07 g/ml Percoll interface and the 1.070- 1.079 g/ml Percoll interface).~4 While the greatest number of basophils is still found to be in 1.070- 1.079 g/ml interface, in some donors the majority of basophils are found in the plasma-1.070 g/ml Percoll interface. From this and other observations, the existence of two populations of basophils has been suggested. These data alone do not prove the existence of multiple populations of basophils, but they do raise the possibility that subpopulations of basophils exist and perhaps have distinct chemical composition and function.
Procedures Utilizing Percoll Density Gradient Centrifugation and Cell Panning Techniques Landry and Findlay ~6 have recently described a basophil purification procedure which combines both Percoll density gradient centrifugation and cell panning techniques, using monoclonal antilymphocyte antibodies. Cell panning techniques negatively select for basophils by the selective removal of unwanted or contaminating cells. In this procedure, stock Percoll (Pharmacia) is prepared by mixing 9 parts commercial Percoll, and 1 part PIPES (10 × buffer) (PIPES 250 mM, NaC1 1180 mM, KC1 50 mM, NaOH 430 mM, and 0.1% 0.1 M EDTA). Leukocyte-rich plasma is prepared by mixing whole blood (anticoagulated with 0.1 M EDTA) with 20% v/v dextran and allowing the mixture to settle at unit gravity for 90 min. The resultant leukocyte preparation is then carefully layered over a discontinuous Percoll gradient prepared by layering 10 ml of a 55% PercoU solution (5.5 ml stock Percoll plus 4.5 ml PIPES 1 × buffer) over 10 ml of a 65% Percoll solution (6.5 ml stock Percoll plus 3.5 ml PIPES 1 × buffer). This gradient is centrifuged at 400 g for 20 min at room temperature. After centrifugation three bands of cells can be seen. The second band of cells found between the 55 and 65% Percoll layers
532
INFLAMMATION
[43]
contains an enriched population of basophils plus contaminating mononuclear cells and neutrophils. This layer is carefully withdrawn, washed twice (250 g for l0 rain at 4 °) with 30 ml of PIPES buffer containing 0.03% human serum albumin (PA), and further purification of basophils is obtained with cell panning techniques. For the preparation of panning plates (see also this series, Vol. 108 [l l ]), plastic petri dishes (Fisher 8-757-12) are coated with 100/d (0.1 mg) of goat anti-mouse IgG (Tago No. 4150) in l0 ml of phosphate-buffered saline (PBS) by incubating at 4 ° for 12 hr. After incubation, the antibody solution is decanted. The plates washed 3 times with l0 ml of PBS after which they are ready for use. Hates may be stored (wrapped in Parafilm) with PBS containing 2% fetal calf serum (FCS) at - 2 0 °. In the panning procedure, cells from band 2 of the Percoll gradient (2-4 × l05) are resuspended in 1 ml of PA buffer. Monoclonal antibodies (anti-Leu-1 and anti-HLA-DR, Becton Dickinson, Sunnyvale, CA) are added to the cells at a final concentration of 1/~g/106 cells, and the mixture is incubated for l hr at 4 °. After incubation, the cells are centrifuged (240 g for 5 min at 4°), resuspended in 3 ml of PBS containing 2% FCS (PBS-FCS, at 4°), added to panning plates, and incubated at 4 ° for 60 min (swirling of plate is performed every 20 min). After this incubation, the unabsorbed cells are decanted, combined with cells obtained by 3 gentle washings of the plate (with 10 ml of PBS-FCS), centrifuged at 240 g for l0 min at 4 °, and resuspended in PA buffer. Counting of basophils is performed using the Alcian blue method. 23 The second cell band in the Percoll gradient obtained from fractionated leukocyte-rich plasma contains basophils of highest purity (mean 14.6 _+ 7.9%)/16 Further purification of these basophils using cell panning techniques increases the purity to a mean of 34 _4- 15% (range 19-54%), with 55% yield of basophils.16 This is approximately a 30-fold purification. The functional capability of the basophils is verified by histamine release using anti-IgE as a secretagogue. Lett-Brown et al. 17 have recently reported a basophil purification technique similar to that described above. The primary difference in their procedure is the Percoll density gradient centrifugation step. In their method, the procedure described by Leonard et al. 13,14 (outlined above) is utilized. Basophil-enriched fractions withdrawn from Percoll are subsequently panned as described above (an OKT 11 monoclonal antibody is used in place of the anti-Leu-1 monoclonal antibody). In selected donors, functional basophils with purities of greater than 90% and yields from blood of approximately 50% may be achieved. 17 The major drawback is the small number of highly purified basophils (5-10 × 105 cells) that can be obtained. While mediator release experiments are feasible with small numbers ofbasophils, biochemical studies usually require larger
[43]
BASOPHILS IN I N F L A M M A T O R Y R E A C T I O N S
533
numbers of pure cells. The use of leukopheresis products may enhance the productivity of these techniques.
