Identification of phosphoproteins associated with human neutrophil granules following chemotactic peptide stimulation

Regulated exocytosis of neutrophil intracellular storage granules is necessary for neutrophil participation in the inflammatory response. The signal transduction pathways that participate in neutrophil exocytosis are complex and poorly defined. Several protein kinases, including p38 MAPK and the nonreceptor tyrosine kinases, Hck and Fgr, participate in this response. However, the downstream targets of these kinases that regulate exocytosis are unknown. The present study combined a novel inhibitor of neutrophil exocytosis with proteomic techniques to identify phosphopeptides and phosphoproteins from a population of gelatinase and specific granules isolated from unstimulated and fMLF-stimulated neutrophils. To prevent loss of granule-associated phosphoproteins upon exocytosis, neutrophils were pretreated with a TAT-fusion protein containing a SNARE domain from SNAP-23 (TAT-SNAP-23), which inhibited fMLF-stimulated CD66b-containing granule exocytosis by 100±10%. Following TAT-SNAP-23 pretreatment, neutrophils were stimulated with the chemotactic peptide fMLF for 0 min, 1 min, and 2 min. Granules were isolated by gradient centrifugation and subjected to proteolytic digestion with trypsin or chymotrypsin to obtain peptides from the outer surface of the granule. Phosphopeptides were enriched by gallium or TiO2 affinity chromatography, and phosphopeptides and phosphorylation sites were identified by reversed phase high performance liquid chromatography-electrospray ionization-tandem MS. This resulted in the identification of 243 unique phosphopeptides corresponding to 235 proteins, including known regulators of vesicle trafficking. The analysis identified 79 phosphoproteins from resting neutrophils, 81 following 1 min of fMLF stimulation, and 118 following 2 min of stimulation. Bioinformatic analysis identified a potential Src tyrosine kinase motif from a phosphopeptide corresponding to G protein coupled receptor kinase 5 (GRK5). Phosphorylation of GRK5 by Src was confirmed by an in vitro kinase reaction and by precursor ion scanning for phospho-tyrosine specific immonium ions containing Tyr251 and Tyr253. Immunoprecipitation of phosphorylated GRK5 from intact cells was reduced by a Src inhibitor. In conclusion, targets of signal transduction pathways were identified that are candidates to regulate neutrophil granule exocytosis.     

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED₅₀ of 15, 31, and 28 nM, respectively, with maximal response at 10^–7 M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC₅₀ for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis. human; cell activation     

Comparison of alternative analytical techniques for the characterisation of the human serum proteome in HUPO Plasma Proteome Project

Based on the same HUPO reference specimen (C1-serum) with the six proteins of highest abundance depleted by immunoaffinity chromatography, we have compared five proteomics approaches, which were (1) intact protein fractionation by anion-exchange chromatography followed by 2-DE-MALDI-TOF-MS/MS for protein identification (2-DE strategy); (2) intact protein fractionation by 2-D HPLC followed by tryptic digestion of each fraction and microcapillary RP-HPLC/microESI-MS/MS identification (protein 2-D HPLC fractionation strategy); (3) protein digestion followed by automated online microcapillary 2-D HPLC (strong cation-exchange chromatography (SCX)-RPC) with IT microESI-MS/MS; (online shotgun strategy); (4) same as (3) with the SCX step performed offline (offline shotgun strategy) and (5) same as (4) with the SCX fractions reanalysed by optimised nanoRP-HPLC-nanoESI-MS/MS (offline shotgun-nanospray strategy). All five approaches yielded complementary sets of protein identifications. The total number of unique proteins identified by each of these five approaches was (1) 78, (2) 179, (3) 131, (4) 224 and (5) 330 respectively. In all, 560 unique proteins were identified. One hundred and sixty-five proteins were identified through two or more peptides, which could be considered a high-confidence identification. Only 37 proteins were identified by all five approaches. The 2-DE approach yielded more information on the pI-altered isoforms of some serum proteins and the relative abundance of identified proteins. The protein prefractionation strategy slightly improved the capacity to detect proteins of lower abundance. Optimising the separation at the peptide level and improving the detection sensitivity of ESI-MS/MS were more effective than fractionation of intact proteins in increasing the total number of proteins identified. Overall, electrophoresis and chromatography, coupled respectively with MALDI-TOF/TOF-MS and ESI-MS/MS, identified complementary sets of serum proteins.     

