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 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 ,* University ofManitoba, not yet entered into a digestive event. They are Winnipeg, Manitoba, Canada; and the Department of mobilized to the surface of the , 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 cytochalasin B.6 Specific (secondary) granules, on dense 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 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 , allowing for chemotaxis, CD63 as an activation marker in neutrophils whereas azurophilic granules predominantly form in- and the first 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- 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 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. The neutrophils were again washed three Localization of Granulophysin in Neutrophils 1373 '/P june 1994. I !. -44. A\n. 6

times. Strepavidin-fluorescein was then added to the A neutrophils, which were incubated for 30 minutes. The cells were washed three times in HBSS with no BSA. Cells were fixed with 2% paraformaldehyde/ 0.1% glutaraldehyde in 0.1 mol/L cacodylate buffer for 1 hour on ice and mounted onto slides in glycerol Tris-buffered saline (TBS) for evaluation using an im- munofluorescence microscope. Those samples requiring permeabilization were fixed in 2% paraformaldehyde in 0.1 mol/L cacody- late buffer for 1 hour on ice. Following this, unreacted aldehyde was neutralized with three rinses of TBS. The neutrophils were permeabilized for 3 minutes with 0.1 % Triton X-1 00 and centrifuge-washed three times with TBS/0.1 % BSA. After this step, subsequent stain- ing procedures were performed as outlined above, using TBS/0.1 % BSA as the buffer rather than HBSS/ 0.1% BSA. Negative controls for each experiment consisted of the above procedure with omission of the primary antibody.

Flow Cytometry

Samples were prepared for flow cytometry as above . . or utilizing directly FITC-conjugated antibodies as we have described previously for .14 The neu- trophils were resuspended in HBSS/0.1% BSA and incubated for 1 hour on ice, in the dark, with D545 (80 ucg/ml) or anti-CD63 (20 ucg/ml) conjugated directly to FITC. Parallel samples were incubated with unla- beled D545 or anti-CD63 in excess before fluorescent labeling to assess nonspecific binding. In addition, experiments were performed in which the neutrophils

Figure 5. R rc/s(i n i(ut,i(, mtr (IC il)/) i/7oit (-_lt01 (/li -iC were incubated with unlabeled anti-CD63 or D545 in IncliWlt dcm ons71.ra0in, bind1)(;i1 OJ D5I i al var{iolls' ti}71(/O,t8/lo(il excess and then incubated with D545-FITC (following FXLslliXti}l{/Sio0i A: zlm.,aiidalllcd(9; nciaropwhll7.s (v) ", v tj9ili W. B: /oiVwlo n, 0) s(cond/s ./ milltl/uiuoil 8.6() posiil MC,l / anti-CD63) or anti-CD63-FITC (following D545). 1 0 6 9i))llowin-) C 5 minulot o .stimidlion ), Q) itivc 1(lJ Samples were fixed with 1 % formaldehyde and ana- _2 _.6,) lyzed using an EPICS Model 753 flow cytometer

Table 1. D 149 lPii(i)7 -( liici(t,nmi(Ilas/c Relcase.(a)1o1( acI(?1C/rrill Release/ .AI/'r .\i ropl)0)1il S1i)illltioii D545 binding f-glucuronidase Lactoferriin (% increase release release Stimulus over baseline) (% of cell content) (% of cell content) Nonstimulated 6.3% 0% 899% FMLP 10-6 mol/L 1 minute* 41 5% 35 2% 9 5% 5 minutes* 35.2% 21.0% 8 0% PMA 10-9 mol/L 5 minutes 7.1% 800% 81 7% PMA 10-8 mol/L 5 minutes 411% 5 0% 78 6% A23187 10-6 mol/L 5 minutes 9.7% 7 2% 28 6% A23187 10-5 mol/L 5 minutes 43.6% 25 8% 64 8%

This is the data from a representative experiment. Experiments were performed at least three times. i FMLP was added following 5-minute pre-incubation with cytochalasin B (5 ucg/ml). 1374 Cham et al AJPJutne 1994, Vol. 144, No. 6

Figure 6. Neutrophils stimulated with FMLP in the presence of cytochalasin B, showing redistribution ofgranulophysin to the plasma membrane (pm), N, tnucleus, (x30,000). Scale bar represents 0.1 umolL.

