and has considerable homology to a family of proteins that includes p24/CD9 (a ... Gerrard JM, Lint D, Sims PJ, Wiedmer T, Fugate RD,. McMillan E, Robertson ...
American journal of Pathology, 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 Dorothy F. Baintont From the Department of Pediatrics and the Manitoba Institute of Cell Biology,* University of Manitoba, Winnipeg, Manitoba, Canada; and the Department of Pathology,t University of California School of Medicine, San Francisco, California
Granulophysin, a protein described in platelet dense granule membranes, has been shown to be similar or identical to CD63, a lysosomal membrane protein. We have previously shown granulophysin to be present in neutrophils using immunofluorescence. We now localize granulophysin to the neutrophil azurophilic granules by fine structural immunocytochemistry. Granulophysin expression on the surface membrane of the neutrophil is increasedfollowing stimulation of the ceUs, demonstrated by flow cytometry and fine structural immunocytochemistry. A similar pattern is shown for an anti-CD63 antibody. Incubation of activated neutrophils with D545, a monoclonal antibody to granulophysin, blocks subsequent binding ofanti-CD63 antibodies to the ceU surface, and anti-CD63 antibodies prevent subsequent binding of D545 as assessed by flow cytometry and immunoblotting. Our results support the homology of CD63 and granulophysin previously demonstrated in platelets and confirm CD63 as an activation marker in neutrophils and the first azurophilic granule membrane marker of neutrophils. (Am J Pathol 1994, 144:
teins, and they are peroxidase positive.3'5 Azurophilic granules are considered to be classic primary lysosomes in that they contain acid hydrolases that have not yet entered into a digestive event. They are mobilized to the surface of the neutrophil, and their contents discharged by formyl-methionyl-leucylphenylalanine (FMLP) following cytochalasin B stimulation, 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 cytochalasin B.6 Specific (secondary) granules, on the other hand, appear later in development and are smaller, but more numerous, than azurophilic granules. They are peroxidase-negative and contain lactoferrin and many other proteins. Considerable heterogeneity exists within the group of peroxidasenegative granules with regard to their content and mobilization. In addition, the neutrophil also contains gelatinase granules and secretory vesicles as reviewed by Borregaard et al.7 Many of these nonperoxidase-containing granules are released early in the inflammatory response and likely allow for cell diapedesis and adhesion via their membrane constituents of C3bi and FMLP receptors. They are translocated to the cell surface in vitro by low concentrations of PMA as well as by low concentrations of FMLP even in the absence of cytochalasin B.6 As a result, it has been speculated that specific granules, and other compartments, mobilize easily and early in the course of inflammation, allowing for chemotaxis, whereas azurophilic granules predominantly form intracellular phagolysosomes and participate in cell
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 identified and characterized by physical and biochemical properties.1" Azurophilic (primary) granules appear earliest in maturation and are the largest granules. Their contents include acid hydrolases, microbicidal enzymes, proteases, and cationic pro-
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 Manitoba Cancer Treatment and Research Foundation, 100 Olivia Street, 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 identical to CD63, a platelet lysosomal protein.10 Using a monoclonal antibody, granulophysin was shown to be present in a granular pattern in many cell types, including endocrine, exocrine, and neuronal tissues, endothelial cells and certain leukocytes.11 We have previously shown this protein to be present in neutrophils using immunofluorescent techniques.9 We now extend our previous observations localizing
granulophysin to the azurophilic granules by fine structural immunocytochemistry.12 D545 was found to co-localize with myeloperoxidase in the azurophilic granules. In addition, D545 was separate in location from lactoferrin, a content marker for specific granules. Binding of D545 to the surface of the neutrophil is increased following stimulation of the cells by cytochalasin B and FMLP, as demonstrated by flow cytometry 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-anlides (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 activated neutrophils, in agreement with a previous report.13 Incubation of activated neutrophils with D545 blocks subsequent binding of anti-CD63 antibodies to the surface of the neutrophil, and anti-CD63 antibodies prevent subsequent binding of D545. Similarly, incubation of neutrophil homogenates with anti-CD63 diminish binding of D545 in immunoblotting studies. Our results are in agreement with the homology of CD63 and granulophysin previously demonstrated in platelets10 and extend the observation that CD63 is an activation marker in neutrophils13 and the first azurophilic granule membrane marker of neutrophils.
