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Subcellular fractionation of human neutrophils on Percoll density gradients
Journal of Immunological Methods 232 Ž1999. 131–143 www.elsevier.nlrlocaterjim
Subcellular fractionation of human neutrophils on Percoll density gradients Lars Kjeldsen ) , Henrik Sengeløv, Niels Borregaard Granulocyte Research Laboratory, Department of Hematology, The Finsen Center, The National UniÕersity Hospital, Rigshospitalet, 9 BlegdamsÕej, 2100 Copenhagen, Denmark
Abstract Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Neutrophil organelles; Percoll; Nitrogen cavitation; Marker proteins; Isolation of granule proteins
1. Introduction In the elucidation of neutrophil morphology and function, immunogold electron microscopy Žcovered by Dr. D.F. Bainton in this issue. and subcellular fractionation have played central roles Žfor review see Bainton, 1975; Borregaard and Cowland, 1997.. Initially, subcellular fractionation was performed by AbbreÕiations: FMLP, N-formylmethionyl-leucyl-phenylalanine; KRG, Krebs–Ringer phosphate with glucose; NGAL, Neutrophil gelatinase-associated lipocalin; P1 , Pellet after centrifugation of neutrophil cavitate; PMA, Phorbol myristate acetate; PMSF, Phenylmethylsulfonylflouride; S1 , Postnuclear supernatant; Vitamin B12 BP, Vitamin B12 binding protein ) Corresponding author. Tel.: q45-35-454046; fax: q45-35454841; e-mail: [email protected]
disrupting neutrophils by hypotonic shock and dounce homogenization, followed by zonal sedimentation or isopycnic gradient centrifugation on sucrose density gradients. A major improvement in the resolution of organelles obtained by subcellular fractionation came with the development of polyvinylpyrrolidone coated silica particles ŽPercoll. as the separating medium ŽPertoft et al., 1978., and with nitrogen cavitation as a gentle and efficient means of disrupting neutrophils ŽKlempner et al., 1980; Borregaard et al., 1983.. Percoll is an inert, self-generating density medium of low viscosity and ionic strength, ideal characteristics for a separating medium ŽPertoft et al., 1978.. Subcellular fractionation experiments have demonstrated heterogeneity within both peroxidasepositive, primary granules and peroxidase-negative
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 1 7 1 - 4
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granules, the former including defensin-rich and defensin-poor azurophil granules and the latter encompassing specific Žalso termed secondary. and gelatinase Žtertiary. granules, respectively ŽBainton, 1975; Borregaard and Cowland, 1997.. In addition, the highly mobilizable secretory vesicles were identified by their content of latent alkaline phosphatase by Borregaard et al. Ž1987.. These granules and secretory vesicles are mobilized in a sequential order, allowing the gradual activation of neutrophils during adhesion, diapedesis, migration and microbial killingrphagocytosis. Not only the liberation of matrix proteins is important for neutrophil activities, it was also realized through subcellular fractionation experiments, that endomembranes play an important role in furnishing the plasma membrane with important functional proteins during neutrophil activation ŽBorregaard and Cowland, 1997.. These proteins include adhesion proteins, receptors for chemotactic peptides and complement factors and in addition the b-cytochrome component of the NADPH-oxidase, which is thereby assembled and activated in the plasma membrane. This paper deals with subcellular fractionation of nitrogen cavitated, unperturbed or activated, human neutrophils on one-, two-, or three-layer Percoll density gradients. The application of subcellular fractionation in the elucidation of neutrophil structural organization, as alluded to above, is described. Furthermore, the use of subcellular fractionation as an initial step in protein purification schemes and in the investigation of binding of cytosolic proteins to isolated granules is exemplified. Appropriate assays for the identification of subcellular organelles are presented.
2. Materials and methods 2.1. Isolation and stimulation of neutrophils Neutrophils are isolated as described ŽBoyum, 1968.. Except for dextran sedimentation of erythrocytes, the procedure is carried out at 48C. Neutrophils are resuspended at the desired cell concentration and either kept on ice or stimulated with the appropriate stimulus for 15 min, after a 5-min preincubation at 378C. After stimulation, the cells are
pelleted and the supernatant, S 0 , decanted. Exocytosis of granule proteins can be calculated as the amount in the supernatant after stimulation ŽS 0 . divided by the total amount measured in supernatant, in postnuclear supernatant and unbroken cells and nuclei ŽS 0rŽS 0 q S1 q P1 ., see below.. 2.2. Disruption of neutrophils by nitrogen caÕitation Isolated neutrophils, either unperturbed or stimulated, are resuspended in KRG at 0.5 = 10 7 to 1.5 = 10 8 cellrml and left for 5 min on ice in 5 mM diisopropylfluorophosphate ŽAldrich Chemical, Milwaukee, WI, USA., followed by centrifugation and resuspension in the initial volume of disruption buffer Žrelaxation buffer minus EGTA, 100 mM KCl, 3 mM NaCl, 1 mM ATPNa 2 , 3.5 mM MgCl 2 , 10 mM Piperazine N, N X-bis2wethan-sulfonic acidx, pH 7.2. containing 0.5 mM PMSF ŽSigma... ATPNa 2 is added just before use from a 50 mM stock solution Žkept in vials at y208C.. Cells are pressurized under nitrogen for 5 min at 380 psi in a nitrogen bomb ŽParr Instrument, Moline, IL, USA.. The cavitate is then collected dropwise into EGTA, to a final concentration of 1.5 mM Ž150 mlr10 ml of cell suspension from a 100-mM stock of EGTA, pH 7.4.. Nuclei and intact cells are pelleted by centrifugation at 400 = g for 15 min. The postnuclear supernatant ŽS 1 . is cautiously decanted, and the pellet ŽP1 . resuspended in 1 ml of disruption buffer Žfor measurement of marker enzymes, if desired.. 2.3. Density centrifugation on Percoll gradients 2.3.1. Preparation of Percoll gradients Percoll solutions are prepared by mixing Percoll stock solution ŽPharmacia. with water and 1r10 the final volume of a 10-fold concentrated relaxation buffer Ž1000 mM KCl, 30 mM NaCl, 10 mM ATPNa 2 , 35 mM MgCl 2 , 12.5 mM EGTA, 100 mM Piperazine N, N X-bis2wethan-sulfonic acidx, pH 7.2. as indicated in Table 1. The constituents are precooled prior to mixing. When 10-fold concentrated relaxation buffer is prepared, the outweighed contents, minus ATPNa 2 , are resuspended to 4r5 of the final volume, since ATPNa 2 is added just before use from a 50-mM stock solution in 1r5, the volume of 10-fold concentrated relaxation buffer. After mixture
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Table 1 Preparation of Percoll solutions of different densities The numbers in the table shows the volumes of water, 10-fold concentrated relaxation buffer Ž10=. and Percoll stock solution Ždensity of 1.129 grml. needed to obtain a Percoll solution of the density given in the first column of the table. If one desires to prepare a solution of a different density, the necessary volumes can be calculated using the following formula: Vp sV0 Ž D 0 y1.0056.rŽ Dp y1., where Vp is the volume of Percoll stock solution, Dp the density of the Percoll stock solution, V0 the final volume of Percoll solution and 1.056 the density of 10-fold concentrated relaxation buffer.
the a-band, containing azurophil granules, the b-band containing the specific and gelatinase granules, and the g-band containing secretory vesicles and plasma membranes ŽFig. 1.. On the three-layer gradient, as opposed to the two-layer gradient, the b-band is split into the b 1-band, which contains specific granules and the b 2-band, which contains gelatinase granules ŽFig. 1.. The clear cytosol is present on top of the upper band, irrespective of the design of the gradient.
Final density Žgrml.
Final volume Žml.
Percoll stock Žml.
10= Žml.
H 2O Žml.