(Coulter Electronics, Hialeah, FL) equipped with an Lactoferrin Assay argon ion laser (500 mW, 488 nm). Fluorescence was detected at 525 nm. Forward and 90-degree light Lactoferrin was measured using a competitive scatter measurements were used to establish gates enzyme-linked immunosorbent assay.16 Nitrocellu- for intact, viable neutrophils. Single parameter, 255- lose plates were coated with a standard amount of channel, log integral green fluorescent histograms lactoferrin (Sigma) and then supernatant, cell soni- were obtained, each based on 1 x 104 gated events. cate, or standard amount of lactoferrin was added along with rabbit anti-lactoferrin antibody (Sigma) and incubated for 2 hours. The plate was washed, ,B-Glucuronidase Assay and goat anti-rabbit antibody conjugated with alka- line phosphatase was then added and incubated for f3-glucuronidase activity was measured in the super- 2 hours. Following plate washing, alkaline phos- natants using 4-methylumbelliferone-j3-glucuronide phatase substrate was added, and the plates were cleavage.15 The results are expressed as a percent developed in a 37 C incubator. Absorbence at 405 nm of the activity in cell sonicates. was read in an enzyme-linked immunosorbent assay Localization of Granulophysin in Neutrophils 1375 AJPJuine 1994, Vol. 144, No. 6

JL I I A ~~~~~~~~~A * ...... ~~~~~~~~~ I

* , I I I -- ,-1,I . , , ,

o f.m ...,,,., .~~~~~~~~

.,, ,,I ,,, ......

Figure 8. Representative data from a single flow cytometry experi- ment demonstrating: A: anti-CD63 binding in unstimuilated neutro- phils (9.3% positive, MCF, 71.4). B: anti-CD63 after 5 minutes of FMLP (48.5%, MCGF 96.3) C: anti-CD63 binding after 5 minutes of FMLP stimulation in the presence of unconjugated HS56 (2.8% posi- tive, MCF, 58.7). D: anti-CD63 binding after 5 minutes of FMLP stimulation in the presence of uncoiqugated D545 (2.900, MCF, 58.5). E: D545 binding in unstimulated neutrophils (9.5% positive, MCF, 61.7). F: D545 binding after 5 minutes of FMLP (74.2% posi- tive, MC, 101.o0). G: D545 binding after 5 minutes ofFMLP stimtula- tion in the presence of unconjugated D545 (15.2% positive, MCGF 65. 8). Anid H: D545 binding after 5 minutes ofFMLP stimiulation in the prcesence of unlconijuigated anti-C'D63 (4.5% positive, MCF, 74.1). (All unconjugated antibodies are present in super sattiratinlg concentra- tion.s)

Figure 7. Representative data from a single flouw cytometry exper!- ment demonstrating binding of anti-CD663 at varous time points fol- lowing FMLP stimulation. A: unstimulated neutrophils (5.0% positive, MCF, 54.2) B: f]llouwing 30 seconds of stimulation (35_50% positive, GAR-5 to detect rabbit polyclonal antibodies. Con- iMGCF, 76.0) C: Jbllowing 5 minutces of stimuilation (52.5% positive, trols consisted of replacement of the primary antibody MCF, 76.4). with normal mouse or rabbit serum respectively. well reader, and the unknown concentrations were then determined by interpolating from a standard Western Blotting curve. Results are expressed as a percentage of a cell sonicate. Western blots were performed as previously de- scribed.9 The protein samples, in a buffer of 2.5% Immunogold Electron Microscopy glycerol, 5% sodium dodecyl sulfate, 125 mmol/L Tris HCI (pH 6.8), were incubated at 37 C for 1 hour and Resting neutrophils or activated cells were fixed in separated by electrophoresis on 10% polyacrylamide equal volume of 2% paraformaldehyde/0.05% glut- gel with a 4% stacking gel according to Laemmli.18 araldehyde in 0.1 mol/L phosphate buffer for 1 hour Lanes were loaded with 30 ucg of protein. Proteins at 4 C. They were then washed three times in 0.1 mol/L were transferred to nitrocellulose at 1 OOV for 1 hour at phosphate buffer with 3% sucrose and processed for room temperature. The nitrocellulose was blocked frozen thin sectioning. Immunocytochemistry was overnight using 10% nonfat powdered milk, washed performed as previously described. 17 The mono- with 0.1% Tween/TBS and incubated with 10 ucg/ml clonal antibody D545 was used at a dilution 1 :100 and of the monoclonal antibody of interest. After washing the polyclonal antibodies to either myeloperoxidase with TweenlTBS, the nitrocellulose was incubated (CalBiochem Corp., San Diego, CA) or lactoferrin with peroxidase-labeled goat anti-mouse immuno- (Davo Corp., Carpinteria, CA) were used at a dilution globulin G (Sigma, 1:3,000 dilution) for 30 minutes at of 1:500. The second step consisted of adding RT. The reaction was developed using an enhanced GAM-10 to detect mouse monoclonal antibody or chemiluminescence. Blocking experiments were per- 1376 Cham et al AJPJune 1994, Vol. 144, No. 6