Defensin-rich granules of human neutrophils: characterization of secretory properties

The various granule subtypes of the human neutrophil differ in propensity for exocytosis. As a rule, granules formed at late stages of myelopoiesis have a higher secretory potential than granules formed in more immature myeloid cells. Neutrophils contain four closely related alpha-defensins, which are stored in a subset of azurophil granules. These defensin-rich azurophil granules (DRG) are formed later than defensin-poor azurophil granules, near the promyelocyte/myelocyte transition. In order to characterize the secretory properties of DRG, we developed a sensitive and accurate ELISA for detection of the neutrophil alpha-defensins HNP 1-3. This allowed us to quantify the exocytosis of alpha-defensins and markers of azurophil (myeloperoxidase), specific (lactoferrin) and gelatinase (gelatinase) granules from neutrophils stimulated with different secretagogues. The release pattern of alpha-defensins correlated perfectly with the release of myeloperoxidase and showed no resemblance to the exocytosis of lactoferrin or gelatinase. This finding was substantiated through subcellular fractionation experiments. In conclusion, despite a distinct profile of biosynthesis, DRG are indistinguishable from defensin-poor azurophil granules with respect to exocytosis. Thus, in contrast to peroxidase-negative granules, azurophil granules display homogeneity in their availability for extracellular release.     

Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils.

In order to identify cytosolic proteins involved in control of granule exocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol from neutrophils in the presence or absence of Ca2+. After washing, proteins were eluted from the organelles by EGTA. Annexins I, II, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily exocytosed organelle of neutrophils. Ca(2+)-dependent association of cytosolic proteins to different granule subsets may control differential exocytosis of granules.     

Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8.

Human neutrophils were found to release a 91-kDa gelatinase that is serologically related to tumor-derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91-kDa active enzyme was further separated from other stainable protein bands by classical SDS PAGE and blotting to a solid support. Amino-terminal sequence analysis of blotted proteins showed that the 91-kDa enzyme is a truncated form of tumor-derived 92-kDa gelatinase (type IV collagenase), lacking eight residues at the NH2-terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin-binding part of the molecule is restricted to the amino-terminal third. Exocytosis of gelatinase-containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12-myristate 13-acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophil-activating peptide interleukin-8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.     

Gelatinase Granules of Neutrophil Granulocytes

The neutrophil contains numerous granules of various composition and structure. For decades, the neutrophil was believed to contain only two granule types, peroxisomes, or peroxidase-positive granules, and peroxidase-negative granules. Later, existence of the third type distinguished by the presence of gelatinase hydrolyzing collagen and gelatin was proposed. Gelatinase was found in the granules that are lighter as compared to the common peroxidase-negative granules and represent their subpopulation. In addition to gelatinase, these granules contain beta-2 microglobulin, cytochrome b ₅₅₈, as well as receptor and adhesion proteins. Upon stimulation by inflammatory mediators, the gelatinase granules are secreted before the common peroxidase-negative granules. Their exocytosis mediates delivery of new adhesion proteins to the plasma membrane, which is required for maintenance of permanent and fast cell adhesion to the endothelium. The released gelatinase allows the neutrophil to penetrate through the basement membrane of the endothelium.     

The Rab27a effectors JFC1/Slp1 and Munc13-4 regulate exocytosis of neutrophil granules

Neutrophil granules contain secretory molecules that contribute to the implementation of all neutrophil functions. The molecular components that regulate the exocytosis of neutrophil granules have not been characterized. In this study, using small interfering RNA gene-targeting approaches and granulocytes from genetically modified mice, we characterized the Rab27a effectors JFC1/Slp1 and Munc13-4 as components of the exocytic machinery of granulocytes. Using total internal reflection fluorescence microscopy analysis, we show that Rab27a and JFC1 colocalize in predocked and docked vesicles in granulocytes. Next, we demonstrate that JFC1-downregulated granulocytes have impaired myeloperoxidase secretion. Using immunological interference, we confirm that JFC1 plays an important role in azurophilic granule exocytosis in human neutrophils. Interference with Rab27a but not with JFC1 impaired gelatinase B secretion in neutrophils, suggesting that a different Rab27a effector modulates this process. In similar studies, we confirmed that Munc13-4 regulates gelatinase secretion. Immunofluorescence analysis indicates that Munc13-4 localizes at secretory organelles in neutrophils. Using neutrophils from a Munc13-4-deficient mouse model (Jinx), we demonstrate that Munc13-4 plays a central role in the regulation of exocytosis of various sets of secretory organelles. However, mobilization of CD11b was not affected in Munc13-4-deficient neutrophils, indicating that secretory defects in these cells are limited to a selective group of exocytosable organelles.     