Procedures Utilizing Flow Microfluorometric Techniques (see also this series, VoL 108 [19]) Flow microfluorometry allows for the isolation of viable and highly purified basophils from blood. This method is the simplest to perform, requiring only commercially available anti-IgE antibody and access to a fluoroescence cell sorter. The technique as described by Weil et al. is consists of two steps. In the first step, blood collected in EDTA (final concentration 5 mM) and diluted with an equal volume of HBSS containing 0.1% bovine serum albumin (BSA) (HBSS-BSA) is layered over a HypaqueFicoll solution (specific gravity 1.077 g/ml) and centrifuged at 800 g for 15 min, and the mononuclear cell layer containing basophils washed 3 times with HBSS-BSA. Washed cells (5 × 107/ml) are incubated with a 1 : 50 dilution of fluorescein isothiocyanate (FITC)-conjugated goat antihuman IgE (epsilon-specific IgG fraction, Cappel Laboratories, Cochranville, PA) at 5 ° for 30 min. Following this incubation, the cells are washed twice with cold HBSS-BSA, and labeled and unlabeled cells are sorted with a FACS II cell sorter (Becton Dickinson) using two sorting rates. The first enrichment sort is performed at a rate of 12 × 103/sec without coincidence constraints. These enriched cells are then sorted at a rate of 4 × 103/sec with coincidence constraints (i.e., only positive cells without cells in adjacent droplets sorted). Basophil purities of 50-65% could be obtained with the first sorting and purities of 97-99% with the second sort. is These cells are viable, and a histamine content of 1.7 pg/cell agrees with other reports published for human basophils. Limitations to this procedure include the following: (1) Sorted cells having been exposed to anti-IgE under conditions preventing histamine release are inactivated and cannot be used to study release using antigen-dependent mechanisms. (2) The number of cells able to be prepared by this technique is small and would not lend itself to biochemical studies.
Procedures Utilizing Centrifugal Elutriation (see also this series, Vol. 108 [20]) All of the methods described above have been successful to some extent in isolating and purifying human basophils. The number of basophils isolated using these procedures is usually low (of the order of 105- 106 cells), and generally the techniques are not suitable or capable of being modified for the isolation and purification of large numbers of basophils. The recent report of DeBoer and Roos ~5 utilizing Percoll density gradient
534
INFLAMMATION
[43 ]
centrifugation, centrifugal elutriation, and increased quantities of donor blood has, however, demonstrated much greater success in isolating and purifying large numbers of human basophils. In this method a stock Percoll solution is prepared by mixing 93 ml of Percoll with 7 ml of a solution containing 1.54 M NaC1 and 0.1 M NaH2PO4 (all solutions are kept at room temperature). Specific Percoll densities are prepared by mixing stock Percoll with PBS (140 m M NaCI, 9.2 m M Na2HPO4, 1.3 m M NaH2PO4, pH 7.4). The final Percoll solution also contains human serum albumin (5 /tg/ml) and trisodium citrate (13 m3/). In the isolation procedure, 1 unit of normal donor blood (500 ml) is collected in a plastic bag containing 75 ml of an ACD solution (containing a hydrous disodium citrate, 100 mM, and anhydrous glucose, 128 mM, pH 5.0). Within 1 hr after collection, the blood is centrifuged in the plastic bag (4000 g, 5 min, room temperature), and the plasma is separated from cells. The upper 50- 100 ml of the packed cells (buffy coat) contains 70% of the leukocytes. This buffy coat is collected ~5and diluted to 200 ml with PBS containing 13 m M trisodium citrate (PBS-TC). Mononuclear cells and basophils are separated from neutrophils, eosinophils, and red blood cells by centrifugation (1000 g, 20 min, room temperature) of the buffy coat over a Percoll solution of 1.077 g/ml density (8 tubes each containing 12 ml of