American journal ofPathology, Vol. 144, No. 6, June 1994Copynght © American Societyfor Investigative Pathology

Granulophysin Is Located in the Membrane ofAzurophilic Granules in Human Neutrophils andMobilizes to the Plasma Membrane FollowingCell Stimulation

Bonnie P. Cham,* Jon M. Gerrard,* andDorothy F. BaintontFrom the Department of Pediatrics and the ManitobaInstitute of Cell Biology,* University ofManitoba,Winnipeg, Manitoba, Canada; and the Department ofPathology,t University of California School ofMedicine, SanFrancisco, California

Granulophysin, a protein described in plateletdense granule membranes, has been shown to besimilar or identical to CD63, a lysosomal mem-brane protein. We have previously shown granu-lophysin to be present in neutrophils usingimmunofluorescence. We now localize granulo-physin to the neutrophil azurophilic granules byfine structural immunocytochemistry. Granulo-physin expression on the surface membrane ofthe neutrophil is increasedfollowing stimulationofthe ceUs, demonstrated byflow cytometry andfine structural immunocytochemistry. A similarpattern is shown for an anti-CD63 antibody. In-cubation of activated neutrophils with D545, amonoclonal antibody to granulophysin, blockssubsequent binding ofanti-CD63 antibodies to theceU surface, and anti-CD63 antibodies preventsubsequent binding ofD545 as assessed byflowcytometry and immunoblotting. Our results sup-port the homology of CD63 and granulophysinpreviously demonstrated inplatelets and confirmCD63 as an activation marker in neutrophilsand the first azurophilic granule membranemarker of neutrophils. (Am J Pathol 1994, 144:1369-1380)

Various subsets of neutrophil granules have beenidentified and characterized by physical and bio-chemical properties.1" Azurophilic (primary) gran-ules appear earliest in maturation and are the largestgranules. Their contents include acid hydrolases, mi-crobicidal enzymes, proteases, and cationic pro-

teins, and they are peroxidase positive.3'5 Azurophilicgranules are considered to be classic primary lyso-somes in that they contain acid hydrolases that havenot yet entered into a digestive event. They aremobilized to the surface of the neutrophil, and theircontents discharged by formyl-methionyl-leucyl-phenylalanine (FMLP) following cytochalasin B stimu-lation, but this translocation occurs only to a minimalextent when the neutrophils are stimulated by phorbolmyristate acetate (PMA) or FMLP in the absence ofcytochalasin B.6 Specific (secondary) granules, onthe other hand, appear later in development and aresmaller, but more numerous, than azurophilic gran-ules. They are peroxidase-negative and containlactoferrin and many other proteins. Considerableheterogeneity exists within the group of peroxidase-negative granules with regard to their content andmobilization. In addition, the neutrophil also containsgelatinase granules and secretory vesicles as re-viewed by Borregaard et al.7 Many of thesenonperoxidase-containing granules are releasedearly in the inflammatory response and likely allow forcell diapedesis and adhesion via their membraneconstituents of C3bi and FMLP receptors. They aretranslocated to the cell surface in vitro by low con-centrations of PMA as well as by low concentrationsof FMLP even in the absence of cytochalasin B.6 Asa result, it has been speculated that specific granules,and other compartments, mobilize easily and early inthe course of inflammation, allowing for chemotaxis,whereas azurophilic granules predominantly form in-tracellular phagolysosomes and participate in cellkilling.6

Granulophysin is a protein originally described aspresent in platelet-dense granule membranes,9

Supported by Children's Hospital Research Foundation, Winnipeg,and NIH grant number DK 10486.

Accepted for publication February 4, 1994.

Address reprint requests to Dr. Bonnie Cham, ON 141 ManitobaCancer Treatment and Research Foundation, 100 Olivia Street,Winnipeg, Manitoba, Canada R3E 0V9.