Beta2 integrins target Rap GTPases to the plasma membrane by means of degranulation

We report the novel observation that engagement of β2 integrins on human neutrophils is accompanied by increased levels of the small GTPases Rap1 and Rap2 in a membrane-enriched fraction and a concomitant decrease of these proteins in a granule-enriched fraction. In parallel, we observed a similar time-dependent decrease of gelatinase B (a marker of specific and gelatinase B-containing granules) but not myeloperoxidase (a marker of azurophil granules) in the granule fraction, and release of lactoferrin (a marker of specific granules) in the extracellular medium. Furthermore, inhibition of Src tyrosine kinases, or phosphoinositide 3-kinase with PP1 or LY294002, respectively, blocked β2 integrin-induced degranulation and the redistribution of Rap1 and Rap2 to a membrane-enriched fraction. Consequently, the β2 integrin-dependent exocytosis of specific and gelatinase B-containing granules occurs via a Src tyrosine kinase/phosphoinositide 3-kinase signaling pathway and is responsible for the translocation of Rap1 and Rap2 to the plasma membrane in human neutrophils.     

A dual role for diacylglycerol kinase generated phosphatidic acid in autoantibody-induced neutrophil exocytosis

Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.     

Annexin I-induced aggregation of human neutrophil gelatinase granules

The ability of neutrophil cytosol to induce the aggregation of gelatinase granules of human neutrophils was studied. The cytosol was found to induce the Ca(2+)-dependent aggregation of granules. The stimulatory effect of cytosol was considerably reduced in the presence of the monoclonal antibody recognizing annexin I. Annexin I is a mediator of Ca(2+)-dependent aggregation of gelatinase granules and probably participate in granule secretion.     

Involvement of Munc18 isoforms in the regulation of granule exocytosis in neutrophils

Human neutrophil granule exocytosis mobilizes a complex set of secretory granules. This involves different combinations of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins to facilitate membrane fusion. The control mechanisms governing the late fusion steps are still poorly understood. Here, we have analyzed SNARE-interacting Sec1/Munc18 (SM) family members. We found that human neutrophils express Munc18-2 and Munc18-3 isoforms and that Munc18-2 interacts with the target-SNARE syntaxin 3. Munc18-2 was associated preferentially with primary granules but could also be found with secondary and tertiary granules, while Munc18-3 was majorily associated with secondary and tertiary granules. Ultrastructural analysis showed that both Munc18-2 and Munc18-3 were often located in close proximity to their respective SNARE-binding partners syntaxin 3 and syntaxin 4. Both isoforms were also found in plasma membrane fractions and in the cytosol, where they associate with cytoskeletal elements. Upon stimulation, Munc18-2 and Munc18-3 redistributed and became enriched on granules and in the plasma membrane. Munc18-2 primary granule exocytosis can be blocked by introduction of Munc18-2-specific antibodies indicating a crucial role in primary granule fusion. Our results suggest that Munc18-2 acts as a regulator of primary granule exocytosis, while Munc18-3 may preferentially regulate the fusion of secondary granules.     

Role of vesicle-associated membrane protein-2, through Q-soluble N-ethylmaleimide-sensitive factor attachment protein receptor/R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor interaction,in the exocytosis of specific and tertiary granules of human neutrophils

We have examined the role of the R-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) synaptobrevin-2/vesicle-associated membrane protein (VAMP)-2 in neutrophil exocytosis. VAMP-2, localized in the membranes of specific and gelatinase-containing tertiary granules in resting human neutrophils, resulted translocated to the cell surface following neutrophil activation under experimental conditions that induced exocytosis of specific and tertiary granules. VAMP-2 was also found on the external membrane region of granules docking to the plasma membrane in activated neutrophils. Specific Abs against VAMP-2 inhibited Ca(2+) and GTP-gamma-S-induced exocytosis of CD66b-enriched specific and tertiary granules, but did not affect exocytosis of CD63-enriched azurophilic granules, in electropermeabilized neutrophils. Tetanus toxin disrupted VAMP-2 and inhibited exocytosis of tertiary and specific granules. Activation of neutrophils led to the interaction of VAMP-2 with the plasma membrane Q-SNARE syntaxin 4, and anti-syntaxin 4 Abs inhibited exocytosis of specific and tertiary granules in electropermeabilized neutrophils. Immunoelectron microscopy showed syntaxin 4 on the plasma membrane contacting with docked granules in activated neutrophils. These data indicate that VAMP-2 mediates exocytosis of specific and tertiary granules, and that Q-SNARE/R-SNARE complexes containing VAMP-2 and syntaxin 4 are involved in neutrophil exocytosis.     

Peroxynitrite regulates exocytosis of neutrophil granules

Peroxynitrite is formed in the organism by activated neutrophils as a result of the enhanced production of nitrogen monoxide and superoxide anion radical in the inflammation foci. Since peroxynitrite modifies the structure of macromolecules, including the elements of actin cytoskeleton, it can influence signal transduction pathways that regulate intracellular granule exocytosis. In this paper we explore a dual effect of peroxynitrite on the processes of neutrophil degranulation by the methods of flow cytometry, light microscopy, and atomic force microscopy. We showed that peroxynitrite at concentrations less than 300 μM activated graded exocytosis of neutrophil intracellular granules, which resulted in the enhancement of neutrophil adhesion to the substrate, cell spreading on the substrate, and activation of neutrophil ability to kill microorganisms. Peroxynitrite at higher concentrations inhibited exocytosis of neutrophil granules and hindered cell adhesion to the substrate. The character of influence of the specific agents, such as colchicine and cytochalasin that selectively disrupt cytoskeletal structures, on peroxynitrite-induced changes in neutrophil morphology indicates an important role of actin cytoskeleton in the regulation of intracellular granule exocytosis induced by peroxynitrite. Our results support the hypothesis suggesting that peroxynitrite is a natural regulator of neutrophil effector functions.     