Materials and Methods Monoclonal Antibodies The monoclonal antibody against granulophysin used in the present study (D545) has been charac-
terized elsewhere.9 Anti-CD63 antibody used for most studies was purchased from Amac Inc. (Westbrook, ME). An additional anti-CD63 antibody (HS56) and fluorescein isothiocyanate (FITC) anti-CD63 were kindly provided by Dr. James Hildreth (The Johns Hopkins University, Baltimore, MD).
Neutrophil Isolation After obtaining consent, blood was drawn from volunteer adult donors into syringes containing ACD 1.5 ml/l0 ml total volume. It was then mixed with 5% dextran in phosphate-buffered saline and allowed to sediment for 30 minutes. The leukocyte-rich plasma was layered onto Ficoll-Paque (Pharmacia) and centrifuged at 400 x g for 30 minutes. Residual erythrocytes in the pellet were lysed with 0.87% ammonium chloride, and the neutrophils were then washed with Hanks' balanced salt solution (HBSS). The cells 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) existing in the same large azturophilic granules (ag) (x 70,000).
co-
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 showing binding of D545 to the plasma membrane of neutrophils
flow cytometry experiment
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 scale. A: unstimulated neutrophils ( 17.77% positive, mean channelflourescence [MCFI, 51.4) B: 5 minutes of FMLP 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/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 thefluorescence is within the positive population).
.
.
.
T
_~~~~~~~~~1c!
.
.
D
.
to an appropriate cell concentration. Where indicated, CaCI2 was then added for a final concentration of 1 mmol/L.
free HBSS. The samples were centrifuged in an Eppendorf micro-centrifuge and the supernatants stored at -20 C for further assays.
Stimulation of Neutrophils
Immunofluorescence
Neutrophils were prewarmed in a 37-C waterbath for 5 minutes. Those samples that were subsequently going to be stimulated with FMLP were incubated with cytochalasin B (Sigma, St. Louis, MO) at a concentration of 5 pg/mI. FMLP, PMA, or A23187 (Sigma) were then added at the indicated concentrations. The cells were incubated in a shaking water bath at 37 C for the time period indicated, and the reaction was stopped by dilution with 600 pl of ice-cold, calcium-
Immunofluorescence was performed essentially as previously described.9 Neutrophil pellets were suspended in primary antibody (D545) at 20 pg/ml in HBSS with 0.1% bovine serum albumin (BSA). This mixture was incubated on ice for 30 minutes. Samples were then centrifuge washed three times with HBSS/ 0.1% BSA. The second antibody, biotinylated goat anti-mouse, was applied in the HBSS/0. 1% BSA for 30 minutes. The neutrophils were again washed three
Localization of Granulophysin in Neutrophils '/P june 1994.
A
I
!.
1373
-44. A\n.
6
times. Strepavidin-fluorescein was then added to the 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 immunofluorescence microscope. Those samples requiring permeabilization were fixed in 2% paraformaldehyde in 0.1 mol/L cacodylate 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 staining 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 utilizing directly FITC-conjugated antibodies as we have described previously for platelets.14 The neutrophils 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 unlabeled D545 or anti-CD63 in excess before fluorescent labeling to assess nonspecific binding. In addition, experiments were performed in which the neutrophils were incubated with unlabeled anti-CD63 or D545 in excess and then incubated with D545-FITC (following anti-CD63) or anti-CD63-FITC (following D545). Samples were fixed with 1 % formaldehyde and analyzed using an EPICS Model 753 flow cytometer or
r R rc/s(i n i(ut,i(, il)/) i/7oit (-_lt01 mt (IC (/li -iC dcm ons71.ra0in, bind1)(;i1 OJ D5I i al var{iolls' ti}71( /O,t8/lo(il FXL slli Xti}l{/Sio0i A: zlm.,aiidalllcd(9; nciaropwhll7.s (v) ", v tj9ili lo n, 0) s(cond/s ./ B: /oiVw l / W. milltl/uiuoil 8 .6() posiil MC, 0 i))llowin- 5 minulot o .stimidlion ), Q) itivc l J
Figure 5. IncliWlt
1
6
9)
C
1(
_2 _.6,)
Table 1.