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1370 Cham et alA/P Julne 1994, Vol. 144, No. 6

which has subsequently been shown to be similar oridentical to CD63, a platelet lysosomal protein.10Using a monoclonal antibody, granulophysin wasshown to be present in a granular pattern in many celltypes, including endocrine, exocrine, and neuronaltissues, endothelial cells and certain leukocytes.11We have previously shown this protein to be presentin neutrophils using immunofluorescent techniques.9We now extend our previous observations localizing

granulophysin to the azurophilic granules by finestructural immunocytochemistry.12 D545 was foundto co-localize with myeloperoxidase in the azurophilicgranules. In addition, D545 was separate in locationfrom lactoferrin, a content marker for specific gran-ules. Binding of D545 to the surface of the neutrophilis increased following stimulation of the cells by cyto-chalasin B and FMLP, as demonstrated by flow cy-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 anitibodyto 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 1371AJPJune 1994, Vol. 144, No. 6

similar pattern is shown for an anti-CD63 antibody inactivated neutrophils, in agreement with a previousreport.13 Incubation of activated neutrophils withD545 blocks subsequent binding of anti-CD63 anti-bodies to the surface of the neutrophil, and anti-CD63antibodies prevent subsequent binding of D545.Similarly, incubation of neutrophil homogenates withanti-CD63 diminish binding of D545 in immunoblot-ting studies. Our results are in agreement with thehomology of CD63 and granulophysin previouslydemonstrated in platelets10 and extend the observa-tion that CD63 is an activation marker in neutrophils13and the first azurophilic granule membrane marker ofneutrophils.

Materials and MethodsMonoclonal Antibodies

The monoclonal antibody against granulophysinused in the present study (D545) has been charac-

terized elsewhere.9 Anti-CD63 antibody used formost studies was purchased from Amac Inc. (West-brook, ME). An additional anti-CD63 antibody (HS56)and fluorescein isothiocyanate (FITC) anti-CD63were kindly provided by Dr. James Hildreth (TheJohns Hopkins University, Baltimore, MD).

Neutrophil Isolation

After obtaining consent, blood was drawn from vol-unteer adult donors into syringes containing ACD 1.5ml/l0 ml total volume. It was then mixed with 5%dextran in phosphate-buffered saline and allowed tosediment for 30 minutes. The leukocyte-rich plasmawas layered onto Ficoll-Paque (Pharmacia) and cen-trifuged at 400 x g for 30 minutes. Residual eryth-rocytes in the pellet were lysed with 0.87% ammo-nium chloride, and the neutrophils were then washedwith Hanks' balanced salt solution (HBSS). The cellswere 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 alA/PJune 1994, Vol. 144, No. 6

Figure 3. Dual staining showing segregation ofD545 (10-nm gold particles) and lactoferrin antibodies (5-nm gold particles) in distinct granulesubsets; (X 70,000).

Figure 4. Representative data from a singleflow cytometry experiment showing binding ofD545 to the plasma membrane of neutrophilsthat have been stimulated with the followingstimuli. The vertical line separates negative andpositive fluorescent populations. Fluorescenceintensity is displayed on a 3-decade loganithmicscale. A: unstimulated neutrophils ( 17.77% posi-tive, mean channelflourescence [MCFI, 51.4) B:5 minutes ofFMLP ic-6 mol/L ( 79.6% positive,MCF, 66.7). C: A23187 5 jmo'L for 5 minutes(80.5% positive, MCF, 85.0) D: PMA 10-9 mol/Lfor 5 minutes (49.6% positive, MCF, 52.9).(Note: results of all flow cytometry experimentsare expressed quantitatively as the percentageof cells that are positive for fluorescence, andthe MCF, is a measure of how intense thefluo-rescence is within the positive population).

to an appropriate cell concentration. Where indi-cated, CaCI2 was then added for a final concentra-tion of 1 mmol/L.