kDa

kDa

106 - -106 s0 - -80

-50 50-

33- -33 24- -28 Figure 9. Western blots of neutrophil (lanes B, C, F, and G), and platelet (lanes A, D, E and H) homogenates. Lanes A and B: D545; lanes -18 C and D: anti CD63; lanes E and F: anti- CD63 followced by D545 conjugated to peroxi- dase; lanes G and H: D545 conjugated to per- ... E. .: ...... oxidase. A (I C D E F a H formed by first incubating with an anti-CD63 antibody, and then analyzed by flow cytometry for binding of subsequently incubating with peroxidase-labeled D545 to the plasma membrane. Flow cytometry dem- D545, and developing using enhanced chemilumi- onstrated maximal surface expression of D545 fol- nescence. lowing stimulation with cytochalasin B and FMLP 10-6 mol/L in the presence of calcium (Figure 4). Binding of D545 was increased to a minimal degree by PMA Results at doses that did not disrupt the cells. The calcium Our previous studies have demonstrated punctate in- ionophore, A23187, also stimulated translocation of tracellular staining suggestive of a granular location the protein, supporting an effect of calcium (Figure 4). of D545 in neutrophils using immunofluorescence.9 Surface expression of granulophysin was found to be Immunogold electron microscopy demonstrates lo- an early event with significant change at 30 seconds, calization of granulophysin in the membranes of large reaching maximal stimulation at 1 to 5 minutes fol- extracted azurophilic granules (Figure 1). No signifi- lowing stimulation (Figure 5). Expression at the cell cant amount of labeling was seen in specific granules surface was generally stable between 1 to 5 minutes or on the plasma membrane. Double labeling of rest- after stimulation. ing neutrophils with D545 and polyclonal serum to The supernatant of neutrophils stimulated for flow myeloperoxidase, a marker for azurophilic granules, cytometry was saved and analyzed for the presence showed co-localization to the same large, clear gran- of ,B-glucuronidase (a marker for azurophilic gran- ules (Figure 2). Whereas only D545 appeared on the ules) and lactoferrin (a marker for specific granules). granule membrane, myeloperoxidase also appeared ,B-glucuronidase release was seen to parallel trans- in the matrix of the granules. Furthermore, dual stain- location of granulophysin to the plasma membrane. ing with anti-lactoferrin antibodies, a marker for spe- Following FMLP stimulation, 35.2% of the cell con- cific granules, showed that lactoferrin and granulo- tents were released; following PMA stimulation, 5 to physin did not co-localize (Figure 3). Flow cytometry 8% were released. With maximal A23187 stimulation, performed on resting neutrophils demonstrated only 25.8% of the cell contents were released (Table 1). minimal surface expression of granulophysin, consis- Lactoferrin release, however, occurred maximally tent with the low level seen by immunocytochemistry with stimulation by PMA 10-9 mol/L, which caused (Figure 4). release of 81.7% of cellular lactoferrin. Secretion of Neutrophils were incubated with cytochalasin B, lactoferrin did not coincide with maximal granulophy- stimulated with FMLP or stimulated by PMA alone, sin surface expression (Table 1). These results further Localization of Granulophysin in Neutrophils 1377 AJPJune 1994, Vol. 144, No. 6

