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*
Department of Immunology and Parasitology, Yamagata University School of Medicine, Yamagata, Japan;
Department of Immunoregulation, Research Institute for Microbial Diseases, Osaka University, Suita, Japan; and
Department of Biochemistry, Fukushima Medical College, Fukushima, Japan
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As a means of studying these problems, we established a mAb, tentatively designated 3H9, that modulates neutrophil adhesion and induces its motility. The 3H9-reactive molecule (3H9RM) is mainly expressed on neutrophils and monocytes that are the most motile and can easily extravasate (8). Activation of ß2 integrin seems to be a prerequisite for modulation of neutrophil function by this mAb, because an increase in intracellular free calcium ([Ca2+]in) evoked by 3H9 treatment was observed only when ß2 integrin had been preactivated through cross-linking of CD18 by a relevant mAb (9). The finding that 3H9 not only inhibits neutrophil adhesion but also induces motility led us to speculate that this mAb may not block the reaction between CAM(s) and their ligand(s), but recognize a molecule that may regulate neutrophil adherence and migration.
In the present report, we describe the molecular cloning of 3H9RM and discuss the biological significance of this molecule in the regulation of adherence and migration of neutrophils.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% heat-inactivated FCS (BioWhittaker, Walkersville, MD) was used as the incubation medium. Reagents and Abs were purchased from the companies in parentheses. These were fibrinogen and FMLP (Sigma, St. Louis, MO); phosphatidylinositol phospholipase C (PIPLC), digoxigenin (DIG)-11-dUTP, alkaline phosphatase-conjugated sheep anti-DIG Fab, and disodium 2-chloro-5-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4yl-1-phenyl phosphate (CDP-Star) (Boehringer Mannheim, Indianapolis, IN); dextran 200,000 and Triton X-100 (Wako, Osaka, Japan); human bone marrow cDNA library (cat. no. HL5005B), Marathon cDNA amplification kit, and human 12-lane multiple tissue Northern (MTN) Blot (cat. no. 7780-1) (Clontech Laboratories, Palo Alto, CA); mouse mAbs to human CD18 (MHM23, DAKO, Glostrup, Denmark); and TS1/18.1.2.11 (American Type Culture Collection, Manassas, VA). 3H9 mAb was obtained by immunizing BALB/c mice with phorbol myristate acetate-treated human peripheral blood neutrophils (8). TCY-3 is an IgG1 mouse mAb used as a control for 3H9 (8). Fab and F(ab')2 was obtained by the methods shown by Hagiwara et al. (10) and Lamoyi and Nisonoff (11), respectively.
Neutrophil adherence assay
Details of this assay have been described elsewhere (12). Briefly, Falcon 3072 plates (Becton Dickinson Labware, Franklin Lakes, NJ) were coated with 50 µg/ml FCS or fibrinogen for 2 h at 37°C and then washed with RPMI 1640 medium. A total of 50 µl of neutrophil suspensions (5 x 105 cells/well) in RPMI 1640, 50 µl of a solution of stimulants, and 100 µl of mAb solutions were added to FCS or fibrinogen-coated plates and incubated for varying periods of time at 37°C. The percentage of adherence inhibition was calculated using the following formula: {percent inhibition = [(OD570 nm in the presence of TCY-3 - OD570 nm in the presence of 3H9)/OD570 nm in the presence of TCY-3] x 100}.
Neutrophil transendothelial migration
We followed the method described by Yong and Linch (13). HUVEC monolayers on cell culture inserts (Nippon Becton Dickinson, Tokyo, Japan) were preincubated for 4 h with human rIL-1ß (10 U/ml) and washed three times with warm PBS before being transferred to wells containing fresh medium (30% FCS, RPMI 1640) for migration experiments. A total of 300 µl of 51Cr-labeled neutrophil suspension (1.5 x 106 cells) containing 10 µg/ml 3H9 or a control mAb, TCY-3, was placed in the upper chamber of cell culture inserts and the culture plate was incubated for varying periods of time at 37°C in a humidified 5% CO2 atmosphere in the presence of 1 x 10-7 M FMLP. At the end of the incubation period, the cell culture inserts were removed and the contents in the lower chamber were collected. After lysis of the migrated cells with 1% Triton X-100, the samples were counted in a gamma-counter (Wallac 1470, Wizard, Turku, Finland). The percentage of migration inhibition was calculated using the following formula: {percent inhibition = [(cpm in the presence of TCY-3 - cpm in the presence of 3H9)/cpm in the presence of TCY-3] x 100}.
