American journal ofPathology, Vol. 144, No. 6, June 1994 Copynght © American Societyfor Investigative Pathology Granulophysin Is Located in the Membrane of Azurophilic Granules in Human Neutrophils and Mobilizes to the Plasma Membrane Following Cell Stimulation Bonnie P. Cham,* Jon M. Gerrard,* and teins, and they are peroxidase positive.3'5 Azurophilic Dorothy F. Baintont granules are considered to be classic primary lyso- From the Department ofPediatrics and the Manitoba somes in that they contain acid hydrolases that have Institute of Cell Biology,* University ofManitoba, not yet entered into a digestive event. They are Winnipeg, Manitoba, Canada; and the Department of mobilized to the surface of the neutrophil, and their Pathology,t University of California School ofMedicine, San contents discharged by formyl-methionyl-leucyl- Francisco, California phenylalanine (FMLP) following cytochalasin B stimu- lation, but this translocation occurs only to a minimal extent when the neutrophils are stimulated by phorbol myristate acetate (PMA) or FMLP in the absence of Granulophysin, a protein described in platelet cytochalasin B.6 Specific (secondary) granules, on dense granule membranes, has been shown to be the other hand, appear later in development and are similar or identical to CD63, a lysosomal mem- smaller, but more numerous, than azurophilic gran- brane protein. We have previously shown granu- ules. They are peroxidase-negative and contain lophysin to be present in neutrophils using lactoferrin and many other proteins. Considerable immunofluorescence. We now localize granulo- heterogeneity exists within the group of peroxidase- physin to the neutrophil azurophilic granules by negative granules with regard to their content and fine structural immunocytochemistry. Granulo- mobilization. In addition, the neutrophil also contains physin expression on the surface membrane of gelatinase granules and secretory vesicles as re- the neutrophil is increasedfollowing stimulation viewed by Borregaard et al.7 Many of these ofthe ceUs, demonstrated byflow cytometry and nonperoxidase-containing granules are released fine structural immunocytochemistry. A similar early in the inflammatory response and likely allow for pattern is shown for an anti-CD63 antibody. In- cell diapedesis and adhesion via their membrane cubation of activated neutrophils with D545, a constituents of C3bi and FMLP receptors. They are monoclonal antibody to granulophysin, blocks translocated to the cell surface in vitro by low con- subsequent binding ofanti-CD63 antibodies to the PMA as as low concentrations ceU surface, and anti-CD63 antibodies prevent centrations of well by subsequent binding ofD545 as assessed byflow of FMLP even in the absence of cytochalasin B.6 As cytometry and immunoblotting. Our results sup- a result, it has been speculated that specific granules, port the homology of CD63 and granulophysin and other compartments, mobilize easily and early in previously demonstrated inplatelets and confirm the course of inflammation, allowing for chemotaxis, CD63 as an activation marker in neutrophils whereas azurophilic granules predominantly form in- and the first azurophilic granule membrane tracellular phagolysosomes and participate in cell marker of neutrophils. (Am J Pathol 1994, 144: killing.6 1369-1380) Granulophysin is a protein originally described as present in platelet-dense granule membranes,9 Various subsets of neutrophil granules have been Supported by Children's Hospital Research Foundation, Winnipeg, identified and characterized by physical and bio- and NIH grant number DK 10486. chemical properties.1" Azurophilic (primary) gran- Accepted for publication February 4, 1994. ules appear earliest in maturation and are the largest Address reprint requests to Dr. Bonnie Cham, ON 141 Manitoba granules. Their contents include acid hydrolases, mi- Cancer Treatment and Research Foundation, 100 Olivia Street, crobicidal enzymes, proteases, and cationic pro- Winnipeg, Manitoba, Canada R3E 0V9. 1369 1370 Cham et al A/P Julne 1994, Vol. 144, No. 6 which has subsequently been shown to be similar or granulophysin to the azurophilic granules by fine identical to CD63, a platelet lysosomal protein.10 structural immunocytochemistry.12 D545 was found Using a monoclonal antibody, granulophysin was to co-localize with myeloperoxidase in the azurophilic shown to be present in a granular pattern in many cell granules. In addition, D545 was separate in location types, including endocrine, exocrine, and neuronal from lactoferrin, a content marker for specific gran- tissues, endothelial cells and certain leukocytes.11 ules. Binding of D545 to the surface of the neutrophil We have previously shown this protein to be present is increased following stimulation