Figure 10. Frozen-tbin section with immunogold cvtochemicalpreparation ofa normal human . The antigen appears on the membrane of most of the crystalloid-containing eosinopbil granules (arrow's) (x 36, 000). support the localization of granulophysin to the azuro- formed in which the stimulated cells were first ex- philic granule membrane. posed to D545 (in excess) and then to anti-CD63 an- Utilizing frozen thin section immuno-electron mi- tibodies conjugated to FITC. This showed nearly croscopy, stimulated neutrophils demonstrated trans- complete blocking of anti-CD63 antibody binding location of granulophysin to the plasma membrane in (Figure 8). Similarly, pre-exposure to anti-CD63 anti- a portion of the neutrophils following incubation with bodies blocked to a significant extent the subsequent cytochalasin B and 2.5 minutes of FMLP stimulation binding of D545 (Figure 8). In parallel experiments, (Figure 6). The redistribution of label to the plasma antibody to lactoferrin was found to mobilize to the cell membrane, with some cells very strongly stained and surface following stimulation by FMLP, but did not others not stained, is similar to the distribution dem- block the subsequent binding of either anti-CD63 or onstrated via flow cytometry, ie, a heterogenous D545. population. Both D545 and anti-CD63 recognize a similar pro- Further studies were undertaken to examine the tein of about 47 kd on Western blots of neutrophil pro- relationship of the proteins recognized by anti-CD63 teins. Staining with anti-CD63 in neutrophils was antibodies and D545. Flow cytometry demonstrated slightly lighter than staining with D545. However, the a similar pattern of translocation of CD63 to that of addition of unlabeled CD63 before peroxidase- D545 following stimulation with FMLP, PMA, and labeled D545 blocked the staining using D545, sug- A23187 (Figure 7). Additional experiments were per- gesting the two antibodies recognized the same or 1378 Cham et al AJPJune 1994, Vol. 144, No. 6

Figure 11. Frozen-thin section with immunogold preparation of a portion of a normal human monoqyte illuistrating the prevsence ofgold in some granules (arrowv) (X 45, 000). nearly identical epitopes (Figure 9). In parallel experi- neutrophils by immunocytochemistry at the light and ments, antibody to lactoferrin did not block the sub- electron microscopic levels. Stimulation of cells re- sequent binding of D545. sults in translocation and incorporation of this mem- In the course of examining neutrophils, D545 bind- brane protein into the plasma membrane. This relo- ing was also observed on the membranes of eosino- cation from storage granules in the resting neutrophil phil granules (Figure 10) and some granules in mono- to the surface membrane in stimulated cells leads us, cytes (Figure 11). We isolated neutrophils from a in agreement with Kuijpers et al,13 to consider granu- patient with Chediak-Higashi syndrome and per- lophysin an activation antigen of neutrophils. formed immunofluorescence studies with D545. The We have shown that this protein is located in the staining in the permeabilized neutrophils demon- azurophilic granules of neutrophils, based on several strated very large, brightly staining granules (Figure lines of evidence. First, maximal translocation is seen 12), significantly different than the diffuse punctate in response to FMLP in the presence of cytochalasin pattern seen in normal neutrophils.9 These large B, with lesser effect in response to PMA with or without granules were similar to the large granules classically cytochalasin B. This selective response to stimulation seen using routine staining and light microscopy in is considered typical of azurophilic granules, as spe- this disorder. cific granules discharge much more readily with PMA.6 Second, immunogold electron microscopy shows localization of this protein to the membrane of Discussion granules that contain myeloperoxidase. Finally, re- We have demonstrated that granulophysin is located lease of f-glucuronidase, a constituent of azurophilic on the membranes of azurophilic granules in human granules,5 to the extracellular medium as determined Localization of Granulophysin in Neutrophils 1379 AJPJuine 1994, Vol. 144, No. 6