EiC3b rosette assay
The details of this assay have been described elsewhere (14). Briefly, to obtain C3b, C3 purified from fresh human serum was incubated with factors B and D. Sensitized sheep erythrocytes were incubated sequentially with Clgp, C4hu, and oxidized C2hu to prepare EAC14oxy2. These complement-coated sheep erythrocytes were then incubated with C3 and used as EC3b. Purified neutrophils (4 x 105) were mixed with EC3b in RPMI 1640 medium containing 1 mg/ml BSA. The tubes were rotated for 30 min at 37°C, and the number of rosette-forming cells was counted under microscopy. Neutrophils bearing more than two RBC were considered rosette-forming cells. The percentage of rosette inhibition was calculated using the following formula: {percent inhibition = [(percent of rosette-forming cells in the presence of TCY-3 and FMLP - percent of rosette-forming cells in the presence of 3H9 and FMLP)/percent of rosette-forming cells in the presence of TCY-3 and FMLP] x 100}.
F actin distribution in cells as seen under confocal microscopy
Human neutrophils (1 x 106/ml) in an incubation buffer (150 mM NaCl, 5 mM KCl, 1 mM MgCl2, 10 mM glucose, 20 mM HEPES, pH 7.4) were treated with 10 µg/ml of 3H9 or control TCY-3 for 15 min on ice. In another experimental group, 50 µg/ml anti-CD18 mAb (TS1/18.1.2.11) were also added to 3H9. The reaction mixtures were then incubated on fibronectin-coated coverslips for 0, 15, or 30 min at 37°C. The reason why we used fibronectin is that this matrix protein was better than fibrinogen to obtain sharp figures in a confocal microscopy, and neutrophil adherence to fibronectin is also ß2 integrin-dependent (15). Following incubation, coverslips were treated for 3 min on ice with University of Wisconsin solution (100 mM lactobionic acid, 30 mM raffinose, 3 mM glutathione, 5 mM adenosine, 1 mM allopurinol, 25 mM KH2PO4, 105 mM KOH, 20 mM NaOH, and 5 mM MgSO4, pH 7.4) containing 0.1% saponin and were then fixed with 4% paraformaldehyde for another 30 min on ice. Samples were stained for 120 min on ice with immunostaining buffer (Dulbecco’s PBS supplemented with 10% heat-inactivated FCS and 2 mg/ml BSA) containing 0.3% rhodamin-phalloidin. The specimens were examined with a confocal laser microscope (TCS, Lica, Hiderberg, Germany).
Screening of a cDNA library, DNA sequencing, and sequence analysis
Using a method described by Mierendorf et al. (16), screening of
a cDNA library constructed from human bone marrow in the phage vector
gt11 (Clontech) was performed using 3H9 mAb as a specific probe.
Positive phage DNA was purified using LambdaSoab (Promega, Madison, WI)
affinity chromatography according to the manufacturer’s protocol.
Before sequencing, the phage DNAs were digested with EcoRI,
purified using low-gelling-temperature agarose gel electrophoresis,
then subcloned into pBluescript II SK(-) phagemid vector (Stratagene,
La Jolla, CA). Deletion mutants were prepared from both strands by
unidirectional digestion with exonuclease III and mung bean nuclease.
All clones were sequenced by the dideoxynucleotide termination method
(17) using a DNA sequencer (Model 373A, Applied Biosystems, Foster
City, CA). To obtain a full-length cDNA, 5' and 3' rapid amplification
of cDNA ends (RACE) (18) were conducted with a Marathon cDNA
amplification kit (Clontech) as follows. Using 1 µg of poly(A) + RNA
from human peripheral blood cells, cDNA was synthesized with avian
myeloblastosis virus reverse transcriptase and the oligo(dT) primer
(5'-TTCTAGAATTCAGCGGCCGC(T)30NN-3'). The cDNA was blunt-ended and
ligated to the adaptor oligonucleotide
(5'-CTAATACGACTCACTAGGGCTCGAGCGGCCGCCCGGGCAGGT-3').