Stimulation of Neutrophils

Neutrophils were prewarmed in a 37-C waterbath for5 minutes. Those samples that were subsequently go-

ing to be stimulated with FMLP were incubated withcytochalasin B (Sigma, St. Louis, MO) at a concen-

tration of 5 pg/mI. FMLP, PMA, or A23187 (Sigma)were then added at the indicated concentrations. Thecells were incubated in a shaking water bath at 37 Cfor the time period indicated, and the reaction was

stopped by dilution with 600 pl of ice-cold, calcium-

free HBSS. The samples were centrifuged in an Ep-pendorf micro-centrifuge and the supernatantsstored at -20 C for further assays.

Immunofluorescence

Immunofluorescence was performed essentially as

previously described.9 Neutrophil pellets were sus-

pended in primary antibody (D545) at 20 pg/ml inHBSS with 0.1% bovine serum albumin (BSA). Thismixture was incubated on ice for 30 minutes. Sampleswere then centrifuge washed three times with HBSS/0.1% BSA. The second antibody, biotinylated goatanti-mouse, was applied in the HBSS/0. 1% BSA for 30minutes. The neutrophils were again washed three

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Localization of Granulophysin in Neutrophils 1373'/P june 1994. I !. -44. A\n. 6

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Figure 5. R rc/s(i n i(ut,i(, rmt (IC il)/) i/7oit (-_lt01 (/li -iCIncliWlt dcm ons71.ra0in, bind1)(;i1 OJ D5I i al var{iolls' ti}71(/O,t8/lo(il

FXLslliXti}l{/Sio0i A: zlm.,aiidalllcd(9; nciaropwhll7.s (v) ", v tj9iliW. B: /o loiVwn, 0) s(cond/s ./ milltl/uiuoil 8.6() posiil MC,l /

106 9 ) C 1(Ji))llowin- 5 minulot o .stimidlion ), Q) itivc l_2 _.6,)

times. Strepavidin-fluorescein was then added to theneutrophils, 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 bufferfor 1 hour on ice and mounted onto slides in glycerolTris-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, unreactedaldehyde was neutralized with three rinses of TBS.The neutrophils were permeabilized for 3 minutes with0.1 % Triton X-1 00 and centrifuge-washed three timeswith 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 experimentconsisted of the above procedure with omission of theprimary antibody.

Flow Cytometry

Samples were prepared for flow cytometry as aboveor utilizing directly FITC-conjugated antibodies as we

have described previously for platelets.14 The neu-

trophils were resuspended in HBSS/0.1% BSA andincubated for 1 hour on ice, in the dark, with D545 (80ucg/ml) or anti-CD63 (20 ucg/ml) conjugated directlyto FITC. Parallel samples were incubated with unla-beled D545 or anti-CD63 in excess before fluorescentlabeling to assess nonspecific binding. In addition,experiments were performed in which the neutrophilswere incubated with unlabeled anti-CD63 or D545 in

excess and then incubated with D545-FITC (followinganti-CD63) or anti-CD63-FITC (following D545).Samples were fixed with 1 % formaldehyde and ana-

lyzed using an EPICS Model 753 flow cytometer

Table 1. D 149 lPii(i)7 -( liici(t,nmi(Ilas/c Relcase.(a) 1(1o acI(?1C/rrill Release/ .AI/'r .\i ropl)0)1il S1i)illltioii

Stimulus

D545 binding(% increase

over baseline)

NonstimulatedFMLP 10-6 mol/L

1 minute*5 minutes*

PMA 10-9 mol/L5 minutes

PMA 10-8 mol/L5 minutes

A23187 10-6 mol/L5 minutes

A23187 10-5 mol/L5 minutes

6.3%

41 5%35.2%

7.1%

411%

9.7%

43.6%

f-glucuronidaserelease

(% of cell content)

0%

35 2%21.0%

800%

5 0%

7 2%

25 8%

Lactoferriinrelease

(% of cell content)

899%

9 5%8 0%

81 7%

78 6%

28 6%

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).