2.4. Fractionation
1.050 1.065 1.090 1.120
30 30 30 30
10.27 13.19 19.58 26.55
3 3 3 3
16.73 13.81 7.42 0.45
of the Percoll solutions, PMSF is added to a final concentration of 0.5 mM and the pH of the Percoll solutions adjusted to pH 7.0 by addition of 1 M HCl. 2.3.2. One-layer Percoll gradient A cushion of 1 ml Percoll, density 1.12 grml, is gently layered under 20 ml of Percoll, density 1.065 grml, through a pleuracentesis needle. 2.3.3. Two-layer percoll gradient Fourteen milliliter of Percoll, density 1.12 grml, is gently layered under 14 ml of Percoll density 1.050 grml. 2.3.4. Three-layer percoll gradient Nine milliliter of Percoll, density 1.090 grml is gently layered under 9 ml of Percoll, density 1.050 grml. Finally, 9 ml Percoll, density 1.12 grml is layered under the 1.090 grml Percoll solution. Ten milliliter of the postnuclear supernatant ŽS 1 . is applied slowly through a fine needle on top of the desired Percoll gradient, avoiding mixture of the S1 with the upper Percoll solution. The gradient is centrifuged at 37,000 = g for 30 min at 48C in an SS34, fixed angle rotor in a Sorvall RC-5B centrifuge. With the one-layer gradient, this gives rise to two distinct bands containing granules and light membranes, respectively. On the two-layer gradient, three bands are formed, from the bottom designated
Fractions are collected at 48C by aspiration from the bottom of the tube, through a glass capillary tube attached to a polyethylene tube connected to a peristaltic pump and a fraction collector. Alternatively, the distinct bands can be harvested by hand through a Pasteur pipette. 2.5. Marker assays Myeloperoxidase is used as a marker for azurophil granules ŽCramer et al., 1985., lactoferrin for specific granules ŽCramer et al., 1985., gelatinase for gelatinase granules ŽKjeldsen et al., 1993, 1994., and HLA for plasma membranes ŽBjerrum and Borregaard, 1990.. All these assays and the assays for NGAL and the a m -subunit CD11b of the b-integrin Mac-1 are ELISAs, as described ŽSengeløv et al., 1993; Kjeldsen et al., 1994.. Latent alkaline phosphatase is used as a marker for secretory vesicles Ži.e., the difference between alkaline phosphatase
Fig. 1. Photo of the three-layer gradient before Žmiddle. and after centrifugation Žright., and of the two-layer gradient after centrifugation Žleft.. The bands formed after centrifugation are marked.
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measured in the presence and absence of a detergent ŽTriton X-100. ŽBorregaard et al., 1987... 2.6. RemoÕal of Percoll For evaluation of the majority of proteins in subcellular fractions by SDS-PAGE and Western blotting or for example in protein purification schemes, it is necessary to remove Percoll. This can be accomplished by ultracentrifugation at 100,000 = g for 45 min for light membranes and 90 min for granules. After centrifugation, the biological material is visible as a disc above the pelleted Percoll, and can easily be collected by a Pasteur pipette and subsequently resuspended in the desired buffer.
3. Results and discussion 3.1. Nitrogen caÕitation The initial and crucial step in subcellular fractionation is disruption of cells, which is accomplished by nitrogen cavitation as described by Klempner et al. Ž1980. and Borregaard et al. Ž1983.. During this procedure, neutrophil plasma membranes break Žand reseal into plasma membrane vesicles., as the cells leave the nitrogen bomb through a valve, going from a 380-psi to atmospheric pressure. Nitrogen cavitation is a reliable and efficient means of disrupting cells, with approximately 81% to 91% of granule marker proteins recovered in the postnuclear supernatant ŽTable 2, Klempner et al., 1980; Borregaard et al., 1983.. There is only a discrete breakage of nuclei as evaluated by measurement of DNA, with approximately 83% being recovered in the pellet after centrifugation of the cavitate ŽBorregaard et al., 1983.. Although modest, this damage to nuclei may be responsible for the observed lower percentage of azurophil granules Žmyeloperoxidase. and plasma membranes ŽHLA. recovered in the postnuclear supernatant ŽTable 2., since nuclear basic proteins may promote adherence of these structures to nuclei ŽKlempner et al., 1980.. The composition of the disruption buffer mimicking the intracellular milieu and its content of MgCl is believed to stabilize nuclei and minimize the problem of both granule and
Table 2 Percentage of marker protein recovered in postnuclear supernatant after nitrogen cavitation and in subcellular fractions after fractionation of density gradients Isolated neutrophils were disrupted by nitrogen cavitation. The cavitate was centrifuged to pellet unbroken cells and nuclei ŽP1 . and the postnuclear supernatant ŽS1 . decanted. Disruption efficiency was calculated for each marker protein as amount measured in S1 in percentage of total amount in S1 and P1 . Ten milliliter postnuclear supernatant was centrifuged on a three-layer Percoll density gradient and fractionated into 37 fractions of 1 ml each. Recovery is calculated as the total content of marker protein in subcellular fractions expressed in percentage of the content in the postnuclear supernatant applied to subcellular fractionation. Numbers are average of 7 to 12 experiments, with the SD given in parenthesis. Marker protein
Assay type
Amount in S1 after disruption Ž%.
Recovery in fractions Ž%.
Myeloperoxidase Lactoferrin Vitamin B12 BP
Elisa Elisa Functional assay Elisa Elisa Functional assay Elisa
83.7 Ž5.3. 90.0 Ž3.2. 92.8 Ž2.3.
99.7 Ž8.5. 102.1 Ž10.6. 93.7 Ž8.9.
90.9 Ž3.5. 89.3 Ž5.1. 90.9 Ž3.8.
91.6 Ž14.8. 95.8 Ž9.6. 97.2 Ž13.5.
80.5 Ž8.9.
82.9 Ž11.1.
Gelatinase Albumin Latent AP HLA
plasma membrane adherence to nuclei and aggregation between different granule and vesicle subsets, thus allowing a better resolution of organelles after density gradient centrifugation. Another important aspect of neutrophil disruption is the preservation of granule integrity, thus avoiding the liberation of potent neutrophil proteases, which may interfere with subsequent analysis of protease sensitive proteins in subcellular fractions Ževen in the presence of the protease inhibitors diisopropylfluorophosphate and phenylmethylsulfonylflouride, which are always included during neutrophil disruption.. When measuring granule proteins in cytosolic fractions after density gradient centrifugation of nitrogen cavitated neutrophils, less than 0.5% of the measured proteins Žmyeloperoxidase Ž0.4%., lactoferrin Ž0.1%., and gelatinase Ž0.5%., average of seven experiments. can be measured in the cytosol ŽKjeldsen et al., 1994., demonstrating that nitrogen cavitation leaves granules largely intact. This is in contrast to disruption by dounce homogenization, where considerable amounts of granule proteins are present in the cytosolic frac-
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tions after fractionation ŽBretz and Baggiolini, 1974; Dewald et al., 1982; Mollinedo and Schneider, 1984.. Due to this problem, the low efficiency, and the need to expose neutrophils to prolonged, hypotonic shock prior to disruption, dounce homogenization of neutrophils may be considered obsolete. 3.2. Percoll Õersus sucrose as a separating medium Sucrose has been widely used over the years as a separating medium in subcellular fractionation. Its high tonicity makes the medium very viscous and leads to shrinkage of the subcellular organelles. This results in alteration of their density and in lack of granule membrane integrity ŽBorregaard et al., 1983.. Percoll has several advantages over sucrose. Its viscosity is very low and the gradient therefore not easily disturbed ŽPertoft et al., 1978.. Besides, the tonicity of Percoll itself is negligible and can be adjusted to isotonicity by addition of buffer, thus avoiding shrinkage of organelles during fractionation ŽPertoft et al., 1978; Borregaard et al., 1983.. Furthermore, Percoll is a self-generating density medium Žin contrast to sucrose., since the Percoll particles sediment during centrifugation, resulting in a continuously increasing density towards the bottom of the tube. Gradients that contain two or more solutions of Percoll of different densities will smooth out during centrifugation, as opposed to discontinuous sucrose gradients. Finally, Percoll allows fast sedimentation of organelles to their isopycnic density with centrifugation times of 30 min or less. 3.3. Assays for marker proteins In our laboratory we prefer immunologically based assays ŽELISAs. for marker proteins of neutrophil subcellular structures, since these assays may be less sensitive to neutrophil proteases ŽKjeldsen et al., 1992.. Furthermore, we have never experienced any interference of Percoll with an ELISA Žunpublished observation., and Percoll is therefore not removed from fractions prior to measurement. Myeloperoxidase is used as a marker for azurophil granules ŽCramer et al., 1985., lactoferrin for specific granules ŽCramer et al., 1985., gelatinase for gelatinase granules Žsome gelatinase is located in specific granules as described below., latent alkaline phosphatase
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ŽBorregaard et al., 1987. or albumin for secretory vesicles ŽBorregaard et al., 1992b., and HLA for plasma membranes ŽBjerrum and Borregaard, 1990. ŽTable 2.. Alternatively, specific granules can be identified by vitamin B 12 binding protein ŽKane and Peters, 1975.. Both alkaline phosphatase and vitamin B 12 binding protein are measured by functional assays, which are unaffected by Percoll. Since most alkaline phosphatase is located intracellularly in secretory vesicles in the resting neutrophil, alkaline phosphatase is not a specific plasma membrane marker, as previously thought. Galactosyl transferase activity can be used as a marker for the Golgi apparatus and was recently identified mainly in the light membrane region of neutrophils ŽMorgan et al., 1997.. The reliability of an assay can be evaluated by calculating the recovery of the protein measured in the subcellular fractions compared to the amount measured in the postnuclear supernatant loaded onto the gradient. Table 2 shows the recoveries for a variety of proteins, including the marker proteins used. The recovery is between 83% and 105%. A recovery invariably exceeding 100% may indicate problems with proteasesrinhibitors of the assays, problems that are overcome by segregation of the proteaserinhibitor from the measured protein after subcellular fractionation. This is especially important to bear in mind with functional assays and has been observed, when gelatinase was measured by a gelatinolytic assay using tritiated gelatine as a substrate ŽDewald et al., 1982.. A low recovery has been observed for the azurophil granule marker b-glucoronidase Žaverage recovery 54%. ŽBorregaard et al., 1983.. This observation was attributed to inhibition of the spectrophotometric assay of b-glucoronidase by Percoll, especially in the densest fractions, which are richest in Percoll. 3.4. Subcellular fractionation on two-layer Percoll gradient Following disruption and centrifugation, the postnuclear supernatant is layered on top of the Percoll gradient and high speed centrifuged at 37,000 = g for 30 min. The first combined use of nitrogen cavitation and Percoll density gradient centrifugation was introduced by Borregaard et al. Ž1983. and
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Fig. 2. Subcellular fractionation of nitrogen-cavitated neutrophils on one-, two-, and three-layer Percoll gradient. Ten milliliter of postnuclear supernatant was applied on top of a one-layer Percoll gradient Ž1.065 grml, panel 1., a two-layer Percoll gradient Ž1.05r1.12 grml, panel 2., or a three-layer Percoll gradient Ž1.05r1.09r1.12 grml, panels 3 and 4.. After centrifugation, the gradients were fractionated by aspiration from the bottom of the tube into the number of fractions given on the x-axis. Each fraction was assayed for the marker enzymes shown, and on the three-layer gradient also for NGAL. The content in each fraction is calculated in percentage of the total content measured in all fractions.