To obtain the 5'-RACE fragment, the cDNA was amplified by PCR with
anchor primer 1 (5'-CCATCCTAATACGACTCACTATAGGGC-3') and the antisense
primer corresponding to nucleotides 367–389 of 3H9RM
(glycosylphosphatidyl-inositol (GPI)-80 from Fig. 3
downward, see
below) cDNA. A nested PCR was then performed with anchor primer 2
(5'-ACTCACTATAGGGCTCGAGCGGC-3') and an antisense primer corresponding
to nucleotides 459–480 of 3H9RM cDNA. To obtain the 3'-RACE fragment,
cDNA was amplified by PCR with anchor primer 1 and a sense primer
corresponding to nucleotides 148–168 of 3H9RM cDNA. Both the 5'-RACE
fragment and the 3'-RACE fragment were fused at the overlapping regions
by PCR, then amplified with adaptor primer 1 and the oligo(dT) primer
to obtain the full length of 3H9RM cDNA. The nucleotide sequence and
the secondary structure of the deduced amino acid sequence were then
compared with those previously reported for other proteins by
computer-aided sequence analysis using the GENETYX-MAC program
(Software Development, Tokyo, Japan).
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Preparation of the DIG-labeled cDNA fragment synthesized by PCR was used as a probe for the detection of mRNA as described previously (19). In brief, 3H9RM (GPI-80) cDNA (see below) was amplified with primer pairs (a sense primer corresponding to nucleotides 148–168 of the 3H9RM (GPI-80) cDNA and an antisense primer corresponding to nucleotides 367–389 of the 3H9RM (GPI-80) cDNA in the presense of DIG-11-dUTP. As the positive control, a human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (20) probe was used. Human 12-lane MTN Blot (Clontech) was hybridized with the DIG-labeled cDNA fragment at 68°C for 6 h as described previously (19). A positive signal(s) was visualized by CDP-Star according to the method described by Engler-Blum et al. (21).
Amino acid sequencing of purified 3H9RM (GPI-80)
Purified neutrophils were lysed in ice-cold 20 mM phosphate buffer, pH 7.0, containing 60 mM N-octyl-ß-D-glucoside, 1 mM PMSF, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. Insoluble fractions were cleared at 13,000 x g for 60 min, and the supernatants were used as a source of crude neutrophil Ags for purification of 3H9RM by affinity chromatography. The supernatants were loaded onto a 3H9-coupled Affi-Gel 10 column (Bio-Rad, Hercules, CA). After washing, bound 3H9RM was eluted with elution buffer (0.1 M acetic acid). After dialyzing against 20 mM phosphate buffer, pH 7.0, 3H9RM-positive fractions were boiled in SDS sample buffer with 2 ME, subjected to electrophoresis through 10% SDS-PAGE, and subsequently blotted onto a polyvinylidene difluoride membrane. The proteins were stained with Coomassie Brilliant Blue R250 and the amino acid sequences were determined using the HPG 1005A protein sequencing system (Hewlett Packard, Meriden, CT).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We examined the effects of 3H9 on FMLP-stimulated neutrophil
adherence to plastic plates precoated with FCS or fibrinogen. These
adherences were ß2 integrin-dependent, because at any
time of incubation the adherence was completely inhibited by
anti-CD18 mAb. In contrast, 3H9 enhanced the early phase (15 min)
of these ß2 integrin-dependent neutrophil adhesions, but
inhibited them at the late phase (60 and 120 min). On the other hand, a
mAb to HLA did not affect this adhesion (Fig. 1A). To observe the
concentration dependency of the effects of 3H9, neutrophils were
incubated for 15 or 60 min in the presence of FMLP and varying
concentrations of 3H9. After 15 min incubation, 3H9 enhanced the
adherence from the point of 100 ng/ml. On the other hand, after
60 min incubation 3H9 inhibited the adherence from the point
of 50 ng/ml (Fig. 1B). Then, we examined whether the whole
IgG molecule was required for the observed phenomena and whether
cross-linking of 3H9 affected the effects of 3H9. When the addition of
5 µg/ml 3H9 whole IgG1 molecule inhibited
41.0 ± 6.4% of neutrophil adhesion in the presence of
10-6 M FMLP at 60 min incubation, the addition of the same
concentrations of its Fab, F(ab')2 and whole
IgG1 in the presence of 5 mg/ml anti-mouse Ig showed
31.8 ± 0.6, 35.8 ± 5.4, and 32.9 ± 4.6% inhibition,
respectively. The result that Fab and F(ab')2 of 3H9 showed
neutrophil adherence inhibition comparable to whole 3H9
IgG1 and the addition of anti-mouse Ig did not augment
the effect of 3H9 suggests that the Fc portion of this mAb or
cross-linking of 3H9RM by 3H9 is not required for the effects of 3H9.