of the cells by cyto- in neutrophils using immunofluorescent techniques.9 chalasin B and FMLP, as demonstrated by flow cy- We now extend our previous observations localizing tometry and fine structural immunocytochemistry. A Figure 1. Frozen-thin sectioni of normal resting PMN labeled uitb D545 as the prinarl antibody anid GAM-10 nin gold as the secondary anitibody to demonstrate the presence of D545 along the membranes oJ large extracted grannules, characteristic of azurophilic grannIes (ag). Specific gr-anl- ides (sg) were lint labeled (AV, Iticlenis) ( X 72,000). Localization of Granulophysin in Neutrophils 1371 AJPJune 1994, Vol. 144, No. 6 similar pattern is shown for an anti-CD63 antibody in terized elsewhere.9 Anti-CD63 antibody used for activated neutrophils, in agreement with a previous most studies was purchased from Amac Inc. (West- report.13 Incubation of activated neutrophils with brook, ME). An additional anti-CD63 antibody (HS56) D545 blocks subsequent binding of anti-CD63 anti- and fluorescein isothiocyanate (FITC) anti-CD63 bodies to the surface of the neutrophil, and anti-CD63 were kindly provided by Dr. James Hildreth (The antibodies prevent subsequent binding of D545. Johns Hopkins University, Baltimore, MD). Similarly, incubation of neutrophil homogenates with anti-CD63 diminish binding of D545 in immunoblot- ting studies. Our results are in agreement with the Neutrophil Isolation homology of CD63 and granulophysin previously demonstrated in platelets10 and extend the observa- After obtaining consent, blood was drawn from vol- tion that CD63 is an activation marker in neutrophils13 unteer adult donors into syringes containing ACD 1.5 and the first azurophilic granule membrane marker of ml/l0 ml total volume. It was then mixed with 5% neutrophils. dextran in phosphate-buffered saline and allowed to sediment for 30 minutes. The leukocyte-rich plasma was layered onto Ficoll-Paque (Pharmacia) and cen- Materials and Methods trifuged at 400 x g for 30 minutes. Residual eryth- Monoclonal Antibodies rocytes in the pellet were lysed with 0.87% ammo- nium chloride, and the neutrophils were then washed The monoclonal antibody against granulophysin with Hanks' balanced salt solution (HBSS). The cells used in the present study (D545) has been charac- were counted by Coulter Counter and resuspended Figure 2. Dual staining showing myeloperoxidase (with the small gold panicles, GAR-O) and D545 (with the large gold particles, GAM-10) co- existing in the same large azturophilic granules (ag) (x 70,000). 1372 Cham et al A/PJune 1994, Vol. 144, No. 6 Figure 3. Dual staining showing segregation of D545 (10-nm gold particles) and lactoferrin antibodies (5-nm gold particles) in distinct granule subsets; (X 70,000). A Figure 4. Representative data from a single flow cytometry experiment showing binding of D545 to the plasma membrane of neutrophils that have been stimulated with the following stimuli. The vertical line separates negative and positive fluorescent populations. Fluorescence intensity is displayed on a 3-decade loganithmic . T . scale. A: unstimulated neutrophils ( 17.77% posi- tive, mean channelflourescence [MCFI, 51.4) B: c! 5 minutes of FMLP ic-6 mol/L ( 79.6% positive, _~~~~~~~~~1 D MCF, 66.7). C: A23187 5 jmo'L for 5 minutes (80.5% positive, MCF, 85.0) D: PMA 10-9 mol/L for 5 minutes (49.6% positive, MCF, 52.9). (Note: results of all flow cytometry experiments are expressed quantitatively as the percentage of cells that are positive for fluorescence, and the MCF, is a measure of how intense thefluo- rescence is within the positive population). to an appropriate cell concentration. Where indi- free HBSS. The samples were centrifuged in an Ep- cated, CaCI2 was then added for a final concentra- pendorf micro-centrifuge and the supernatants tion of 1 mmol/L. stored at -20 C for further assays. Stimulation of Neutrophils Immunofluorescence Neutrophils were prewarmed in a 37-C waterbath for Immunofluorescence was performed essentially as 5 minutes. Those samples that were subsequently go- previously described.9 Neutrophil pellets were sus- ing to be stimulated with FMLP were incubated with pended in primary antibody (D545) at 20 pg/ml in cytochalasin B (Sigma, St. Louis, MO) at a concen- HBSS with 0.1% bovine serum albumin (BSA). This tration of 5 pg/mI. FMLP, PMA, or A23187 (Sigma) mixture was incubated on ice for 30 minutes. Samples were then added at the indicated concentrations. The were then centrifuge washed three times with HBSS/ cells were incubated in a shaking water bath at 37 C 0.1% BSA. The second antibody, biotinylated goat for the time period indicated, and the reaction was anti-mouse, was applied in the HBSS/0. 1% BSA for 30 stopped by dilution with 600 pl of ice-cold, calcium- minutes.