A

1374 Cham et alAJPJutne 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 anargon ion laser (500 mW, 488 nm). Fluorescence wasdetected at 525 nm. Forward and 90-degree lightscatter measurements were used to establish gatesfor intact, viable neutrophils. Single parameter, 255-channel, log integral green fluorescent histogramswere obtained, each based on 1 x 104 gated events.

,B-Glucuronidase Assay

f3-glucuronidase activity was measured in the super-natants using 4-methylumbelliferone-j3-glucuronidecleavage.15 The results are expressed as a percentof the activity in cell sonicates.

Lactoferrin Assay

Lactoferrin was measured using a competitiveenzyme-linked immunosorbent assay.16 Nitrocellu-lose plates were coated with a standard amount oflactoferrin (Sigma) and then supernatant, cell soni-cate, or standard amount of lactoferrin was addedalong with rabbit anti-lactoferrin antibody (Sigma)and incubated for 2 hours. The plate was washed,and goat anti-rabbit antibody conjugated with alka-line phosphatase was then added and incubated for2 hours. Following plate washing, alkaline phos-phatase substrate was added, and the plates weredeveloped in a 37 C incubator. Absorbence at 405 nmwas read in an enzyme-linked immunosorbent assay

Localization of Granulophysin in Neutrophils 1375AJPJuine 1994, Vol. 144, No. 6

I A

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

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,iMGCF, 76.0) C: Jbllowing 5 minutces of stimuilation (52.5% positive,MCF, 76.4).

well reader, and the unknown concentrations were

then determined by interpolating from a standardcurve. Results are expressed as a percentage of a

cell sonicate.

Immunogold Electron Microscopy

Resting neutrophils or activated cells were fixed inequal volume of 2% paraformaldehyde/0.05% glut-araldehyde in 0.1 mol/L phosphate buffer for 1 hourat 4 C. They were then washed three times in 0.1 mol/Lphosphate buffer with 3% sucrose and processed forfrozen thin sectioning. Immunocytochemistry was

performed as previously described. 17 The mono-

clonal antibody D545 was used at a dilution 1 :100 andthe polyclonal antibodies to either myeloperoxidase(CalBiochem Corp., San Diego, CA) or lactoferrin(Davo Corp., Carpinteria, CA) were used at a dilutionof 1:500. The second step consisted of addingGAM-10 to detect mouse monoclonal antibody or

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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 ofFMLP (48.5%, MCGF 96.3) C: anti-CD63 binding after 5 minutes ofFMLP stimulation in the presence of unconjugated HS56 (2.8% posi-tive, MCF, 58.7). D: anti-CD63 binding after 5 minutes of FMLPstimulation 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 theprcesence of unlconijuigated anti-C'D63 (4.5% positive, MCF, 74.1). (Allunconjugated antibodies are present in super sattiratinlg concentra-tion.s)

GAR-5 to detect rabbit polyclonal antibodies. Con-trols consisted of replacement of the primary antibodywith normal mouse or rabbit serum respectively.

Western Blotting

Western blots were performed as previously de-scribed.9 The protein samples, in a buffer of 2.5%glycerol, 5% sodium dodecyl sulfate, 125 mmol/L TrisHCI (pH 6.8), were incubated at 37 C for 1 hour andseparated by electrophoresis on 10% polyacrylamidegel with a 4% stacking gel according to Laemmli.18Lanes were loaded with 30 ucg of protein. Proteinswere transferred to nitrocellulose at 1 OOV for 1 hour atroom temperature. The nitrocellulose was blockedovernight using 10% nonfat powdered milk, washedwith 0.1% Tween/TBS and incubated with 10 ucg/mlof the monoclonal antibody of interest. After washingwith TweenlTBS, the nitrocellulose was incubatedwith peroxidase-labeled goat anti-mouse immuno-globulin G (Sigma, 1:3,000 dilution) for 30 minutes atRT. The reaction was developed using an enhancedchemiluminescence. Blocking experiments were per-