D
149 lPii(i)7
-(
liici(t,nmi(Ilas/c Relcase. (a)1o acI(?1C /rrill Release/ .AI/'r 1(
D545 binding
(% Stimulus
Nonstimulated FMLP 10-6 mol/L 1 minute* 5 minutes* PMA 10-9 mol/L 5 minutes PMA 10-8 mol/L 5 minutes A23187 10-6 mol/L 5 minutes A23187 10-5 mol/L 5 minutes
increase
over
baseline)
.\i
ropl)0)1il S1i)illltioii
f-glucuronidase release (% of cell content)
Lactoferriin release (% of cell content)
6.3%
0%
899%
41 5% 35.2%
35 2% 21.0%
9 5% 8 0%
7.1%
800%
81 7%
411%
5 0%
78 6%
9.7%
7 2%
28 6%
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 of granulophysin to the plasma membrane (pm), N, tnucleus, (x30,000). Scale bar represents 0.1 umolL.
(Coulter Electronics, Hialeah, FL) equipped with an argon ion laser (500 mW, 488 nm). Fluorescence was
Lactoferrin Assay
detected at 525 nm. Forward and 90-degree light scatter measurements were used to establish gates for intact, viable neutrophils. Single parameter, 255channel, log integral green fluorescent histograms were obtained, each based on 1 x 104 gated events.
Lactoferrin was measured using a competitive enzyme-linked immunosorbent assay.16 Nitrocellulose plates were coated with a standard amount of lactoferrin (Sigma) and then supernatant, cell sonicate, or standard amount of lactoferrin was added along with rabbit anti-lactoferrin antibody (Sigma) and incubated for 2 hours. The plate was washed, and goat anti-rabbit antibody conjugated with alkaline phosphatase was then added and incubated for 2 hours. Following plate washing, alkaline phosphatase substrate was added, and the plates were developed in a 37 C incubator. Absorbence at 405 nm was read in an enzyme-linked immunosorbent assay
,B-Glucuronidase Assay f3-glucuronidase activity was measured in the supernatants using 4-methylumbelliferone-j3-glucuronide cleavage.15 The results are expressed as a percent of the activity in cell sonicates.
~ ~ ~A
Localization of Granulophysin in Neutrophils
JL
I
I
A
1375
AJPJuine 1994, Vol. 144, No. 6
*
..............
.~~~~~~~~~ I * ,
I
I
I
--
,-1 ,I
.
,
,
,
o f.m
...,,,.,
.~~~~~~~~
.,,
,,I
,,,
. . . ..
..................
Figure 8. Representative data from a single flow cytometry experiment demonstrating: A: anti-CD63 binding in unstimuilated neutrophils (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% positive, 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% positive, MC, 101.o0). G: D545 binding after 5 minutes of FMLP stimtulation in the presence of unconjugated D545 (15.2% positive, MCGF 65. 8). Anid H: D545 binding after 5 minutes of FMLP 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 following 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 standard curve. Results are expressed as a percentage of a cell sonicate.
