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(Journal of Leukocyte Biology. 2001;70:776-782.)
© 2001 by Society for Leukocyte Biology

Regulation of the very late antigen-4-mediated adhesive activity of normal and nonreleaser basophils: roles for Src, Syk, and phosphatidylinositol 3-kinase

Ronald P. Andrews, Christopher L. Kepley, Lama Youssef, Bridget S. Wilson and Janet M. Oliver

Department of Pathology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131

Correspondence: Ronald P. Andrews, Department of Pathology, University of New Mexico, School of Medicine, CRF Building, Rm. 203, 2325 Camino de Salud, Albuquerque, NM 87131. Email: randrews{at}salud.unm.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Normal human basophils express the integrin, VLA-4, and cross-linking their high-affinity IgE receptor, Fc{varepsilon}RI, increases their VLA-4-dependent adhesion to VCAM-1-transfected Chinese hamster ovary (CHO) cells. Here we show that the Fc{varepsilon}RI-mediated up-regulation of normal basophil VLA-4 adhesion is abolished by the Src inhibitor, PP1, the Syk inhibitor, ER-27319, and the phosphatidylinositol 3-kinase inhibitor, wortmannin. PP1, but not ER-27319 or wortmannin, also reduces basal adhesion and adhesion stimulated by chemotactic peptide, by Ca++ ionophores, and by phorbol myristate acetate (PMA). Nonreleaser basophils (the consistently Syk-deficient, variably Lyn-deficient, severely degranulation-impaired cells found in about 10% of donors) share the PP1 phenotype of lowered basal adhesion, no Fc{varepsilon}RI-mediated adhesion up-regulation, and reduced adhesive responses to chemoattractant ionophores and PMA. These results implicate Src kinases in the control of basal VLA-4 activity and place Syk and phosphatidylinositol 3-kinase in the pathway linking Fc{varepsilon}RI cross-linking to VLA-4 up-regulation. Both Src and Syk-regulated components of adhesion may be impaired in nonreleaser basophils.

Key Words: signal transduction • tyrosine kinase • Lyn • human


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Integrins are heterodimeric adhesion molecules whose ligand binding activity can be modulated rapidly, allowing cells to specify the timing and location of integrin-mediated adhesive events. Regulating the activity of very late antigen-4 (VLA-4), the {alpha}4ß1 integrin expressed on a wide range of leukocytes, is particularly complex and interesting. Most leukocytes maintain a constitutive level of VLA-4 activation and can be induced to increase this activity both by stimulation from the outside, such as in response to incubation with Mn2+ or with certain anti-VLA-4 monocloanl antibodies (mAbs) (8A2, T/S16) [1 , 2 ], or from the inside, such as in response to the engagement of signaling receptors or activating agents like the phorbol ester; phorbol myristate acetate (PMA); and the Ca2+ ionophores, ionomycin and A23187 [3 , 4 ]. Recent studies have shown that VLA-4 may occur in multiple activation states on individual cells and that low-affinity forms of VLA-4 may be conformationally responsive to ligand [5 , 6 ]. Despite this increasing understanding of integrin properties, no clear definition of how cells modulate VLA-4 activity and maintain differences in VLA-4 activation states is yet available.

The regulation of VLA-4 activity has been studied most extensively in T cells [7 , 8 ]. However, VLA-4 is also a prominent integrin of human basophils and these cells, too, are selectively recruited from the peripheral circulation during inflammation in part by the interaction of their VLA-4 with its counter-receptor, vascular cell adhesion molecule (VCAM)-1, expressed on activated endothelium [9 , 10 ]. Following extravasation, interactions between basophil VLA-4 and the separate VLA-4 counter-receptor, fibronectin, are very likely involved in retaining these cells at specific tissue sites [11 , 12 ]. In particular, basophils, generally rare cells in the circulation, are accumulated in the lung during late-phase allergic responses [13 ] and are particularly prominent in lungs of people who died from asthma (Kepley et al., unpublished results). Thus, a greater understanding of the regulation of basophil VLA-4-mediated adhesion is likely to be of practical importance for treating allergy and asthma.

Our previous work in human basophils focused primarily on the early events that initiate signaling through the high-affinity IgE receptor, Fc{varepsilon}RI. In these cells, cross-linking the tetrameric {alpha}ß{gamma}2 IgE receptor, Fc{varepsilon}RI, activates the signal-initiating kinase, Lyn, that phosphorylates Fc{varepsilon}RI ß and {gamma} subunit tyrosines, creating binding sites for the signal-propagating kinase, Syk. Activated Syk in turn interacts with multiple downstream enzymes and adaptors that ultimately stimulate functional responses, including Ca2+ mobilization, secretion, ruffling, and cytokine production [14 15 16 17 ].