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C).
To explore the mechanisms involved in the above phenomena, we used the
iC3b rosette assay to examine the effect of 3H9 on the binding avidity
of FMLP-stimulated neutrophils to iC3b, a ligand to Mac-1 and p150, 95.
The number of iC3b neutrophils forming rosettes in the presence of FMLP
was reduced by the addition of anti-CD18 mAb. In contrast, when 3H9
was added to FMLP, it enhanced iC3b binding activity of neutrophils at
5 and 15 min but inhibited it at 30 and 60 min (Fig. 1D).
The effects of 3H9 on iC3b neutrophil binding were similar to those
seen with ß2 integrin-dependent neutrophil adhesion in
that 3H9 enhanced binding in the early phase, but inhibited it in the
late phase. 3H9 modulation of the iC3b binding avidity of neutrophils
did not occur via a change in the level of expression of
ß2 integrin on these cells, because treatment with 3H9
did not affect the expression of CD18 on neutrophil surfaces (data not
shown).
3H9 induces a change in F actin distribution in neutrophils
Our previous results showed that 3H9 induced neutrophil motility,
although the total F actin content of these cells did not change with
3H9 treatment, unlike treatment with FMLP (9). To assess the effect of
3H9 on the cytoskeleton, we examined F actin localization in
neutrophils treated with this mAb alone, because if treatment were
conducted in the presence of FMLP, it would surely make the results
more difficult to interpret inasmuch as FMLP itself induces changes in
F actin localization. Many protrusions were observed in the F actin of
cells incubated with this Ab for 30 min at 37°C, but only
small changes were detected in cells treated with a control Ab, TCY-3,
at this point in time. The change in F actin distribution induced by
3H9 was ß2 integrin-dependent because it was inhibited by
anti-CD18 mAb (Fig. 2). This result
suggests that 3H9RM is involved in ß2 integrin-dependent
cytoskeletal remodeling.
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An attempt was then made to isolate the cDNA, which encodes 3H9RM
using a human bone marrow cDNA expression library with 3H9 mAb as the
probe. A clone isolated using molecular cloning techniques, encodes a
522 amino acid sequence (Fig. 3).
Distribution of the 3H9RM message was examined by Northern blot
analysis as shown in Fig. 4. A single
signal was observed mainly at the position of 
2.0 kb in mRNA
obtained from human peripheral blood leukocytes. This value is similar
to the total number of nucleotides (2034 bp) in the 3H9RM cDNA. Spleen,
lung, placenta, colon, and liver, which contain relatively large
amounts of blood, also showed a very faint signal.
Hydrophobicity plot analysis revealed a strong hydrophobicity at the
N-terminal portion of the deduced polypeptide (data not shown). These
first 23 amino acids form a hydrophobic core, typical of the signal
sequence of an integral membrane protein. Using the sliding
window/matrix scoring method and the -1, -3 rule for predicting
signal cleavage sites (22, 23), the amino acid positions between Gln-23
and Asp-24 were suggested as cleavage sites for the signal peptidase.