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Fig. 2 Žcontinued..
involved a two-layer Percoll gradient. After centrifugation, three distinct bands are observed from the bottom designated the a-band, the b-band, and the g-band with the clear cytosol on top ŽFig. 1.. The a-band contains the majority of the azurophil, peroxidase-positive granules, identified by the marker myeloperoxidase, well separated from peroxidasenegative granules represented by the overlapping profiles of the markers lactoferrin and gelatinase, both contained within the b-band ŽFig. 2.. There is a minor myeloperoxidase peak within the b-band. The g-band contains the plasma membranes identified by HLA class 1 and the secretory vesicles, identified by latent alkaline phosphatase, i.e., activity only measurable in the presence of a detergent ŽFig. 2.. Above the g-band is the cytosol, identified by lactate dehydrogenase Žnot shown.. There is a considerable overlap between the distribution profiles of the latent alkaline phosphatase and HLA, although in most cases alkaline phosphatase peaks one fraction below HLA. Furthermore, it is clearly visible, that latent alkaline phosphatase displays a shoulder to the left of the peak that extends somewhat into the b-band region of the gradient, thereby overlapping with markers of peroxidase-negative granules. In the search for other constituents of secretory vesicles, distribution profiles similar to latent alkaline phosphatase were found for complement receptor 1, tetranectin and albumin, the latter two indicating the endocytic origin of secretory vesicles ŽBorregaard et
al., 1990, 1992b; Sengeløv et al., 1994.. It should be stressed, that isolation of neutrophils at 48C throughout the procedure is absolutely crucial in the preservation of the easily mobilizable secretory vesicles Žsee below.. 3.5. Subcellular fractionation on one-layer Percoll gradient In order to obtain a more ‘‘clean’’ preparation of light membranes with minimal contamination of granules, a one-layer gradient with a Percoll density of 1.065 grml can be used ŽSengeløv et al., 1994.. A 1-ml cusion of Percoll with a density of 1.12 grml is placed in the bottom of the tube. With this gradient, most granule markers are recovered in the first three fractions, well separated from the light membranes identified by latent alkaline phosphatase and HLA ŽFig. 2.. Although this gradient is useful in the preparation of ‘‘clean’’ light membranes devoid of granules, the difference in distribution profiles of HLA and latent alkaline phosphatase is less pronounced than on the two-layer gradient. The similar density of plasma membranes and secretory vesicles precludes their complete separation by means of density centrifugation. Instead, the light membranes, as for instance obtained by the one-layer gradient, can be separated by differences in their surface charge by free flow electrophoresis ŽSengeløv et al., 1992, 1994 and the contribution by Dr. Sengeløv in this issue.. Recently, a Percoll based, flotation gradi-
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Fig. 3. Subcellular fractionation of resting, FMLP- and PMA-stimulated neutrophils on three-layer Percoll gradients. Prior to disruption, cells were either kept on ice Žcontrol. or stimulated for 15 min at 378C with FMLP Ž10 nM. or PMA Ž2 mgrml.. After washing and disruption of the cells, the postnuclear supernatants were centrifuged on three-layer Percoll gradients and fractionated as described in Section 2. Fractions were assayed for myeloperoxidase, lactoferrin, gelatinase, latent alkaline phosphatase Žlatent AP., albumin, HLA and CD11b, the a m subunit of Mac-1.
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Fig. 3 Žcontinued..
ent has been developed by Dahlgren et al. Ž1995., that allows a more efficient, yet not complete, separation of plasma membranes from secretory vesicles,
than can be obtained by the ‘‘classical’’ Percoll gradients. This can be useful, when free flow electrophoresis is not available.
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Fig. 3 Žcontinued..
3.6. Subcellular fractionation on three-layer Percoll density gradients On the two-layer gradient, a considerable overlap exists in the distribution profiles of lactoferrin and gelatinase within the b-band, as observed in Fig. 2. By introducing a third layer of Percoll of intermediate density Ž1.090 grml., the efficient separating volume of the gradient is improved and the b-band split into a denser b 1-band and a b 2-band ŽFig. 1.. The b 1-band contains the majority of lactoferrin, whereas the b 2-band contains the peak of gelatinase separated from the lactoferrin peak by several fractions ŽFig. 2.. As also observed from Fig. 2, peroxidase-negative granules exist as a continuum of granules rich in lactoferrin and devoid of gelatinase Ždefined as specific granules. to granules enriched in gelatinase but containing very little lactoferrin. Approximately 66% of all peroxidase-negative granules contain both lactoferrin and gelatinase, as demonstrated by double-labeling immunogold electron microscopy ŽKjeldsen et al., 1993., and a complete separation of the two proteins is thus physically impossible. Gelatinase granules are arbitrarily defined as all granules contained in fraction 16 and higher, albeit these granules contain 6% of total lactoferrin. The a- and g-bands are largely unchanged on the three-layer as compared to the twolayer gradient ŽFig. 2., although the ‘‘shoulder’’ of latent alkaline phosphatase Žthe secretory vesicles. does not extend as far into the b-band as is the case on the two-layer gradient.