JL I

1376 Cham et alAJPJune 1994, Vol. 144, No. 6

kDa

kDa

106 -

s0 -

50-

33-

24-

Figure 9. Western blots of neutrophil (lanes B,C, F, and G), and platelet (lanes A, D, E andH) homogenates. Lanes A and B: D545; lanesC 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-oxidase.

formed by first incubating with an anti-CD63 antibody,subsequently incubating with peroxidase-labeledD545, and developing using enhanced chemilumi-nescence.

ResultsOur previous studies have demonstrated punctate in-tracellular staining suggestive of a granular locationof D545 in neutrophils using immunofluorescence.9Immunogold electron microscopy demonstrates lo-calization of granulophysin in the membranes of largeextracted azurophilic granules (Figure 1). No signifi-cant amount of labeling was seen in specific granulesor on the plasma membrane. Double labeling of rest-ing neutrophils with D545 and polyclonal serum tomyeloperoxidase, a marker for azurophilic granules,showed co-localization to the same large, clear gran-ules (Figure 2). Whereas only D545 appeared on thegranule membrane, myeloperoxidase also appearedin the matrix of the granules. Furthermore, dual stain-ing with anti-lactoferrin antibodies, a marker for spe-cific granules, showed that lactoferrin and granulo-physin did not co-localize (Figure 3). Flow cytometryperformed on resting neutrophils demonstrated onlyminimal surface expression of granulophysin, consis-tent with the low level seen by immunocytochemistry(Figure 4).

Neutrophils were incubated with cytochalasin B,stimulated with FMLP or stimulated by PMA alone,

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E. .: ... ... .... ...

A (I C D E F a H

and then analyzed by flow cytometry for binding ofD545 to the plasma membrane. Flow cytometry dem-onstrated maximal surface expression of D545 fol-lowing stimulation with cytochalasin B and FMLP 10-6mol/L in the presence of calcium (Figure 4). Bindingof D545 was increased to a minimal degree by PMAat doses that did not disrupt the cells. The calciumionophore, A23187, also stimulated translocation ofthe protein, supporting an effect of calcium (Figure 4).Surface expression of granulophysin was found to bean early event with significant change at 30 seconds,reaching maximal stimulation at 1 to 5 minutes fol-lowing stimulation (Figure 5). Expression at the cellsurface was generally stable between 1 to 5 minutesafter stimulation.

The supernatant of neutrophils stimulated for flowcytometry was saved and analyzed for the presenceof ,B-glucuronidase (a marker for azurophilic gran-ules) and lactoferrin (a marker for specific granules).,B-glucuronidase release was seen to parallel trans-location of granulophysin to the plasma membrane.Following FMLP stimulation, 35.2% of the cell con-tents were released; following PMA stimulation, 5 to8% were released. With maximal A23187 stimulation,25.8% of the cell contents were released (Table 1).Lactoferrin release, however, occurred maximallywith stimulation by PMA 10-9 mol/L, which causedrelease of 81.7% of cellular lactoferrin. Secretion oflactoferrin did not coincide with maximal granulophy-sin surface expression (Table 1). These results further

Localization of Granulophysin in Neutrophils 1377AJPJune 1994, Vol. 144, No. 6

Figure 10. Frozen-tbin section with immunogold cvtochemicalpreparation ofa normal human eosinophil. The antigen appears on the membraneof most of the crystalloid-containing eosinopbil granules (arrow's) (x 36, 000).

support the localization of granulophysin to the azuro-philic granule membrane.

Utilizing frozen thin section immuno-electron mi-croscopy, stimulated neutrophils demonstrated trans-location of granulophysin to the plasma membrane ina portion of the neutrophils following incubation withcytochalasin B and 2.5 minutes of FMLP stimulation(Figure 6). The redistribution of label to the plasmamembrane, with some cells very strongly stained andothers not stained, is similar to the distribution dem-onstrated via flow cytometry, ie, a heterogenouspopulation.