Percoll solution). The supernatant autologous plasma (SP) from this centrifugation is saved, and the cells at the plasma-Percoll interface are diluted to 200 ml with PBS-TC and centrifuged at 400 g for 5 min at room temperature. To remove platelets, the resultant cell pellets are resuspended first in 100 ml then in 50 ml of the SP solution. After each resuspension of cells in SP, the cells are centrifuged at 400 g for 5 min at room temperature. The final cell pellet is resuspended in 10-20 ml of the SP solution and injected into a JE-6 elutriation rotor powered by a J-2 I C centrifuge (Beckman). The elutriation medium is PBS-TC plus 5 mg/ml human serum albumin. To separate the various cell populations, the flow rate is maintained at 20 ml/min while the rotor speed is diminished from 4000 to 0 rpm. Fractions of 100-200 ml of the outflow are collected at rotor speeds of 3700, 2600, 2500, and 0 rpm. The last fraction (0 rpm) contains 80% of the basophils present in the starting preparation. This fraction is centrifuged (400 g, 5 min, room temperature), resuspended in 4 ml of Percoll 1.067 g/ml), and layered over 2 ml of Percoll of 1.075 g/ml density. A top layer of 0.2 ml of PBS-TC containing 5 mg/ml of human serum albumin is added, and the gradient is centrifuged at 1000 g for 10 min at room temperature. After centrifugation, two interfaces containing cells are observed. The lower Percoll interface (1.067-1.075 g/ml densities) contains the basophils. Basophil purity of 70% (range 45- 87%, as measured by Alcian blue)
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
535
and yield of 60% from the buffy coat have been reported. ~5 More importantly, however, 4 - 19 X 106 basophils can be obtained using this procedure. Histamine content (1-2 pg/cells) and releasability (30-50% to antiIgE) have been shown in these preparations. ~5The success and consistency of this procedure has been predicated on the following: (1) the capabilities of centi'ifugal elutriation; (2) suspension of the cells in Percoll (1.067 g/ml density) before applying to the Percoll solution of higher density (this prevents carryover of cells into a layer with a higher specific gravity than the cells); and (3) prevention of partial degranulation of basophils in the procedure (this is avoided by preventing platelet aggregation), accomplished by platelet removal with a low-speed centrifugation in autologous plasma. Methods for the Isolation of Basophils from Other Species Although present, basophils in rodents are much less abundant and, therefore, are less well studied. ~9In dogs, basophils make up less than 1% of the leukocyte population, and their concentration can be enhanced to 1-2% using double-density Hypaque-Ficoll centrifugation.25 Naturally abundant basophils have been observed in the snapping turtle Chelydra serpentina. 2° Normal circulating basophil levels of 50-63% have been reported, and these cells have been shown to release histamine in response to a rabbit anti-turtle immunoglobulin. Studies aimed at purifying and further characterizing these cells have not as yet been reported. To date, basophil function and techniques for isolation from blood have been most thoroughly studied in humans and guinea pigs .21'22Procedures for isolation of guinea pig basophils are discussed in detail below. Procedure
Techniques for purification of guinea pig basophils utilizing Ficoll density gradient centrifugation2! and negative selection using a rabbit antileukocyte antibody and Percoll density gradient centrifugation have been reported. 22 However, the most consistent and reliable technique utilized for guinea pig basophil purification involves both centrifugal elutriation and Percoll density gradient centrifugation.26 This procedure is described below.
25 D. Austin, S. Chan, A. Malley, J. Hanifin, and C. Hirshman, J. Lab. Clin. Med. 17, 53 (1985). 26 B. Undem, J. Brendel, T. Hirth, C. Buckner, and F. M. Graziano, Am. Rev. Respir. Dis. 133, 763 (1986).