A three-layer Percoll density gradient Ž1.07r 1.12r1.14 grml, 10 ml of each density. has also been developed by Nitsch et al. Ž1990.. After centrifugation, this gave rise to nine visible bands including three bands enriched in azurophil granule constituents, and four bands mainly containing peroxidase-negative granule proteins. The lightest of the latter four bands was relatively enriched in gelatinase, thus resembling the lactoferrin-poor, gelatinase-rich, peroxidase-negative granules Žgelatinase granules., described above. Isolation of a similar gelatinase-rich peroxidase-negative granule subpopulation has also been obtained on discontinuous sucrose density gradients ŽJones et al., 1990; Graves et al., 1992.. 3.7. Subcellular fractionation of resting and actiÕated neutrophils A drawback of subcellular fractionation is that separation of subcellular structures is based solely on differences in density. This implies that two different structures of equal density will colocalize on the gradient. However, discrimination between different organelles, that band at the same density, can be achieved, if these are mobilized differently upon stimulation of the neutrophils. This can be investigated by comparison of distribution profiles in unperturbed neutrophils and in neutrophils stimulated prior to nitrogen cavitation and density gradient centrifugation. Fig. 3 shows subcellular fractionation of unstimulated neutrophils and neutrophils stimulated with either the bacterial chemotactic peptide FMLP
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or the phorbol-ester PMA. It is observed, that following FMLP-stimulation, an almost complete mobilization of secretory vesicles occurs with parallel disappearance of latent alkaline phosphatase and albumin within the g-band. Hardly any azurophil or specific granules are mobilized by FMLP, whereas approximately 25% of gelatinase is exocytosed, preferentially from the lightest peroxidase-negative granules in fraction 16 and above, i.e., gelatinase granules ŽFig. 3.. When neutrophils are stimulated with PMA, a complete exocytosis of gelatinase and a considerable specific granule mobilization is observed Ž50%– 60%., whereas only around 1r3 of myeloperoxidase is lost from the a-bandrazurophil granules. When evaluating a membrane associated protein, in Fig. 3 exemplified by the a m subunit CD11b of the b 2-integrin Mac-1, the protein translocates to the plasma membrane following stimulation in parallel with its disappearance from granules and secretory vesicles. After stimulation with FMLP there is an increase in CD11b in the plasma membrane, recruited in part from gelatinase granules in fractions 16 and above, and in part from secretory vesicles within the g-band, where approximately 15% to 20% of total Mac-1 is located in unperturbed neutrophils ŽSengeløv et al., 1993; Kjeldsen et al., 1994.. If one focuses only on the total content of Mac-1 within the g-band before and after stimulation, one overlooks this translocation from secretory vesicles to plasma membrane. Only after stimulation with PMA, there is a parallel loss of CD11b and lactoferrin from the b 1-region, indicating translocation of CD11b from specific granules to the plasma membrane. 3.8. Strategy in inÕestigation of the subcellular localization of a constituent of neutrophils When facing the task of determining the subcellular localization of a constituent of human neutrophils, an initial approach would be to evaluate its presence in peroxidase-positive and -negative granules and in light membranes separated by fractionation on the two-layer Percoll gradient. If the protein is mainly located in peroxidase-negative granules, its distribution between specific and gelatinase granules and the mobilization from these subsets Žfollowing FMLP- and PMA-stimulation. should be investigated on the three-layer gradient. If the protein of interest is detected within the g-band, its distribution profile
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should be compared to that of markers for secretory vesicles and plasma membranes in both unperturbed and FMLP-activated neutrophils. These findings can be corroborated by fractionation on the flotation Percoll gradient described above ŽDahlgren et al., 1995., or by subsequent free flow electrophoresis of the g-band Žas described by Dr. Sengeløv in this issue.. When investigating the subcellular localization of a constituent of the neutrophil, it should ideally be measured in each fraction by a quantitative assay, since this allows the comparison of the distribution profile of the protein with the profile of well established and specific markers for subcellular organelles. Exact matching profiles strongly indicate a common subcellular localization as for example seen with the specific granule marker lactoferrin and NGAL ŽFig. 2.. If a quantitative assay is not available, the presence of the protein in subcellular organelles can be evaluated by Western blotting. In this case, Percoll must be removed from fractions by ultracentrifugation Žsee Section 2. prior to SDSPAGE, unless the abundance of the protein under investigation allows 20-fold or more dilution of the Percoll containing subcellular fraction, in which case Percoll will not affect the quality of gel electrophoresis. Findings obtained by subcellular fractionation should preferentially be validated by double labeling immunogold electron microscopy given that an antibody against the protein is available. Electron microscopy can be performed either on intact neutrophils or on subcellular fractions, as described herein by Dr. Bainton. 3.9. Use of subcellular fractionation in isolation of granule proteins Besides being an invaluable tool in the investigation of the subcellular localization of neutrophil constituents, subcellular fractionation has also been used in the isolation of granule proteins. For preparative purposes, the number of cells loaded on each gradient can be increased to about 1.5 = 10 9 , without affecting the resolution of subcellular organelles on the gradient. It is an efficient initial step in purification schemes resulting in several fold increases in the specific activity of the purified protein. The band that contains the protein can be harvested from the
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gradients by hand by aspiration through a pasteur pipette. After removal of Percoll by ultracentrifugation, the proteins can be solubilized by an appropriate detergent, or granules can be repetitively freeze thawed, resulting in liberation of granule matrix proteins to the supernatant after pelleting of the membranes by centrifugation. Subcellular fractionation has been used as an initial step in the purification of proteinase 3 ŽStummann and Wiik, 1997., the defensin HNP4 ŽWilde et al., 1989., myeloperoxidase ŽBorregaard, unpublished., and in purification from the specific granules of the cathelicidin, hCAP18 ŽSørensen et al., 1999.. hCAP-18 is especially sensitive to degradation by proteases from azurophil granules, and subcellular fractionation was therefore an important initial step in the preservation of intact hCAP-18 during the purification procedure. 3.10. Subcellular fractionation in the inÕestigation of in Õitro translocation of cytosolic proteins to isolated granule and membrane fractions Subcellular fractionation has been used to obtain isolated azurophil and specific granules and light membranes for use in the investigation of in vitro translocation of cytosolic proteins to the respective membranes. This approach was used in the identification of a 28-kDa protein, later identified as grancalcin, which reversibly bound to secretory vesicles and plasma membranes in the presence of calcium ŽBorregaard et al., 1992a.. In addition, several annexins were also found to bind to isolated granule and vesicle membranes in the presence of calcium, and their differential calcium-dependency and affinity for granule subsets was delineated by the same approach ŽBorregaard et al., 1992a; Sjolin et al., 1994, 1997.. Also, the in vitro translocation of protein kinase C to isolated granules and light membranes was investigated in the presence of PMA ŽChristiansen and Borregaard, 1989..
4. Conclusion Subcellular fractionation of nitrogen cavitated neutrophils on Percoll density gradients is an easy, fast and very reproducible technique. The combined use of subcellular fractionation and immunogold
electron microscopy has contributed considerably to the understanding of neutrophil structural organization including the dynamics of exocytosis of granules and secretory vesicles. Subcellular fractionation will also be beyond doubt an invaluable tool in the future study of neutrophil cell biology.
Acknowledgements This work was supported by The Alfred Benzon Fund, The Danish Cancer Society, The Danish Medical Research Council, The Lundbeck Fund, Emil C. Hertz’s Fund, The Novo Fund, Amalie Jørgensen’s Fund, Brøchner-Mortensen’s Fund, Anders Hasselbalch’s Fund, Ane Kathrine Plesner’s Fund.
References Bainton, D.F., 1975. Neutrophil granules. Br. J. Haematol. 29, 17. Bjerrum, O.W., Borregaard, N., 1990. Mixed enzyme-linked immunosorbent assay ŽMELISA. for HLA class I antigen: a plasma membrane marker. Scand. J. Immunol. 31, 305. Borregaard, N., Cowland, J.B., 1997. Granules of the human polymorphonuclear leukocyte. Blood 89, 3503. Borregaard, N., Heiple, J.M., Simons, E.R., Clark, R.A., 1983. Subcellular localization of the b-cytochrome component of the neutrophil microbicidal oxidase: translocation during activation. J. Cell Biol. 97, 52. Borregaard, N., Miller, L.J., Springer, T.A., 1987. Chemoattractant-regulated mobilization of a novel intracellular compartment in human neutrophils. Science 237, 1204. Borregaard, N., Christensen, L., Bjerrum, O.W., Birgens, H.S., Clemmensen, I., 1990. Identification of a highly mobilizable subset of human neutrophil intracellular vesicles that contains tetranectin and latent alkaline phosphatase. J. Clin. Invest. 85, 408. Borregaard, N., Kjeldsen, L., Lollike, K., Sengeløv, H., 1992a. Ca2q-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from neutrophils. FEBS Lett. 304, 195. Borregaard, N., Kjeldsen, L., Rygaard, K., Bastholm, L., Nielsen, M.H., Sengeløv, H., Bjerrum, O.W., Johnsen, A.H., 1992b. Stimulus-dependent secretion of plasma proteins from human neutrophils. J. Clin. Invest. 90, 86. Boyum, A.S., 1968. Separation of leukocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21, 77, Suppl. Bretz, U., Baggiolini, M., 1974. Biochemical and morphological characterization of azurophil and specific granules of human neutrophilic polymorphonuclear leukocytes. J. Cell Biol. 63, 251.
L. Kjeldsen et al.r Journal of Immunological Methods 232 (1999) 131–143 Christiansen, N.O., Borregaard, N., 1989. Translocation of protein kinase C to subcellular fractions of human neutrophils. Scand. J. Immunol. 29, 409. Cramer, E., Pryzwansky, K.B., Villeval, J.-L., Testa, U., BretonGorius, J., 1985. Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold. Blood 65, 423. Dahlgren, C., Carlsson, S.R., Karlsson, A., Lundqvist, H., Sjoelin, C., 1995. The lysosomal membrane glycoproteins Lamp-1 and Lamp-2 are present in mobilizable organelles, but are absent from the azurophil granules of human neutrophils. Biochem. J. 311, 667. Dewald, B., Bretz, U., Baggiolini, M., 1982. Release of gelatinase from a novel secretory compartment of human neutrophils. J. Clin. Invest. 70, 518. Graves, V., Gabig, T., McCarthy, L., Strour, E.F., Leemhuis, T., English, D., 1992. Simultaneous mobilization of Mac-1 ŽCD11brCD18. and formyl peptide chemoattractant receptors in human neutrophils. Blood 80, 776. Jones, H.J., Schmalsteig, F.C., Dempsey, K., Krater, S.S., Nannen, D.D., Smith, C.W., Anderson, D.C., 1990. Subcellular distribution and mobilization of Mac-1 ŽCD11brCD18. in neonatal neutrophils. Blood 75, 488. Kane, S.P., Peters, T.J., 1975. Analytical subcellular fractionation of human granulocytes with reference to the localization of vitamin B12 binding protein. Clin. Sci. Mol. Med. 49, 171. Kjeldsen, L., Bjerrum, O.W., Hovgaard, D., Johnsen, A.H., Sehested, M., Borregaard, N., 1992. Human neutrophil gelatinase: a marker for circulating blood neutrophils. Purification and quantitation by enzyme linked immunosorbent assay. Eur. J. Haematol. 49, 180. Kjeldsen, L., Bainton, D.F., Sengeløv, H., Borregaard, N., 1993. Structural and functional heterogeneity among peroxidase negative granules in human neutrophils: identification of a distinct gelatinase containing granule subset by combined immunocytochemistry and subcellular fractionation. Blood 82, 3183. Kjeldsen, L., Sengeløv, H., Lollike, K., Nielsen, M.H., Borregaard, N., 1994. Isolation and characterization of gelatinase granules from human neutrophils. Blood 83, 1640. Klempner, M.S., Mikkelsen, R.B., Corfman, D.H., Andre´ Schwartz, J., 1980. Neutrophil plasma membranes: I. High yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation. J. Cell Biol. 86, 21.