Further studies were undertaken to examine therelationship of the proteins recognized by anti-CD63antibodies and D545. Flow cytometry demonstrateda similar pattern of translocation of CD63 to that ofD545 following stimulation with FMLP, PMA, andA23187 (Figure 7). Additional experiments were per-

formed in which the stimulated cells were first ex-posed to D545 (in excess) and then to anti-CD63 an-tibodies conjugated to FITC. This showed nearlycomplete blocking of anti-CD63 antibody binding(Figure 8). Similarly, pre-exposure to anti-CD63 anti-bodies blocked to a significant extent the subsequentbinding of D545 (Figure 8). In parallel experiments,antibody to lactoferrin was found to mobilize to the cellsurface following stimulation by FMLP, but did notblock the subsequent binding of either anti-CD63 orD545.

Both D545 and anti-CD63 recognize a similar pro-tein of about 47 kd on Western blots of neutrophil pro-teins. Staining with anti-CD63 in neutrophils wasslightly lighter than staining with D545. However, theaddition of unlabeled CD63 before peroxidase-labeled D545 blocked the staining using D545, sug-gesting the two antibodies recognized the same or

1378 Cham et alAJPJune 1994, Vol. 144, No. 6

Figure 11. Frozen-thin section with immunogold preparation ofa portion ofa normal human monoqyte illuistrating the prevsence ofgold in somemonocyte granules (arrowv) (X 45, 000).

nearly identical epitopes (Figure 9). In parallel experi-ments, antibody to lactoferrin did not block the sub-sequent binding of D545.

In the course of examining neutrophils, D545 bind-ing was also observed on the membranes of eosino-phil granules (Figure 10) and some granules in mono-cytes (Figure 11). We isolated neutrophils from apatient with Chediak-Higashi syndrome and per-formed immunofluorescence studies with D545. Thestaining in the permeabilized neutrophils demon-strated very large, brightly staining granules (Figure12), significantly different than the diffuse punctatepattern seen in normal neutrophils.9 These largegranules were similar to the large granules classicallyseen using routine staining and light microscopy inthis disorder.

DiscussionWe have demonstrated that granulophysin is locatedon the membranes of azurophilic granules in human

neutrophils by immunocytochemistry at the light andelectron microscopic levels. Stimulation of cells re-sults in translocation and incorporation of this mem-brane protein into the plasma membrane. This relo-cation from storage granules in the resting neutrophilto the surface membrane in stimulated cells leads us,in agreement with Kuijpers et al,13 to consider granu-lophysin an activation antigen of neutrophils.We have shown that this protein is located in the

azurophilic granules of neutrophils, based on severallines of evidence. First, maximal translocation is seenin response to FMLP in the presence of cytochalasinB, with lesser effect in response to PMA with or withoutcytochalasin B. This selective response to stimulationis considered typical of azurophilic granules, as spe-cific granules discharge much more readily withPMA.6 Second, immunogold electron microscopyshows localization of this protein to the membrane ofgranules that contain myeloperoxidase. Finally, re-lease of f-glucuronidase, a constituent of azurophilicgranules,5 to the extracellular medium as determined

Localization of Granulophysin in Neutrophils 1379AJPJuine 1994, Vol. 144, No. 6

Figure 12. Immunofluorescent staining withD545 of neutrophils from a patient uw)ithChediak-Higashi Ks'ndrome, demonstratingver large, sparse, brightly staining granules.

by biochemical assay, parallels translocation ofgranulophysin to the plasma membrane as deter-mined by flow cytometry.