536
INFLAMMATION
[43]
Basophils comprise only a small percentage of the total nucleated cells in guinea pig blood. However, the animals can be treated to increase the number of basophils which, in turn, facilitates purification. A striking basophilia can be obtained in guinea pigs sensitized with whole sheep blood for 12 days (daily i.p. injections of 2 ml whole blood diluted 1 : 2 with PBS). 21,22On day 13, blood obtained from six animals and containing 0.1 M EDTA is thoroughly mixed with 3% gelatin (2 parts blood to 1 part gelatin) in a test tube and placed at an angle of 30 ° in a 37 ° water bath. After incubation for 45 min, the leukocyte-rich plasma is withdrawn and centrifuged at 400 g (room temperature) for 30 min. Pelleted cells are washed once in HG (HEPES 10 mM, NaC1 137 mM, KC1 5 mM, 0.5 mg/ml gelatin, 5.5 m M glucose, pH adjusted to 7.4 with NaOH) containing 4 m M EDTA, and residual red cells are removed using hypotonic lysis (see this series, Vol. 108 [6]). Leukocyte preparations (containing an average of 6% basophils) 22'26 are then fractionated using a JE-6 elutriator rotor driven by a J-21B Centrifuge (Beckman Instruments, Palo Alto, CA). Rotor speed is controlled with a 10-turn potentiometer which allows for accurate rpm selection (within 10 rpm). Before each run, the system is washed sequentially with ethanol (200 ml), distilled water (1000 ml), and PBS (200 ml). Blood leukocyte suspensions containing basophils are loaded into the elutriator with a rotor speed of 4150 rpm, and the flow rate is adjusted to and maintained at 30 ml/min. Leukocytes are fractionated by incrementally decreasing the rotor speed to 3600, 2930, 2760, 2100, and 0 rpm. After each adjustment in rotor speed, two to four 45-ml fractions are collected. An additional 90-ml collection is obtained at the end of each run with the centrifuge turned off. Elutriated fractions containing the highest percentage of basophils (fractions collected after reduction in rotor speed to 2760 and 2100 rpm) are pooled, centrifuged, resuspended in l ml of HG (10-50 × 106 cells/ml), and layered onto a discontinuous Percoll gradient (1.06, 1.07, and 1.08 g/ml). 26 Centrifugation is carried out at 400 g for 20 min at room temperature, and cell bands are carefully removed and washed with HG. Total basophil counts are performed at each step during the purification procedure using the Alcian blue method. 23 Differential counts are obtained on cytocentrifuged (Cytospin, Shandon Elliot Corp.) smears stained with Wright stain. Cell viability (90-98%) is monitored at each step during the isolation procedure by Trypan blue dye exclusion. Guinea pig basophils (5 - 10 × l06 basophils) of greater than 90% purity and 40-50% yield from whole blood are consistently observed at the 1,070- 1.080 g/ml Percoll interface using this procedure. 26 Use of centrifugal elutriation alone will consistently give basophil purity of 40-50%. 26 Morphologically, the cells appear to be intact (see Fig. 1), and, functionally, histamine release from these cells can be demonstrated with antigen
[43]
BASOPHILS IN INFLAMMATORY REACTIONS
537
(sheep ~,-globulin), concanavalin A, rabbit anti-IgG~ antibody, and 12-0tetradecanoylphorbol 13-acetate (TPA). Rat Basophilic Leukemia Cells The problems inherent in studying normal basophils (obtaining large quantities of pure cells for biochemical studies, possible heterogeneity of basophil populations) discussed above have focused attention on the study of a rat basophilic leukemia tumor cell line. In 1973 Eccleston et al. 27 first described the development of basophilic leukemia in a Wistar rat fed fl-chlorethylamine (a potent inducer of myeloid or lymphatic leukemia). Leukemic cells obtained from such animals (RBL cells) can be serially transplanted by intraperitoneal injection into rats, resulting in the development of a solid t u m o r . 27 A number of investigators have shown these tumor cells to contain histamine and serotonin and have used them for study of the structure and binding characteristics of IgE surface receptors present on these cells. 