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Mollinedo, F., Schneider, D.L., 1984. Subcellular localization of cytochrome b and ubiquinone in a tertiary granule of resting human neutrophils and evidence for a proton pump ATPase. J. Biol. Chem. 259, 7143. Morgan, C.P., Sengeløv, H., Whatmore, J., Borregaard, N., Cockcroft, S., 1997. ADP-ribosylation-factor-regulated phospholipase D activity localizes to secretory vesicles and mobilizes to the plasma membrane following N-formylmethionyl-leucylphenylalanine stimulation of human neutrophils. Biochem. J. 325, 581. Nitsch, M., Gabrijelcic, D., Tschesche, H., 1990. Separation of granule subpopulations in human polymorphonuclear leukocytes. Biol. Chem. Hoppe-Seyler 371, 611. Pertoft, H., Laurent, T.C., Laas, L., 1978. Density ˚˚ T., Kagedal, ˚ gradients prepared from colloidal silica particles coated with polyvinylpyrrolidone ŽPercoll.. Anal. Biochem. 88, 271. Sengeløv, H., Nielsen, M.H., Borregaard, N., 1992. Separation of human neutrophil plasma membrane from intracellular vesicles containing alkaline phosphatase and NADPH-oxidase activity by free flow electrophoresis. J. Biol. Chem. 267, 14912. Sengeløv, H., Kjeldsen, L., Diamond, M.S., Springer, T.A., Borregaard, N., 1993. Subcellular localization and dynamics of Mac-1 Ž a m b 2 . in human neutrophils. J. Clin. Invest. 92, 1467. Sengeløv, H., Kjeldsen, L., Kroeze, W., Berger, M., Borregaard, N., 1994. Secretory vesicles are the intracellular reservoir of complement receptor 1 in human neutrophils. J. Immunol. 153, 804. Sjolin, C., Stendahl, O., Dahlgren, C., 1994. Calcium-induced translocation of annexins to subcellular organelles of human neutrophils. Biochem. J. 300, 325. Sjolin, C., Movitz, C., Lundquist, H., Dahlgren, C., 1997. Translocation of annexin XI to neutrophil subcellular organelles. Biochim. Biophys. Acta 1326, 149. Sørensen, O., Bratt, T., Johnsen, A.H., Madsen, MT, Borregaard, N., 1999. The human antibacterial cathelicidin, hCAP-18, is bound to lipoprotein in serum. J. Biol. Chem. 274, 22445. Stummann, L., Wiik, A., 1997. A simple high yield procedure for purification of human proteinase 3, the main molecular target for cANCA. J. Immunol. Methods 206, 35. Wilde, C.G., Griffith, J.E., Marra, M.N., Snable, J.L., Scott, R.W., 1989. Purification and characterization of human neutrophil peptide 4, a novel member of the defensin family. J. Biol. Chem. 264, 11200.
Subcellular fractionation of human neutrophils on Percoll density gradients Lars Kjeldsen ) , Henrik Sengeløv, Niels Borregaard Granulocyte Research Laboratory, Department of Hematology, The Finsen Center, The National UniÕersity Hospital, Rigshospitalet, 9 BlegdamsÕej, 2100 Copenhagen, Denmark
Abstract Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Neutrophil organelles; Percoll; Nitrogen cavitation; Marker proteins; Isolation of granule proteins
1. Introduction In the elucidation of neutrophil morphology and function, immunogold electron microscopy Žcovered by Dr. D.F. Bainton in this issue. and subcellular fractionation have played central roles Žfor review see Bainton, 1975; Borregaard and Cowland, 1997.. Initially, subcellular fractionation was performed by AbbreÕiations: FMLP, N-formylmethionyl-leucyl-phenylalanine; KRG, Krebs–Ringer phosphate with glucose; NGAL, Neutrophil gelatinase-associated lipocalin; P1 , Pellet after centrifugation of neutrophil cavitate; PMA, Phorbol myristate acetate; PMSF, Phenylmethylsulfonylflouride; S1 , Postnuclear supernatant; Vitamin B12 BP, Vitamin B12 binding protein ) Corresponding author. Tel.: q45-35-454046; fax: q45-35454841; e-mail: [email protected]
disrupting neutrophils by hypotonic shock and dounce homogenization, followed by zonal sedimentation or isopycnic gradient centrifugation on sucrose density gradients. A major improvement in the resolution of organelles obtained by subcellular fractionation came with the development of polyvinylpyrrolidone coated silica particles ŽPercoll. as the separating medium ŽPertoft et al., 1978., and with nitrogen cavitation as a gentle and efficient means of disrupting neutrophils ŽKlempner et al., 1980; Borregaard et al., 1983.. Percoll is an inert, self-generating density medium of low viscosity and ionic strength, ideal characteristics for a separating medium ŽPertoft et al., 1978.. Subcellular fractionation experiments have demonstrated heterogeneity within both peroxidasepositive, primary granules and peroxidase-negative
0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 Ž 9 9 . 0 0 1 7 1 - 4
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granules, the former including defensin-rich and defensin-poor azurophil granules and the latter encompassing specific Žalso termed secondary. and gelatinase Žtertiary. granules, respectively ŽBainton, 1975; Borregaard and Cowland, 1997.. In addition, the highly mobilizable secretory vesicles were identified by their content of latent alkaline phosphatase by Borregaard et al. Ž1987.. These granules and secretory vesicles are mobilized in a sequential order, allowing the gradual activation of neutrophils during adhesion, diapedesis, migration and microbial killingrphagocytosis. Not only the liberation of matrix proteins is important for neutrophil activities, it was also realized through subcellular fractionation experiments, that endomembranes play an important role in furnishing the plasma membrane with important functional proteins during neutrophil activation ŽBorregaard and Cowland, 1997.. These proteins include adhesion proteins, receptors for chemotactic peptides and complement factors and in addition the b-cytochrome component of the NADPH-oxidase, which is thereby assembled and activated in the plasma membrane. This paper deals with subcellular fractionation of nitrogen cavitated, unperturbed or activated, human neutrophils on one-, two-, or three-layer Percoll density gradients. The application of subcellular fractionation in the elucidation of neutrophil structural organization, as alluded to above, is described. Furthermore, the use of subcellular fractionation as an initial step in protein purification schemes and in the investigation of binding of cytosolic proteins to isolated granules is exemplified. Appropriate assays for the identification of subcellular organelles are presented.