Proteomic analysis of synaptosomes using isotope-coded affinity tags and mass spectrometry

Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.     

Combinatorial SNARE complexes modulate the secretion of cytoplasmic granules in human neutrophils

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.     

Low molecular weight GTP-binding proteins in human neutrophil granule membranes

Degranulation of neutrophils involves the differential regulation of the exocytosis of at least two populations of granules. Low molecular weight GTP-binding proteins (LMW-GBPs) have been implicated in the regulation of vesicular traffic in the secretory pathways of several types of cells. In the present study we identify distinct subsets of LMW-GBPs associated with the membranes of neutrophil-specific and azurophilic granules. Ninety-four percent of total [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was equally distributed between the plasma membrane and cytosol with the remaining 6% localized in the granules. In contrast, the cytosol contained only 10% of the total GTPase activity while the specific granules accounted for 13%. [alpha-32P]GTP binding to proteins transferred to nitrocellulose revealed LMW-GBPs in all fractions except the azurophilic granules. The specific granules contained three out of four bands which were found in the plasma membrane; these ranged from 20 to 23 kDa and all were resistant to alkaline extraction. Photoaffinity labeling with [alpha-32P]8-azido-GTP in the presence of micromolar Al3+ identified proteins of 25 and 26 kDa unique to azurophilic granules; these could not be labeled with [alpha-32P]8-azido-ATP and could be extracted by acidic but not alkaline pH. Botulinum C3-mediated [32P]ADP-ribosylation identified proteins of 16, 20, and 24 kDa both in plasma membranes and those of specific granules. An anti-ras monoclonal antibody, 142-24E5, recognized a 20-kDa protein localized to the plasma and specific granule membranes which could not be extracted by alkaline pH, was not a substrate for botulinum C3 ADP-ribosyltransferase, and was translocated from specific granules to plasma membrane after exposure of neutrophils to phorbol myristate acetate. We conclude that neutrophil-specific and azurophilic granules contain distinct subsets of LMW-GBPs which are uniquely situated to regulate the differential exocytosis of these two compartments.     

Intracellular organelle motility and membrane fusion processes in human neutrophils upon cell activation

Release and subcellular fractionation experiments indicate that fusion of a novel tertiary granule with the plasma membrane is concomitant with human neutrophil activation. Phorbol 12-myristate 13-acetate (PMA) induced a respiratory burst in human neutrophils as well as a high release of gelatinase, a marker of the tertiary granule. Preincubation of neutrophils with cytochalasin E induced a partially activated or 'primed' state, in which cells were unable to generate superoxide anion, but showed a reduced latency period for this activity. Fusion of tertiary granules with the cell surface also occurred during priming, although to a lesser extent than in PMA stimulation. The rapid tertiary granule degranulation, preceding that of specifics and azurophilics, seems to play an important role in the functionality and secretory properties of human neutrophils.     

Identification of CD66a and CD66b as the major galectin-3 receptor candidates in human neutrophils.

The mammalian lectin galectin-3 is a potent stimulus of human neutrophils, provided that the receptor(s) for the lectin has been mobilized to the cell surface before activation. We have recently shown that the receptors for galectin-3 are stored in intracellular mobilizable granules. Here we show supportive evidence for this in that DMSO-differentiated (neutrophil-like) HL-60 cells, which lack gelatinase and specific granules, are nonresponsive when exposed to galectin-3. Neutrophil granules were subsequently used for isolation of galectin-3 receptors by affinity chromatography. Proteins eluted from a galectin-3-Sepharose column by lactose were analyzed on SDS-polyacrylamide gels and showed two major bands of 100 and 160 kDa and a minor band of 120 kDa. By immunoblotting, these proteins were shown to correspond to CD66a (160 kDa), CD66b (100 kDa), and lysosome-associated membrane glycoprotein-1 and -2 (Lamp-1 and -2; 120 kDa). The unresponsive HL-60 cells lacked the CD66 Ags but contained the Lamps, implying that neutrophil CD66a and/or CD66b may be the functional galectin-3 receptors. This conclusion was supported by the subcellular localization of the CD66 proteins to the gelatinase and specific granules in resting neutrophils.