Immunogold Electron Microscopy Resting neutrophils or activated cells were fixed in equal volume of 2% paraformaldehyde/0.05% glutaraldehyde in 0.1 mol/L phosphate buffer for 1 hour at 4 C. They were then washed three times in 0.1 mol/L phosphate buffer with 3% sucrose and processed for frozen thin sectioning. Immunocytochemistry was performed as previously described. 17 The monoclonal antibody D545 was used at a dilution 1 :100 and the polyclonal antibodies to either myeloperoxidase (CalBiochem Corp., San Diego, CA) or lactoferrin (Davo Corp., Carpinteria, CA) were used at a dilution of 1:500. The second step consisted of adding GAM-10 to detect mouse monoclonal antibody or
GAR-5 to detect rabbit polyclonal antibodies. Controls consisted of replacement of the primary antibody with normal mouse or rabbit serum respectively.
Western Blotting Western blots were performed as previously described.9 The protein samples, in a buffer of 2.5% glycerol, 5% sodium dodecyl sulfate, 125 mmol/L Tris HCI (pH 6.8), were incubated at 37 C for 1 hour and separated by electrophoresis on 10% polyacrylamide gel with a 4% stacking gel according to Laemmli.18 Lanes were loaded with 30 ucg of protein. Proteins were transferred to nitrocellulose at 1 OOV for 1 hour at room temperature. The nitrocellulose was blocked overnight using 10% nonfat powdered milk, washed with 0.1% Tween/TBS and incubated with 10 ucg/ml of the monoclonal antibody of interest. After washing with TweenlTBS, the nitrocellulose was incubated with peroxidase-labeled goat anti-mouse immunoglobulin G (Sigma, 1:3,000 dilution) for 30 minutes at RT. The reaction was developed using an enhanced chemiluminescence. Blocking experiments were per-
1376
Cham et al
AJPJune 1994, Vol. 144, No. 6
kDa
kDa
106 -
-106
-80
s0 -
-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 C and D: anti CD63; lanes E and F: antiCD63 followced by D545 conjugated to peroxidase; lanes G and H: D545 conjugated to peroxidase.
-18
A
formed by first incubating with an anti-CD63 antibody, subsequently incubating with peroxidase-labeled D545, and developing using enhanced chemiluminescence.
Results Our previous studies have demonstrated punctate intracellular staining suggestive of a granular location of D545 in neutrophils using immunofluorescence.9 Immunogold electron microscopy demonstrates localization of granulophysin in the membranes of large extracted azurophilic granules (Figure 1). No significant amount of labeling was seen in specific granules or on the plasma membrane. Double labeling of resting neutrophils with D545 and polyclonal serum to myeloperoxidase, a marker for azurophilic granules, showed co-localization to the same large, clear granules (Figure 2). Whereas only D545 appeared on the granule membrane, myeloperoxidase also appeared in the matrix of the granules. Furthermore, dual staining with anti-lactoferrin antibodies, a marker for specific granules, showed that lactoferrin and granulophysin did not co-localize (Figure 3). Flow cytometry performed on resting neutrophils demonstrated only minimal surface expression of granulophysin, consistent with the low level seen by immunocytochemistry (Figure 4). Neutrophils were incubated with cytochalasin B, stimulated with FMLP or stimulated by PMA alone,
(I
C
D
E.
.:
...
...
E
....
F
a
...