2. Materials and methods 2.1. Isolation and stimulation of neutrophils Neutrophils are isolated as described ŽBoyum, 1968.. Except for dextran sedimentation of erythrocytes, the procedure is carried out at 48C. Neutrophils are resuspended at the desired cell concentration and either kept on ice or stimulated with the appropriate stimulus for 15 min, after a 5-min preincubation at 378C. After stimulation, the cells are
pelleted and the supernatant, S 0 , decanted. Exocytosis of granule proteins can be calculated as the amount in the supernatant after stimulation ŽS 0 . divided by the total amount measured in supernatant, in postnuclear supernatant and unbroken cells and nuclei ŽS 0rŽS 0 q S1 q P1 ., see below.. 2.2. Disruption of neutrophils by nitrogen caÕitation Isolated neutrophils, either unperturbed or stimulated, are resuspended in KRG at 0.5 = 10 7 to 1.5 = 10 8 cellrml and left for 5 min on ice in 5 mM diisopropylfluorophosphate ŽAldrich Chemical, Milwaukee, WI, USA., followed by centrifugation and resuspension in the initial volume of disruption buffer Žrelaxation buffer minus EGTA, 100 mM KCl, 3 mM NaCl, 1 mM ATPNa 2 , 3.5 mM MgCl 2 , 10 mM Piperazine N, N X-bis2wethan-sulfonic acidx, pH 7.2. containing 0.5 mM PMSF ŽSigma... ATPNa 2 is added just before use from a 50 mM stock solution Žkept in vials at y208C.. Cells are pressurized under nitrogen for 5 min at 380 psi in a nitrogen bomb ŽParr Instrument, Moline, IL, USA.. The cavitate is then collected dropwise into EGTA, to a final concentration of 1.5 mM Ž150 mlr10 ml of cell suspension from a 100-mM stock of EGTA, pH 7.4.. Nuclei and intact cells are pelleted by centrifugation at 400 = g for 15 min. The postnuclear supernatant ŽS 1 . is cautiously decanted, and the pellet ŽP1 . resuspended in 1 ml of disruption buffer Žfor measurement of marker enzymes, if desired.. 2.3. Density centrifugation on Percoll gradients 2.3.1. Preparation of Percoll gradients Percoll solutions are prepared by mixing Percoll stock solution ŽPharmacia. with water and 1r10 the final volume of a 10-fold concentrated relaxation buffer Ž1000 mM KCl, 30 mM NaCl, 10 mM ATPNa 2 , 35 mM MgCl 2 , 12.5 mM EGTA, 100 mM Piperazine N, N X-bis2wethan-sulfonic acidx, pH 7.2. as indicated in Table 1. The constituents are precooled prior to mixing. When 10-fold concentrated relaxation buffer is prepared, the outweighed contents, minus ATPNa 2 , are resuspended to 4r5 of the final volume, since ATPNa 2 is added just before use from a 50-mM stock solution in 1r5, the volume of 10-fold concentrated relaxation buffer. After mixture
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Table 1 Preparation of Percoll solutions of different densities The numbers in the table shows the volumes of water, 10-fold concentrated relaxation buffer Ž10=. and Percoll stock solution Ždensity of 1.129 grml. needed to obtain a Percoll solution of the density given in the first column of the table. If one desires to prepare a solution of a different density, the necessary volumes can be calculated using the following formula: Vp sV0 Ž D 0 y1.0056.rŽ Dp y1., where Vp is the volume of Percoll stock solution, Dp the density of the Percoll stock solution, V0 the final volume of Percoll solution and 1.056 the density of 10-fold concentrated relaxation buffer.
the a-band, containing azurophil granules, the b-band containing the specific and gelatinase granules, and the g-band containing secretory vesicles and plasma membranes ŽFig. 1.. On the three-layer gradient, as opposed to the two-layer gradient, the b-band is split into the b 1-band, which contains specific granules and the b 2-band, which contains gelatinase granules ŽFig. 1.. The clear cytosol is present on top of the upper band, irrespective of the design of the gradient.
Final density Žgrml.
Final volume Žml.
Percoll stock Žml.
10= Žml.
H 2O Žml.
2.4. Fractionation
1.050 1.065 1.090 1.120
30 30 30 30
10.27 13.19 19.58 26.55
3 3 3 3
16.73 13.81 7.42 0.45
of the Percoll solutions, PMSF is added to a final concentration of 0.5 mM and the pH of the Percoll solutions adjusted to pH 7.0 by addition of 1 M HCl. 2.3.2. One-layer Percoll gradient A cushion of 1 ml Percoll, density 1.12 grml, is gently layered under 20 ml of Percoll, density 1.065 grml, through a pleuracentesis needle. 2.3.3. Two-layer percoll gradient Fourteen milliliter of Percoll, density 1.12 grml, is gently layered under 14 ml of Percoll density 1.050 grml. 2.3.4. Three-layer percoll gradient Nine milliliter of Percoll, density 1.090 grml is gently layered under 9 ml of Percoll, density 1.050 grml. Finally, 9 ml Percoll, density 1.12 grml is layered under the 1.090 grml Percoll solution. Ten milliliter of the postnuclear supernatant ŽS 1 . is applied slowly through a fine needle on top of the desired Percoll gradient, avoiding mixture of the S1 with the upper Percoll solution. The gradient is centrifuged at 37,000 = g for 30 min at 48C in an SS34, fixed angle rotor in a Sorvall RC-5B centrifuge. With the one-layer gradient, this gives rise to two distinct bands containing granules and light membranes, respectively. On the two-layer gradient, three bands are formed, from the bottom designated
Fractions are collected at 48C by aspiration from the bottom of the tube, through a glass capillary tube attached to a polyethylene tube connected to a peristaltic pump and a fraction collector. Alternatively, the distinct bands can be harvested by hand through a Pasteur pipette. 2.5. Marker assays Myeloperoxidase is used as a marker for azurophil granules ŽCramer et al., 1985., lactoferrin for specific granules ŽCramer et al., 1985., gelatinase for gelatinase granules ŽKjeldsen et al., 1993, 1994., and HLA for plasma membranes ŽBjerrum and Borregaard, 1990.. All these assays and the assays for NGAL and the a m -subunit CD11b of the b-integrin Mac-1 are ELISAs, as described ŽSengeløv et al., 1993; Kjeldsen et al., 1994.. Latent alkaline phosphatase is used as a marker for secretory vesicles Ži.e., the difference between alkaline phosphatase
Fig. 1. Photo of the three-layer gradient before Žmiddle. and after centrifugation Žright., and of the two-layer gradient after centrifugation Žleft.. The bands formed after centrifugation are marked.
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measured in the presence and absence of a detergent ŽTriton X-100. ŽBorregaard et al., 1987... 2.6. RemoÕal of Percoll For evaluation of the majority of proteins in subcellular fractions by SDS-PAGE and Western blotting or for example in protein purification schemes, it is necessary to remove Percoll. This can be accomplished by ultracentrifugation at 100,000 = g for 45 min for light membranes and 90 min for granules. After centrifugation, the biological material is visible as a disc above the pelleted Percoll, and can easily be collected by a Pasteur pipette and subsequently resuspended in the desired buffer.
3. Results and discussion 3.1. Nitrogen caÕitation The initial and crucial step in subcellular fractionation is disruption of cells, which is accomplished by nitrogen cavitation as described by Klempner et al. Ž1980. and Borregaard et al. Ž1983.. During this procedure, neutrophil plasma membranes break Žand reseal into plasma membrane vesicles., as the cells leave the nitrogen bomb through a valve, going from a 380-psi to atmospheric pressure. Nitrogen cavitation is a reliable and efficient means of disrupting cells, with approximately 81% to 91% of granule marker proteins recovered in the postnuclear supernatant ŽTable 2, Klempner et al., 1980; Borregaard et al., 1983.. There is only a discrete breakage of nuclei as evaluated by measurement of DNA, with approximately 83% being recovered in the pellet after centrifugation of the cavitate ŽBorregaard et al., 1983.. Although modest, this damage to nuclei may be responsible for the observed lower percentage of azurophil granules Žmyeloperoxidase. and plasma membranes ŽHLA. recovered in the postnuclear supernatant ŽTable 2., since nuclear basic proteins may promote adherence of these structures to nuclei ŽKlempner et al., 1980.. The composition of the disruption buffer mimicking the intracellular milieu and its content of MgCl is believed to stabilize nuclei and minimize the problem of both granule and
Table 2 Percentage of marker protein recovered in postnuclear supernatant after nitrogen cavitation and in subcellular fractions after fractionation of density gradients Isolated neutrophils were disrupted by nitrogen cavitation. The cavitate was centrifuged to pellet unbroken cells and nuclei ŽP1 . and the postnuclear supernatant ŽS1 . decanted. Disruption efficiency was calculated for each marker protein as amount measured in S1 in percentage of total amount in S1 and P1 . Ten milliliter postnuclear supernatant was centrifuged on a three-layer Percoll density gradient and fractionated into 37 fractions of 1 ml each. Recovery is calculated as the total content of marker protein in subcellular fractions expressed in percentage of the content in the postnuclear supernatant applied to subcellular fractionation. Numbers are average of 7 to 12 experiments, with the SD given in parenthesis. Marker protein
Assay type
Amount in S1 after disruption Ž%.
Recovery in fractions Ž%.
Myeloperoxidase Lactoferrin Vitamin B12 BP
Elisa Elisa Functional assay Elisa Elisa Functional assay Elisa
83.7 Ž5.3. 90.0 Ž3.2. 92.8 Ž2.3.
99.7 Ž8.5. 102.1 Ž10.6. 93.7 Ž8.9.
90.9 Ž3.5. 89.3 Ž5.1. 90.9 Ž3.8.
91.6 Ž14.8. 95.8 Ž9.6. 97.2 Ž13.5.
80.5 Ž8.9.
82.9 Ž11.1.