27-31 RBL cells have been adapted to suspension cell culture and have been maintained for prolonged periods of time with little morphologic or functional change in cell characteristics compared to original minced tumor cells. 2s The original cell lines, while useful in studying the IgE receptor, failed to be triggered for IgE-mediated histamine release and, therefore, were not useful in studying the mechanisms of this process.27,2s Subsequently, several cloned histamine-releasing cell lines and nonhistamine-releasing cloned variant sublines defective at different steps in the histaminereleasing process have been reported) 2-~ The defects at different sites of the release pathway have allowed for a dissection of the biochemical events involved in histamine release. 33-3s RBL cell lines have been and will 27 E. Eccleston, J. Leonard, S. Lowe, and H. Welford, Nature (New Biol.} 244, 73 (1973). 2s A. Kulczycki, C. Isersky, and H. Metzger, J. Exp. Med. 139, 600 (1974). 29 A. Froese, R. Helm, D. Conrad, C. Isersky, T. Ishizaka, and A. Kulczycki, Immunology 46, 107 (1981). 30 H. Metzger, Immunol. Rev. 41, 186 (1979). 31 H. Metzger, A. Goetze, J. Kanellopoulos, D. Holowka, and C. Fewtrell, Fed. Proc., Fed. Am. Soc. Exp. Biol. 41, 8 (1982). 32 E. Barsumian, C. Isersky, M. Petrino, and R. Siraganian, Eur. J. Irnmunol. 11, 317 (1981). 33A. McGivney, F. Crews, F. Hiraha, J. Axelrod, and R. Siraganian, Proc. Natl. Acad. Sci. U.S.A. 78, 6176 (1981). R. Siraganian, A. McGivney, E. Barsumian, F. Crews, F. Hiraha, and J. Axelrod, Fed. Proc., Fed. Am. Soc. Exp. Biol. 41, 30 (1982). 35 C. Meyer, L. Wahl, B. Shader, and R. Siraganian, J. Immunol. 131, 911 (1983). 36 j. Rivera, J. M. Mullins, K. Furuichi, and C. Isersky, J. Immunol. 136, 623 (1986). 37 M. Garcia-Gil and R. Siraganian, J. Immunol. 136, 259 (1986). 3s M. Gareia-Gil and R. Siraganian, J. Imrnunol. 136, 3825 (1986).
538
INFLAMMATION
[44]
continue to be a convenient model for study of the mechanisms of mediator release. A rat basophilic leukemia cell line (CRL 1378-RBL-l) is available from the American Type Culture Collection, Rockville, MD. M e d i a t o r R e l e a s e b y Basophils Mediator release (e.g., histamine, arachidonic acid metabolites) from isolated and purified basophils has been documented in humans s- 15,39and other species. 19-21,25,26In the general procedure for mediator release, purified cells (generally 5 - 10 × 104 basophils per assay tube) in 0 . 5 - 1 ml of isotonic buffer (e.g., HEPES or PIPES buffer) containing 1 m M calcium and magnesium are reacted with varying concentrations of secretagogue, e.g., anti-IgE (0.01- 1 gg/ml) s (Calbiochem-Behring Corp.), antigen (e.g., sheep ),-globulin, 0.1 - 10 gg/ml), 22,26 or concanavalin A (0.1 - 3 #g/ml). 4° Mediators released into the supernatant fluid can be measured with one of the available methods (e.g., this volume [42]). 39R. Schleimer, C. Fox, R. Naclerio, M. Plaut, P. Creticos, A. Togias, J. Warner, A KageySobotka, and L. Lichtenstein, J. Allergy Clin. Immunol. 76, 369 (1985). 4op. Siraganian and R. Siraganian, J. Immunol. 112, 2117 (1974).
[44] H u m a n
Neutrophil
Degranulation
By DANIEL G. WRIGHT Introduction Neutrophils are wandering, phagocytic white blood cells that are critical to host defenses against bacterial and fungal infections. They are also the first circulating leukocytes to arrive at a site of acute inflammation. Indeed, the extravascular accumulation of these cells in tissues is a fundamental histologic hallmark of the acute inflammatory response. Inflammatory neutrophils are recognized to play a key role in mediating the dissolution and remodeling of connective tissues at sites of inflammation. Neutrophils injure or destroy connective tissue cells and digest or alter extracellular matrix proteins by at least two mechanisms, generation of toxic oxygen metabolites (see this series, Vols. 105 and 132) and secretion of proteolytic enzymes. Unlike the monocyte/macrophage family of phagocytes, neutrophils retain a very limited capacity for d e n o v o protein synthesis once they are METHODS IN ENZYMOLOGY, VOL. 162
Copyright © 1988 by Academic Press, Inc. All fights of reproduction in any form r-~-~rved.