H
and then analyzed by flow cytometry for binding of D545 to the plasma membrane. Flow cytometry demonstrated maximal surface expression of D545 following 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 at doses that did not disrupt the cells. The calcium ionophore, A23187, also stimulated translocation of the protein, supporting an effect of calcium (Figure 4). Surface expression of granulophysin was found to be an early event with significant change at 30 seconds, reaching maximal stimulation at 1 to 5 minutes following stimulation (Figure 5). Expression at the cell surface was generally stable between 1 to 5 minutes after stimulation. The supernatant of neutrophils stimulated for flow cytometry was saved and analyzed for the presence of ,B-glucuronidase (a marker for azurophilic granules) and lactoferrin (a marker for specific granules). ,B-glucuronidase release was seen to parallel translocation of granulophysin to the plasma membrane. Following FMLP stimulation, 35.2% of the cell contents were released; following PMA stimulation, 5 to 8% were released. With maximal A23187 stimulation, 25.8% of the cell contents were released (Table 1). Lactoferrin release, however, occurred maximally with stimulation by PMA 10-9 mol/L, which caused release of 81.7% of cellular lactoferrin. Secretion of lactoferrin did not coincide with maximal granulophysin 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 of a normal human eosinophil. 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 azurophilic granule membrane. Utilizing frozen thin section immuno-electron microscopy, stimulated neutrophils demonstrated translocation of granulophysin to the plasma membrane in a portion of the neutrophils following incubation with cytochalasin B and 2.5 minutes of FMLP stimulation (Figure 6). The redistribution of label to the plasma membrane, with some cells very strongly stained and others not stained, is similar to the distribution demonstrated via flow cytometry, ie, a heterogenous population. Further studies were undertaken to examine the relationship of the proteins recognized by anti-CD63 antibodies and D545. Flow cytometry demonstrated a similar pattern of translocation of CD63 to that of D545 following stimulation with FMLP, PMA, and A23187 (Figure 7). Additional experiments were per-
formed in which the stimulated cells were first exposed to D545 (in excess) and then to anti-CD63 antibodies conjugated to FITC. This showed nearly complete blocking of anti-CD63 antibody binding (Figure 8). Similarly, pre-exposure to anti-CD63 antibodies blocked to a significant extent the subsequent binding of D545 (Figure 8). In parallel experiments, antibody to lactoferrin was found to mobilize to the cell surface following stimulation by FMLP, but did not block the subsequent binding of either anti-CD63 or D545. Both D545 and anti-CD63 recognize a similar protein of about 47 kd on Western blots of neutrophil proteins. Staining with anti-CD63 in neutrophils was slightly lighter than staining with D545. However, the addition of unlabeled CD63 before peroxidaselabeled D545 blocked the staining using D545, suggesting 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 of gold in some monocyte granules (arrowv) (X 45, 000).
nearly identical epitopes (Figure 9). In parallel experiments, antibody to lactoferrin did not block the subsequent binding of D545. In the course of examining neutrophils, D545 binding was also observed on the membranes of eosinophil granules (Figure 10) and some granules in monocytes (Figure 11). We isolated neutrophils from a patient with Chediak-Higashi syndrome and performed immunofluorescence studies with D545. The staining in the permeabilized neutrophils demonstrated very large, brightly staining granules (Figure 12), significantly different than the diffuse punctate pattern seen in normal neutrophils.9 These large granules were similar to the large granules classically seen using routine staining and light microscopy in this disorder.
Discussion We have demonstrated that granulophysin is located on the membranes of azurophilic granules in human