Gelatinase Albumin Latent AP HLA
plasma membrane adherence to nuclei and aggregation between different granule and vesicle subsets, thus allowing a better resolution of organelles after density gradient centrifugation. Another important aspect of neutrophil disruption is the preservation of granule integrity, thus avoiding the liberation of potent neutrophil proteases, which may interfere with subsequent analysis of protease sensitive proteins in subcellular fractions Ževen in the presence of the protease inhibitors diisopropylfluorophosphate and phenylmethylsulfonylflouride, which are always included during neutrophil disruption.. When measuring granule proteins in cytosolic fractions after density gradient centrifugation of nitrogen cavitated neutrophils, less than 0.5% of the measured proteins Žmyeloperoxidase Ž0.4%., lactoferrin Ž0.1%., and gelatinase Ž0.5%., average of seven experiments. can be measured in the cytosol ŽKjeldsen et al., 1994., demonstrating that nitrogen cavitation leaves granules largely intact. This is in contrast to disruption by dounce homogenization, where considerable amounts of granule proteins are present in the cytosolic frac-
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tions after fractionation ŽBretz and Baggiolini, 1974; Dewald et al., 1982; Mollinedo and Schneider, 1984.. Due to this problem, the low efficiency, and the need to expose neutrophils to prolonged, hypotonic shock prior to disruption, dounce homogenization of neutrophils may be considered obsolete. 3.2. Percoll Õersus sucrose as a separating medium Sucrose has been widely used over the years as a separating medium in subcellular fractionation. Its high tonicity makes the medium very viscous and leads to shrinkage of the subcellular organelles. This results in alteration of their density and in lack of granule membrane integrity ŽBorregaard et al., 1983.. Percoll has several advantages over sucrose. Its viscosity is very low and the gradient therefore not easily disturbed ŽPertoft et al., 1978.. Besides, the tonicity of Percoll itself is negligible and can be adjusted to isotonicity by addition of buffer, thus avoiding shrinkage of organelles during fractionation ŽPertoft et al., 1978; Borregaard et al., 1983.. Furthermore, Percoll is a self-generating density medium Žin contrast to sucrose., since the Percoll particles sediment during centrifugation, resulting in a continuously increasing density towards the bottom of the tube. Gradients that contain two or more solutions of Percoll of different densities will smooth out during centrifugation, as opposed to discontinuous sucrose gradients. Finally, Percoll allows fast sedimentation of organelles to their isopycnic density with centrifugation times of 30 min or less. 3.3. Assays for marker proteins In our laboratory we prefer immunologically based assays ŽELISAs. for marker proteins of neutrophil subcellular structures, since these assays may be less sensitive to neutrophil proteases ŽKjeldsen et al., 1992.. Furthermore, we have never experienced any interference of Percoll with an ELISA Žunpublished observation., and Percoll is therefore not removed from fractions prior to measurement. Myeloperoxidase is used as a marker for azurophil granules ŽCramer et al., 1985., lactoferrin for specific granules ŽCramer et al., 1985., gelatinase for gelatinase granules Žsome gelatinase is located in specific granules as described below., latent alkaline phosphatase
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ŽBorregaard et al., 1987. or albumin for secretory vesicles ŽBorregaard et al., 1992b., and HLA for plasma membranes ŽBjerrum and Borregaard, 1990. ŽTable 2.. Alternatively, specific granules can be identified by vitamin B 12 binding protein ŽKane and Peters, 1975.. Both alkaline phosphatase and vitamin B 12 binding protein are measured by functional assays, which are unaffected by Percoll. Since most alkaline phosphatase is located intracellularly in secretory vesicles in the resting neutrophil, alkaline phosphatase is not a specific plasma membrane marker, as previously thought. Galactosyl transferase activity can be used as a marker for the Golgi apparatus and was recently identified mainly in the light membrane region of neutrophils ŽMorgan et al., 1997.. The reliability of an assay can be evaluated by calculating the recovery of the protein measured in the subcellular fractions compared to the amount measured in the postnuclear supernatant loaded onto the gradient. Table 2 shows the recoveries for a variety of proteins, including the marker proteins used. The recovery is between 83% and 105%. A recovery invariably exceeding 100% may indicate problems with proteasesrinhibitors of the assays, problems that are overcome by segregation of the proteaserinhibitor from the measured protein after subcellular fractionation. This is especially important to bear in mind with functional assays and has been observed, when gelatinase was measured by a gelatinolytic assay using tritiated gelatine as a substrate ŽDewald et al., 1982.. A low recovery has been observed for the azurophil granule marker b-glucoronidase Žaverage recovery 54%. ŽBorregaard et al., 1983.. This observation was attributed to inhibition of the spectrophotometric assay of b-glucoronidase by Percoll, especially in the densest fractions, which are richest in Percoll. 3.4. Subcellular fractionation on two-layer Percoll gradient Following disruption and centrifugation, the postnuclear supernatant is layered on top of the Percoll gradient and high speed centrifuged at 37,000 = g for 30 min. The first combined use of nitrogen cavitation and Percoll density gradient centrifugation was introduced by Borregaard et al. Ž1983. and
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Fig. 2. Subcellular fractionation of nitrogen-cavitated neutrophils on one-, two-, and three-layer Percoll gradient. Ten milliliter of postnuclear supernatant was applied on top of a one-layer Percoll gradient Ž1.065 grml, panel 1., a two-layer Percoll gradient Ž1.05r1.12 grml, panel 2., or a three-layer Percoll gradient Ž1.05r1.09r1.12 grml, panels 3 and 4.. After centrifugation, the gradients were fractionated by aspiration from the bottom of the tube into the number of fractions given on the x-axis. Each fraction was assayed for the marker enzymes shown, and on the three-layer gradient also for NGAL. The content in each fraction is calculated in percentage of the total content measured in all fractions.
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137
Fig. 2 Žcontinued..
involved a two-layer Percoll gradient. After centrifugation, three distinct bands are observed from the bottom designated the a-band, the b-band, and the g-band with the clear cytosol on top ŽFig. 1.. The a-band contains the majority of the azurophil, peroxidase-positive granules, identified by the marker myeloperoxidase, well separated from peroxidasenegative granules represented by the overlapping profiles of the markers lactoferrin and gelatinase, both contained within the b-band ŽFig. 2.. There is a minor myeloperoxidase peak within the b-band. The g-band contains the plasma membranes identified by HLA class 1 and the secretory vesicles, identified by latent alkaline phosphatase, i.e., activity only measurable in the presence of a detergent ŽFig. 2.. Above the g-band is the cytosol, identified by lactate dehydrogenase Žnot shown.. There is a considerable overlap between the distribution profiles of the latent alkaline phosphatase and HLA, although in most cases alkaline phosphatase peaks one fraction below HLA. Furthermore, it is clearly visible, that latent alkaline phosphatase displays a shoulder to the left of the peak that extends somewhat into the b-band region of the gradient, thereby overlapping with markers of peroxidase-negative granules. In the search for other constituents of secretory vesicles, distribution profiles similar to latent alkaline phosphatase were found for complement receptor 1, tetranectin and albumin, the latter two indicating the endocytic origin of secretory vesicles ŽBorregaard et
al., 1990, 1992b; Sengeløv et al., 1994.. It should be stressed, that isolation of neutrophils at 48C throughout the procedure is absolutely crucial in the preservation of the easily mobilizable secretory vesicles Žsee below.. 3.5. Subcellular fractionation on one-layer Percoll gradient In order to obtain a more ‘‘clean’’ preparation of light membranes with minimal contamination of granules, a one-layer gradient with a Percoll density of 1.065 grml can be used ŽSengeløv et al., 1994.. A 1-ml cusion of Percoll with a density of 1.12 grml is placed in the bottom of the tube. With this gradient, most granule markers are recovered in the first three fractions, well separated from the light membranes identified by latent alkaline phosphatase and HLA ŽFig. 2.. Although this gradient is useful in the preparation of ‘‘clean’’ light membranes devoid of granules, the difference in distribution profiles of HLA and latent alkaline phosphatase is less pronounced than on the two-layer gradient. The similar density of plasma membranes and secretory vesicles precludes their complete separation by means of density centrifugation. Instead, the light membranes, as for instance obtained by the one-layer gradient, can be separated by differences in their surface charge by free flow electrophoresis ŽSengeløv et al., 1992, 1994 and the contribution by Dr. Sengeløv in this issue.. Recently, a Percoll based, flotation gradi-
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Fig. 3. Subcellular fractionation of resting, FMLP- and PMA-stimulated neutrophils on three-layer Percoll gradients. Prior to disruption, cells were either kept on ice Žcontrol. or stimulated for 15 min at 378C with FMLP Ž10 nM. or PMA Ž2 mgrml.. After washing and disruption of the cells, the postnuclear supernatants were centrifuged on three-layer Percoll gradients and fractionated as described in Section 2. Fractions were assayed for myeloperoxidase, lactoferrin, gelatinase, latent alkaline phosphatase Žlatent AP., albumin, HLA and CD11b, the a m subunit of Mac-1.
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139
Fig. 3 Žcontinued..
ent has been developed by Dahlgren et al. Ž1995., that allows a more efficient, yet not complete, separation of plasma membranes from secretory vesicles,
than can be obtained by the ‘‘classical’’ Percoll gradients. This can be useful, when free flow electrophoresis is not available.
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Fig. 3 Žcontinued..