The N-terminal amino acid sequence of the affinity-purified 3H9RM is
shown in Table I. These results allowed
us to conclude that the cDNA sequence consists of a full-length mature
3H9RM coding region (499 amino acids) with a leader peptide of 23 amino
acids. Furthermore, the m.w. deduced from this clone is 58,000, which
is identical with that obtained by Western blotting after treatment of
3H9RM with N-glycanase (data not shown). Hydropathy plot
analysis revealed two signal sequences at the N- and C-terminals of the
molecule, suggesting that 3H9RM is a GPI-anchored protein. We examined
neutrophil expression of 3H9RM after treatment with PIPLC and found it
to have been almost completely abolished (Fig. 5). We also examined the expression of
this molecule on neutrophils of patients with paroxysmal nocturnal
hemoglobinuria (PNH), which is a typical GPI-anchored protein-deficient
disease. In two of these patients, 50% of neutrophils lacked 3H9
expression, a finding quite similar to that obtained for CD59, a
GPI-anchored protein (Fig. 6). Based on
the above mentioned results, this novel GPI-anchored protein is now
designated as GPI-80 for 3H9RM. An amino acid homology search revealed
that GPI-80 is 39% homologous to human biotinidase (24) (Fig. 7). Furthermore, GPI-80 has 59% amino
acid sequence homology with Vanin-1, a recently reported GPI-anchored
perivascular molecule involved in thymus homing in mice (25) (Fig. 7),
which also showed 40% molecular homology with biotinidase. The
position of cysteine in these proteins is completely conserved.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular mechanisms involved in integrin-mediated cell adhesion
have been previously discussed from various standpoints (26, 27). Based
on the finding that mutations that eliminate the highly conserved
cytoplasmic domain of the 
subunit of IIbß2
integrin increase ligand-binding affinity, inside-out signaling has
been proposed as essential for the regulation of integrin functions
(28, 29), and a cytoplasmic regulator of LFA-1 integrin, cytohesin-1,
has been reported as a likely candidate for this inside-out signaling
(30). On the other hand, intermolecular reactions outside the plasma
membrane between the ectodomain of ß2 integrin and
GPI-anchored proteins such as urokinase-type plasminogen activator
(uPA) receptor (uPAR) (31, 32), Fc
RIIIB (CD16b) (33, 34, 35), and CD14
(36) have recently been recognized as important in the regulation of
ß2 integrin function. Furthermore, the ß2
integrin-mediated intermolecular reaction is considered to be a
prerequisite for signal transduction through these GPI-anchored
proteins into the cytoplasm (37, 38). In addition, it has been proven
that uPAR is also involved in the regulation of ß1
integrin-dependent cell adhesion and migration (39).
The present finding that GPI-80 is a GPI-anchored protein that may be involved in the regulation of ß2 integrin-mediated adherence of neutrophils and our previous results showing that preactivation of ß2 integrin is required for a [Ca2+]in increase by cross-linking with 3H9 (9) are consistent with the fact that the [Ca2+]in increase induced by uPAR stimulation with uPA is only observable when ß2 integrin is also present on cell surfaces (31). Our preliminary results from light (Sakurai et al., unpublished observations) and electron microscopy (Sato et al., unpublished observations) showing that GPI-80 and ß2 integrin are colocalized on neutrophil surfaces may support the assumption that the effect of 3H9 is proximal to the functions of ß2 integrin. However, because we have no definitive evidence yet showing physical association of 3H9RM with ß2 integrins as shown for other integrin-associated GPI-anchored proteins, we do not know the mode of reaction of 3H9RM with integrins at this time.