neutrophils by immunocytochemistry at the light and electron microscopic levels. Stimulation of cells results in translocation and incorporation of this membrane protein into the plasma membrane. This relocation from storage granules in the resting neutrophil to the surface membrane in stimulated cells leads us, in agreement with Kuijpers et al,13 to consider granulophysin an activation antigen of neutrophils. We have shown that this protein is located in the azurophilic granules of neutrophils, based on several lines of evidence. First, maximal translocation is seen in response to FMLP in the presence of cytochalasin B, with lesser effect in response to PMA with or without cytochalasin B. This selective response to stimulation is considered typical of azurophilic granules, as specific granules discharge much more readily with PMA.6 Second, immunogold electron microscopy shows localization of this protein to the membrane of granules that contain myeloperoxidase. Finally, release of f-glucuronidase, a constituent of azurophilic 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 granulophysin to the plasma membrane as determined by flow cytometry. Recently, CD63, an antigen originally described as being present on platelet lysosomal membranes, has also been shown to be an activation antigen of neutrophils.13 Evidence from our laboratory suggests that CD63 and granulophysin may be the same protein.10 This is based on amino acid sequencing and platelet localization studies. We have demonstrated cross-reactivity between antibodies recognizing CD63 and granulophysin in neutrophils. CD63 has been shown to co-localize with myeloperoxidase in azurophilic granules and to translocate to the plasma membrane following preincubation with cytochalasin B and stimulation with FMLP. This translocation correlated with release of ,B-glucuronidase.13 Extrapolating from the data we have published regarding these proteins in platelets, it is likely that anti-CD63 antibodies and D545 are recognizing closely related epitopes, and possibly identical proteins. This is the first specific marker described for exocytosis of azurophilic granules. CD63 is a protein originally described as present in platelet lysosomes.1920 It was found to be identical to ME491, a melanoma-associated antigen.20'21 It is an integral membrane protein of platelet lysosomes and has considerable homology to a family of proteins that includes p24/CD9 (a surface marker present on a wide variety of hemopoietic and nonhemopoietic tissues), the leukocyte antigens CD37 and CD53, and TAPA-1 (target of an antiproliferative antibody-1). No
biological function is known for any of these molecules, although several functions have been proposed. Antibodies to p24/CD9 have been shown to cause platelet activation and aggregation; those to CD37 have been shown to modulate activation of B lymphocytes; anti-CD63 antibodies have been reported to inhibit monocyte adherence to serumcoated surfaces and aggregation of T and B lymphocytes.22 These studies have suggested a possible role for this family of proteins in signal transduction.23 Little is known about additional granule membrane proteins of azurophilic neutrophil granules. The only other known membrane component is CD68,24 a 11 0-kd transmembrane glycoprotein whose proximal domain has homology with lysosomal associated membrane protein-1 (LAMP-1 ).2526 However, Bainton and August27 found LAMP-1 and LAMP-2 in vesicles, not in the azurophil granules. The adhesion molecule, Mac-1, is present in the membrane of specific granules (75%) including gelatinase granules and 20% in secretory vesicles.28 Its role in adhesion and phagocytosis is well documented and has enabled specific granules to be studied in greater detail than azurophilic granules. In summary, we have identified granulophysin as an activation marker of neutrophils localized in the granule membrane of azurophilic granules. This will enable us to study the contribution of azurophilic granule exocytosis in the physiological functions of the neutrophil. In addition, it will be of interest to look at in vivo activation of neutrophils in inflammatory disorders such as adult respiratory distress syndrome,
1380
Cham et al
AJPJune 1994, Vol. 144, No. 6
rheumatoid arthritis, and immune complex disorders in which activation of neutrophils may play a role in pathophysiology of disease.
14.
Acknowledgments
15.
We thank Ms. Yvonne Jacques for her excellent technical assistance and Dr. E. Rector for expert assistance with flow cytometry analysis.
16. 17.
References 1. Rice WG, Kinkade JM Jr, Parmley RT: High resolution of heterogeneity among human neutrophil granules:
2.
3.
4.
5.
6.
7.
8. 9.
10.
11. 12.
13.