3.6. Subcellular fractionation on three-layer Percoll density gradients On the two-layer gradient, a considerable overlap exists in the distribution profiles of lactoferrin and gelatinase within the b-band, as observed in Fig. 2. By introducing a third layer of Percoll of intermediate density Ž1.090 grml., the efficient separating volume of the gradient is improved and the b-band split into a denser b 1-band and a b 2-band ŽFig. 1.. The b 1-band contains the majority of lactoferrin, whereas the b 2-band contains the peak of gelatinase separated from the lactoferrin peak by several fractions ŽFig. 2.. As also observed from Fig. 2, peroxidase-negative granules exist as a continuum of granules rich in lactoferrin and devoid of gelatinase Ždefined as specific granules. to granules enriched in gelatinase but containing very little lactoferrin. Approximately 66% of all peroxidase-negative granules contain both lactoferrin and gelatinase, as demonstrated by double-labeling immunogold electron microscopy ŽKjeldsen et al., 1993., and a complete separation of the two proteins is thus physically impossible. Gelatinase granules are arbitrarily defined as all granules contained in fraction 16 and higher, albeit these granules contain 6% of total lactoferrin. The a- and g-bands are largely unchanged on the three-layer as compared to the twolayer gradient ŽFig. 2., although the ‘‘shoulder’’ of latent alkaline phosphatase Žthe secretory vesicles. does not extend as far into the b-band as is the case on the two-layer gradient.
A three-layer Percoll density gradient Ž1.07r 1.12r1.14 grml, 10 ml of each density. has also been developed by Nitsch et al. Ž1990.. After centrifugation, this gave rise to nine visible bands including three bands enriched in azurophil granule constituents, and four bands mainly containing peroxidase-negative granule proteins. The lightest of the latter four bands was relatively enriched in gelatinase, thus resembling the lactoferrin-poor, gelatinase-rich, peroxidase-negative granules Žgelatinase granules., described above. Isolation of a similar gelatinase-rich peroxidase-negative granule subpopulation has also been obtained on discontinuous sucrose density gradients ŽJones et al., 1990; Graves et al., 1992.. 3.7. Subcellular fractionation of resting and actiÕated neutrophils A drawback of subcellular fractionation is that separation of subcellular structures is based solely on differences in density. This implies that two different structures of equal density will colocalize on the gradient. However, discrimination between different organelles, that band at the same density, can be achieved, if these are mobilized differently upon stimulation of the neutrophils. This can be investigated by comparison of distribution profiles in unperturbed neutrophils and in neutrophils stimulated prior to nitrogen cavitation and density gradient centrifugation. Fig. 3 shows subcellular fractionation of unstimulated neutrophils and neutrophils stimulated with either the bacterial chemotactic peptide FMLP
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or the phorbol-ester PMA. It is observed, that following FMLP-stimulation, an almost complete mobilization of secretory vesicles occurs with parallel disappearance of latent alkaline phosphatase and albumin within the g-band. Hardly any azurophil or specific granules are mobilized by FMLP, whereas approximately 25% of gelatinase is exocytosed, preferentially from the lightest peroxidase-negative granules in fraction 16 and above, i.e., gelatinase granules ŽFig. 3.. When neutrophils are stimulated with PMA, a complete exocytosis of gelatinase and a considerable specific granule mobilization is observed Ž50%– 60%., whereas only around 1r3 of myeloperoxidase is lost from the a-bandrazurophil granules. When evaluating a membrane associated protein, in Fig. 3 exemplified by the a m subunit CD11b of the b 2-integrin Mac-1, the protein translocates to the plasma membrane following stimulation in parallel with its disappearance from granules and secretory vesicles. After stimulation with FMLP there is an increase in CD11b in the plasma membrane, recruited in part from gelatinase granules in fractions 16 and above, and in part from secretory vesicles within the g-band, where approximately 15% to 20% of total Mac-1 is located in unperturbed neutrophils ŽSengeløv et al., 1993; Kjeldsen et al., 1994.. If one focuses only on the total content of Mac-1 within the g-band before and after stimulation, one overlooks this translocation from secretory vesicles to plasma membrane. Only after stimulation with PMA, there is a parallel loss of CD11b and lactoferrin from the b 1-region, indicating translocation of CD11b from specific granules to the plasma membrane. 3.8. Strategy in inÕestigation of the subcellular localization of a constituent of neutrophils When facing the task of determining the subcellular localization of a constituent of human neutrophils, an initial approach would be to evaluate its presence in peroxidase-positive and -negative granules and in light membranes separated by fractionation on the two-layer Percoll gradient. If the protein is mainly located in peroxidase-negative granules, its distribution between specific and gelatinase granules and the mobilization from these subsets Žfollowing FMLP- and PMA-stimulation. should be investigated on the three-layer gradient. If the protein of interest is detected within the g-band, its distribution profile
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should be compared to that of markers for secretory vesicles and plasma membranes in both unperturbed and FMLP-activated neutrophils. These findings can be corroborated by fractionation on the flotation Percoll gradient described above ŽDahlgren et al., 1995., or by subsequent free flow electrophoresis of the g-band Žas described by Dr. Sengeløv in this issue.. When investigating the subcellular localization of a constituent of the neutrophil, it should ideally be measured in each fraction by a quantitative assay, since this allows the comparison of the distribution profile of the protein with the profile of well established and specific markers for subcellular organelles. Exact matching profiles strongly indicate a common subcellular localization as for example seen with the specific granule marker lactoferrin and NGAL ŽFig. 2.. If a quantitative assay is not available, the presence of the protein in subcellular organelles can be evaluated by Western blotting. In this case, Percoll must be removed from fractions by ultracentrifugation Žsee Section 2. prior to SDSPAGE, unless the abundance of the protein under investigation allows 20-fold or more dilution of the Percoll containing subcellular fraction, in which case Percoll will not affect the quality of gel electrophoresis. Findings obtained by subcellular fractionation should preferentially be validated by double labeling immunogold electron microscopy given that an antibody against the protein is available. Electron microscopy can be performed either on intact neutrophils or on subcellular fractions, as described herein by Dr. Bainton. 3.9. Use of subcellular fractionation in isolation of granule proteins Besides being an invaluable tool in the investigation of the subcellular localization of neutrophil constituents, subcellular fractionation has also been used in the isolation of granule proteins. For preparative purposes, the number of cells loaded on each gradient can be increased to about 1.5 = 10 9 , without affecting the resolution of subcellular organelles on the gradient. It is an efficient initial step in purification schemes resulting in several fold increases in the specific activity of the purified protein. The band that contains the protein can be harvested from the
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gradients by hand by aspiration through a pasteur pipette. After removal of Percoll by ultracentrifugation, the proteins can be solubilized by an appropriate detergent, or granules can be repetitively freeze thawed, resulting in liberation of granule matrix proteins to the supernatant after pelleting of the membranes by centrifugation. Subcellular fractionation has been used as an initial step in the purification of proteinase 3 ŽStummann and Wiik, 1997., the defensin HNP4 ŽWilde et al., 1989., myeloperoxidase ŽBorregaard, unpublished., and in purification from the specific granules of the cathelicidin, hCAP18 ŽSørensen et al., 1999.. hCAP-18 is especially sensitive to degradation by proteases from azurophil granules, and subcellular fractionation was therefore an important initial step in the preservation of intact hCAP-18 during the purification procedure. 3.10. Subcellular fractionation in the inÕestigation of in Õitro translocation of cytosolic proteins to isolated granule and membrane fractions Subcellular fractionation has been used to obtain isolated azurophil and specific granules and light membranes for use in the investigation of in vitro translocation of cytosolic proteins to the respective membranes. This approach was used in the identification of a 28-kDa protein, later identified as grancalcin, which reversibly bound to secretory vesicles and plasma membranes in the presence of calcium ŽBorregaard et al., 1992a.. In addition, several annexins were also found to bind to isolated granule and vesicle membranes in the presence of calcium, and their differential calcium-dependency and affinity for granule subsets was delineated by the same approach ŽBorregaard et al., 1992a; Sjolin et al., 1994, 1997.. Also, the in vitro translocation of protein kinase C to isolated granules and light membranes was investigated in the presence of PMA ŽChristiansen and Borregaard, 1989..
4. Conclusion Subcellular fractionation of nitrogen cavitated neutrophils on Percoll density gradients is an easy, fast and very reproducible technique. The combined use of subcellular fractionation and immunogold
electron microscopy has contributed considerably to the understanding of neutrophil structural organization including the dynamics of exocytosis of granules and secretory vesicles. Subcellular fractionation will also be beyond doubt an invaluable tool in the future study of neutrophil cell biology.
Acknowledgements This work was supported by The Alfred Benzon Fund, The Danish Cancer Society, The Danish Medical Research Council, The Lundbeck Fund, Emil C. Hertz’s Fund, The Novo Fund, Amalie Jørgensen’s Fund, Brøchner-Mortensen’s Fund, Anders Hasselbalch’s Fund, Ane Kathrine Plesner’s Fund.
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