Recently, CD63, an antigen originally describedas being present on platelet lysosomal membranes,has also been shown to be an activation antigen ofneutrophils.13 Evidence from our laboratory sug-gests that CD63 and granulophysin may be thesame protein.10 This is based on amino acid se-quencing and platelet localization studies. We havedemonstrated cross-reactivity between antibodiesrecognizing CD63 and granulophysin in neutrophils.CD63 has been shown to co-localize with myeloper-oxidase in azurophilic granules and to translocate tothe plasma membrane following preincubation withcytochalasin B and stimulation with FMLP. This trans-location correlated with release of ,B-glucuroni-dase.13 Extrapolating from the data we have pub-lished regarding these proteins in platelets, it is likelythat anti-CD63 antibodies and D545 are recognizingclosely related epitopes, and possibly identical pro-teins. This is the first specific marker described forexocytosis of azurophilic granules.CD63 is a protein originally described as present

in platelet lysosomes.1920 It was found to be identicalto ME491, a melanoma-associated antigen.20'21 It isan integral membrane protein of platelet lysosomesand has considerable homology to a family of proteinsthat includes p24/CD9 (a surface marker present ona wide variety of hemopoietic and nonhemopoietictissues), the leukocyte antigens CD37 and CD53, andTAPA-1 (target of an antiproliferative antibody-1). No

biological function is known for any of these mol-ecules, although several functions have been pro-posed. Antibodies to p24/CD9 have been shown tocause platelet activation and aggregation; those toCD37 have been shown to modulate activation of Blymphocytes; anti-CD63 antibodies have been re-ported to inhibit monocyte adherence to serum-coated surfaces and aggregation of T and B lympho-cytes.22 These studies have suggested a possiblerole for this family of proteins in signal transduction.23

Little is known about additional granule membraneproteins of azurophilic neutrophil granules. The onlyother known membrane component is CD68,24 a110-kd transmembrane glycoprotein whose proximaldomain has homology with lysosomal associatedmembrane protein-1 (LAMP-1 ).2526 However, Bain-ton and August27 found LAMP-1 and LAMP-2 invesicles, not in the azurophil granules. The adhesionmolecule, Mac-1, is present in the membrane of spe-cific granules (75%) including gelatinase granulesand 20% in secretory vesicles.28 Its role in adhesionand phagocytosis is well documented and has en-abled specific granules to be studied in greater detailthan azurophilic granules.

In summary, we have identified granulophysin asan activation marker of neutrophils localized in thegranule membrane of azurophilic granules. This willenable us to study the contribution of azurophilicgranule exocytosis in the physiological functions ofthe neutrophil. In addition, it will be of interest to lookat in vivo activation of neutrophils in inflammatory dis-orders such as adult respiratory distress syndrome,

1380 Cham et alAJPJune 1994, Vol. 144, No. 6

rheumatoid arthritis, and immune complex disordersin which activation of neutrophils may play a role inpathophysiology of disease.

AcknowledgmentsWe thank Ms. Yvonne Jacques for her excellent tech-nical assistance and Dr. E. Rector for expert assis-tance with flow cytometry analysis.

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4. Bainton DF, Farquhar MG: Segregation and packagingof granule enzymes in eosinophilic leukocytes. J CellBiol 1970, 45:54-73

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9. Gerrard JM, Lint D, Sims PJ, Wiedmer T, Fugate RD,McMillan E, Robertson C, Israels SJ: Identification of aplatelet dense granule membrane protein that is defi-cient in a patient with the Hermannsky-Pudlak syn-drome. Blood 1991, 77:101-112

10. Nishibori M, Cham B, McNicol A, Shalev A, GerrardJM: CD63 is present in platelet dense granules, is de-ficient in a patient with the Hermansky-Pudlak syn-drome, and appears identical to granulophysin. J ClinInvest 1993, 91:1775-1782

11. Hatskelzon L, Dalal BI, Shalev A, Robertson C, GerrardJM: Wide distribution of granulophysin epitopes in gran-ules of human tissues. Lab Invest 1993, 68:509-519

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