physical, biochemical, and ultrastructural properties of isolated fractions. Blood 1986, 68:541-555 Bainton DF, Farquhar MG: Origin of granules in polymorphonuclear leukocytes: two types derived from opposite faces of the Golgi complex in developing granulocytes. J Cell Biol 1966, 28:277-301 Bainton DF, Ullyot JL, Farquhar MG: The development of neutrophilic polymorphonuclear leukocytes in human bone marrow: origin and content of azurophil and specific granules. J Exp Med 1971, 134:907-934 Bainton DF, Farquhar MG: Segregation and packaging of granule enzymes in eosinophilic leukocytes. J Cell Biol 1970, 45:54-73 Boxer LA, Smolen JE: Neutrophil granule constituents and their release in health and disease. Hematology/ Oncology Clinics of North America, 1988, 2:101-134 Estensen RD, White JG, Holmes B: Specific degranulation of human polymorphonuclear leukocytes. Nature 1974, 248:347-348 Borregaard N, Lollike K, Kjeldsen L, Sengelov H, Bastholm L, Nielsen MH, Bainton DF: Human neutrophil granules and secretory vesicles. Eur J Haem 1993, 51: 189-198 Gallin JI: Neutrophil specific granules: a fuse that ignites the inflammatory response. Clin Res 1984, 32:320-328 Gerrard JM, Lint D, Sims PJ, Wiedmer T, Fugate RD, McMillan E, Robertson C, Israels SJ: Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermannsky-Pudlak syndrome. Blood 1991, 77:101-112 Nishibori M, Cham B, McNicol A, Shalev A, Gerrard JM: CD63 is present in platelet dense granules, is deficient in a patient with the Hermansky-Pudlak syndrome, and appears identical to granulophysin. J Clin Invest 1993, 91:1775-1782 Hatskelzon L, Dalal BI, Shalev A, Robertson C, Gerrard JM: Wide distribution of granulophysin epitopes in granules of human tissues. Lab Invest 1993, 68:509-519 Bainton DF, Gerrard JM: Granulophysin (GP) is a marker for the membranes of human neutrophil azurophilic granules (AG). J Cell Biol 1991, 115:304a Kuijpers TW, Tool ATJ, van der Schoot CE, Onderwater JJM, Roos D, Verhoeven AJ: Membrane surface antigen expression on neutrophils: a reappraisal of the
18.
19.
20.
21.
22.
23. 24.
25. 26. 27.
28.
use of surface markers for neutrophil activation. Blood 1991, 78:1105-1111 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 Hoehn SK, Kanfer JN: L-ascorbic acid and lysosomal acid hydrolase activities of guinea pig liver and brain. Can J Biochem 1978, 56:352-356 Hetherington SV, Spitznagel JK, Quie PG: An enzyme linked immunoassay (ELISA) for measurement of lactoferrin. J Immunol Methods 1983, 65:183-190 Bainton DF, Miller LJ, Kishimoto TK, Springer TA: Leukocyte adhesion receptors are stored in peroxidasenegative granules of human neutrophils. J Exp Med 1987, 166:1641-1653 Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227:680-685 Nieuwenhius HK, Van Oosterhout JJG, Rozemuller E, van lwaarden F, Sixma JJ: Studies with a monoclonal antibody against activated platelets: evidence that a secreted 53,000-mw lysosome-like granule protein is exposed on the surface of activated platelets in the circulation. Blood 1987, 70:838-845 Metzelaar MJ, Winjgaard PLJ, Peters PJ, Sixma JJ, Nieuwenhuis HK, Clevers HC: CD63 antigen: a novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. J Biol Chem 1991, 266:3239-3245 Hotta H, Ross AH, Huebner K, Isobe M, Wendeborn S, Chao MV, Ricciardi RP, Tsujimoto Y, Croce CM, Koprowski H: Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression. Cancer Res 1988, 48:2955-2962 Horejsi V, Vleek C: Novel structurally distinct family of leukocyte surface glycoproteins including CD9, CD37, CD53, and CD63. FEBS Lett 1991, 288:1-4 Metzelaar MJ, Clevers HC: Lysosomal membrane glycoproteins in platelets. Thromb Haemost 1992, 68: 378-382 Salto N, Pulford KAF, Breton-Gorius J, Masse JM, Mason DY, Cramer EM: Ultrastructural localization of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes. Am J Pathol 1991, 139:1053-1059 Holness CL, Simmons DL: Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 1993, 81:1607-1613 Fukuda M: Lysosomal membrane glycoproteins, structure, biosynthesis, and intracellular trafficking. J Biol Chem 1991, 266:21327-21330 Bainton DF, August JT: Multivesicular bodies of human neutrophils (pmn) not granules, immunolabel with the two major lysosomal membrane glycoproteins, hLAMP-1 and h-LAMP 2. J Histochem Cytochem (abst) 1988, 36:453 Sengelov H, Kjeldsen L, Diamond MS, Springer TA, Borregaard N: Subcellular localization and dynamics of Mac-1 in human neutrophils. J Clin Invest 1993, 92: 1467-1476