Figure 12. Immunofluorescent staining with D545 of neutrophils from a patient uw)ith Chediak-Higashi Ks'ndrome, demonstrating ver large, sparse, brightly staining granules. by biochemical assay, parallels translocation of biological function is known for any of these mol- granulophysin to the plasma membrane as deter- ecules, although several functions have been pro- mined by flow cytometry. posed. Antibodies to p24/CD9 have been shown to Recently, CD63, an antigen originally described cause platelet activation and aggregation; those to as being present on platelet lysosomal membranes, CD37 have been shown to modulate activation of B has also been shown to be an activation antigen of lymphocytes; anti-CD63 antibodies have been re- neutrophils.13 Evidence from our laboratory sug- ported to inhibit monocyte adherence to serum- gests that CD63 and granulophysin may be the coated surfaces and aggregation of T and B lympho- same protein.10 This is based on amino acid se- cytes.22 These studies have suggested a possible quencing and platelet localization studies. We have role for this family of proteins in signal transduction.23 demonstrated cross-reactivity between antibodies Little is known about additional granule membrane recognizing CD63 and granulophysin in neutrophils. proteins of azurophilic neutrophil granules. The only CD63 has been shown to co-localize with myeloper- other known membrane component is CD68,24 a oxidase in azurophilic granules and to translocate to 110-kd transmembrane glycoprotein whose proximal the plasma membrane following preincubation with domain has homology with lysosomal associated cytochalasin B and stimulation with FMLP. This trans- membrane protein-1 (LAMP-1).2526 However, Bain- location correlated with release of ,B-glucuroni- ton and August27 found LAMP-1 and LAMP-2 in dase.13 Extrapolating from the data we have pub- vesicles, not in the azurophil granules. The adhesion lished regarding these proteins in platelets, it is likely molecule, Mac-1, is present in the membrane of spe- that anti-CD63 antibodies and D545 are recognizing cific granules (75%) including gelatinase granules closely related epitopes, and possibly identical pro- and 20% in secretory vesicles.28 Its role in adhesion teins. This is the first specific marker described for and is well documented and has en- exocytosis of azurophilic granules. abled specific granules to be studied in greater detail CD63 is a protein originally described as present than azurophilic granules. in platelet .1920 It was found to be identical In summary, we have identified granulophysin as to ME491, a melanoma-associated antigen.20'21 It is an activation marker of neutrophils localized in the an integral membrane protein of platelet lysosomes granule membrane of azurophilic granules. This will and has considerable homology to a family of proteins enable us to study the contribution of azurophilic that includes p24/CD9 (a surface marker present on granule exocytosis in the physiological functions of a wide variety of hemopoietic and nonhemopoietic the neutrophil. In addition, it will be of interest to look tissues), the leukocyte antigens CD37 and CD53, and at in vivo activation of neutrophils in inflammatory dis- TAPA-1 (target of an antiproliferative antibody-1). No orders such as adult respiratory distress syndrome, 1380 Cham et al AJPJune 1994, Vol. 144, No. 6 rheumatoid arthritis, and immune complex disorders use of surface markers for neutrophil activation. Blood in which activation of neutrophils may play a role in 1991, 78:1105-1111 pathophysiology of disease. 14. Israels SJ, Gerrard JM, Jacques YV, McNicol A, Cham B, Nishibori M, Bainton DF: Platelet dense granule membranes contain both granulophysin and p-selectin (GMP-140). Blood 1992, 80:143-152 Acknowledgments 15. Hoehn SK, Kanfer JN: L-ascorbic acid and lysosomal brain. We thank Ms. Yvonne Jacques for her excellent tech- activities of guinea pig liver and Can J Biochem 1978, 56:352-356 nical assistance and Dr. E. Rector for expert assis- 16. Hetherington SV, Spitznagel JK, Quie PG: An enzyme tance with flow cytometry analysis. linked immunoassay (ELISA) for measurement of lactoferrin. J Immunol Methods 1983, 65:183-190 17. Bainton DF, Miller LJ, Kishimoto TK, Springer TA: Leu- References kocyte adhesion receptors are stored in peroxidase- negative granules of human neutrophils. J Exp Med 1. Rice WG, Kinkade JM Jr, Parmley RT: High resolution 1987, 166:1641-1653 of heterogeneity among human neutrophil granules: 18. Laemmli UK: Cleavage of structural proteins during physical, biochemical, and ultrastructural properties of the assembly of the head of bacteriophage T4. Nature isolated fractions. Blood 1986, 68:541-555 1970, 227:680-685 2. Bainton DF, Farquhar MG: Origin of granules in poly- 19. 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