Although little is known of the precise mechanisms involved in the sequential up- and down-regulation of ß2 integrin-dependent neutrophil adherence and transendothelial migration induced by 3H9 treatment, these may be at least partially due to the modulation of ß2 integrin avidity of the neutrophil for its ligand because the kinetics of cell adhesion, transendothelial migration, and iC3b rosette formation were similar in the sense that all these activities were enhanced at an early stage of 3H9 exposure and were inhibited at a late stage. Furthermore, these opposite effects of 3H9 may eliminate the possibility that it is the kind of mAb that activates ß2-dependent cell adhesion (40). Inasmuch as the ligands for GPI-anchored proteins that have relationships with integrin functions, uPAR, CD16b, and CD14; uPA, IgG, and LPS/LPS binding protein complex, respectively, are in plasma or sera, the GPI-80 ligand may also be in plasma or sera. Activity of the mAb, 3H9 may mimic the function of this unknown GPI-80 ligand to stimulate neutrophils. Regarding the possible mechanisms of GPI-80-mediated regulation of ß2 integrin-dependent cell adhesion and locomotion, although speculative, molecular interactions between GPI-80 and its hypothetical ligand(s) may modulate ß2 integrin-mediated functions of neutrophils, similar to the interactions between uPA and uPAR in myeloid cells (31, 32). Very recently, it has been reported that leukocyte recruitment via ß2 integrin in vivo is impaired in uPAR-deficient mice, suggesting that integrin associated GPI-anchored proteins are important for regulation of leukocyte recruitment (41). We would like to examine whether GPI-80 has some roles in vivo for the regulation of leukocyte extravasation through the establishment of GPI-80-overproducing or -deficient animals.
The homology between GPI-80 and Vanin-1 and homology of these two
molecules with biotinidase (Fig. 7) presents us with a puzzling but
interesting problem. We are unable at this point to speculate on the
involvement of biotinidase-like-functions in GPI-80 regulation of
integrin-mediated cell adhesion and motility, as already reported in
the case of Vanin-1, which has no biotinidase activity (25). However,
there is 60% molecular homology between GPI-80 and Vanin-1, and
these two molecules share a similar function in the sense that both may
be involved in the regulation of leukocyte trafficking. This suggests
the existence of a family of molecules that regulates leukocyte
extravasation through as yet unknown mechanisms. We are now searching
for other potential members of this family using genetic engineering
techniques.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Fujiro Sendo, Department of Immunology and Parasitology, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan. E-mail address:
3 Abbreviations used in this paper: CAM, cell adhesion molecule; GPI, glycosylphosphatidyl-inositol; 3H9, a mAb that reacts with GPI-80 and affects various neutrophil functions; 3H9RM, 3H9 reactive molecule; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; [Ca2+]in, intracellular Ca2+; PIPLC, phosphatidylinositol phospholipase C; PNH, paroxysmal nocturnal hemoglobinuria; DIG, digoxigenin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RACE, rapid amplification of cDNA end.
Received for publication August 6, 1998. Accepted for publication December 22, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gt11 libraries with antibodies. Methods Enzymol. 152:458.[Medline]
Lß2 integrin/LFA-1 binding to ICAM-1 induced by cytohesin-1, a cytoplasmic regulatory molecule. Cell 86:233.[Medline]
receptor type III (FcRIII, CD16) triggers cell activation through interaction with complement receptors. J. Immunol. 157:1184.[Abstract]
receptor type IIIB with complement receptor type III in fibroblast transfectants: evidence from lateral diffusion and resonance energy transfer studies. J. Mol. Biol. 247:597.[Medline]
III receptor in human neutrophils. Possible role of lectin-like interaction. J. Immunol. 150:3030.[Abstract]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, 
), a mAb to
monomorphic HLA-A.B.C (MB40.5, American Type Culture Collection) (
),
and TCY-3. OD values of FCS-coated group at 570 nm in the presence of
the control mAb, TCY-3, were 0.88 ± 0.04, 0.55 ± 0.08, or
0.34 ± 0.02 at 15, 60, or 120 min, respectively
(n = 3). OD values of the fibrinogen-coated group
in the presence of TCY-3 were 1.23 ± 0.06, 0.99 ± 0.05, or
0.50 ± 0.06 at 15, 60, or 120 min of incubation, respectively
(n = 3). Percentage of adherence inhibition was
calculated as follows: {percent inhibition of adherence = [(OD
values in the presence of the TCY-3 - OD values in the presence
of 3H9, anti-CD18 mAb or anti-HLA mAb)/OD values in the
presence of TCY-3] x 100}. *, p < 0.05.
B, 3H9 effects on neutrophil adherence at different
concentrations. Human neutrophils in plates precoated with FCS in RPMI
1640 were incubated with 10-6 M FMLP for 15 (•) or 60
(