Two discoveries, both reported in Kepley et al. [17 ] stimulated this study of the regulation of VLA-4 adhesive activity in normal and nonreleaser basophils. The first discovery was that cross-linking the high-affinity IgE receptor, Fc{varepsilon}RI, on normal basophils increases their VLA-4-dependent adhesive activity toward Chinese hamster ovary (CHO) cells transfected with the VLA-4 counter-receptor, VCAM-1, as measured in a conjugate assay [17 ]. In contrast, previous investigators had focused primarily on the contribution of ß2 integrin family members to the anti-IgE-mediated up-regulation of basophil adherence both to other basophils and to endothelium [18 , 19 ]. The second discovery was that VLA-4 levels are normal, but antigen-stimulated VLA-4-mediated adhesion to VCAM-1-CHO cells is impaired, in the "nonreleaser" basophils present in about 10% of normal blood donors. Impaired adhesion is only one of multiple defects in these cells. Other abnormalities include severely impaired secretory and ruffling responses to Fc{varepsilon}RI cross-linking, the absence of Fc{varepsilon}RI-mediated IL-4 production, and—very likely the reason for most of the functional defects—the absence of detectable protein levels of the tyrosine kinase, Syk, despite apparently normal Syk mRNA levels.

To establish specific requirements for Lyn, Syk, and PI 3-kinase in the control of basophil basal and stimulated VLA-4 adhesive activity, we used PP1, which inhibits Lyn and other Src kinase family members [20 , 21 ]; ER-27319, which prevents the interaction of Syk with Fc{varepsilon}RI [22 ]; and wortmannin, which inhibits phosphatidylinositol 3-kinase (PI 3-kinase) [23 , 24 ]. Our results implicate a Src kinase(s) in the control of basal VLA-4 activity and place Syk and PI 3-kinase in the pathway linking Fc{varepsilon}RI cross-linking to VLA-4 up-regulation. Src- and Syk-regulated components of adhesion may be impaired in nonreleaser basophils.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Monoclonal antibody HP2/1, a blocking antibody to the {alpha}4 subunit of the human {alpha}4ß1 integrin, VLA-4, was from Immunotech (Marseilles, France). The Ca2+ ionophores A23187 and ionomycin, fMET peptide (N-formyl-methionyl-leucyl-phenylalanine), PMA, wortmannin, interleukin-3 (IL-3), RPMI-1640, Ham’s F-12, and Iscove’s-modified Dulbecco’s media were from Sigma (St. Louis, MO). PP1 was obtained from Calbiochem (San Diego, CA). Monoclonal antibody 22E7 to the Fc{varepsilon}RI-{alpha} chain was from Dr. J. Kochan, Hoffman-LaRoche Inc. (Nutley, NJ) [25 ]; the stimulatory mAb 8A2 to human VLA-4, was from Dr. J. Harlan, University of Washington (Seattle, WA); the mAb 4B9 to human VCAM-1 was a gift from Dr. R. Larson (University of New Mexico, Albuquerque), and the Syk-selective inhibitor ER27319 was from Dr. J. Rivera (National Institutes of Health, Bethesda, MD) [22 ]. Human VCAM-1-transfected CHO cells were obtained from Dr. D. Leavesley (Hanson Cancer Center, Adelaide, Australia) [26 ].

Isolation of peripheral blood cells
We used a protocol approved by the Human Research Review Committee, University of New Mexico, to obtain venous blood from normal donors with no history of allergy symptoms. Basophils were obtained by Percoll gradient centrifugation as described [16 , 27 ]. Purities from this initial step ranged from 15% to 66%. Basophil purity was routinely increased to 95–99% using a negative selection cocktail from StemCell Technologies (Vancouver, B.C.) and MidiMacs (Miltenyi Biotec, Auburn, CA) magnetic columns (described in [16 ]).

VLA-4-mediated adhesive activity
The adhesive activity of basophils was measured using a modification of the assay of Leavesley et al. [26 ]. Percoll-enriched, negatively selected IgE-primed basophils (5 x 105 basophils/mL) were suspended in RPMI-FBS medium and fluorescence-labeled by incubation for 45 min with 4 mg/mL dihydroethidium (Molecular Probes, Eugene, OR) in a 5% CO2 incubator. In parallel, VCAM-CHO cells (2 x 106 cells/mL) were suspended in 1 mL of Ham’s F-12 Nutrient Mixture, 10% FBS, 200 mM L-glutamine, penicillin–streptomycin, and 1% Na pyruvate (Ham’s-FBS medium) and fluorescence-labeled with 1.5 mM Fluo-3 AM (Molecular Probes, Eugene, OR). Cells were washed once in the medium used for fluorescence labeling and once in adhesion buffer (modified Hanks’ buffered salt solution with 0.9 mM calcium and 0.35 mM magnesium; HBSS+). Each group of cells was then suspended in 400 µL adhesion buffer at 37°C. For adhesion assays, cells were combined at a ratio of 1:3.5 basophils:VCAM–CHO cells in adhesion buffer at 37°C to a final volume of 400 µL, and duplicate samples were incubated on a hematology mixer (Fisher Scientific, Pittsburgh, PA) in the presence of no addition, anti-IgE (1.0 µg/mL) or anti-Fc{varepsilon}RI mAb 22E7 (3.0 µg/mL), and other activating agents. In preliminary experiments, this ratio consistently yielded substantial differences between resting and anti-IgE or 22E7-activated basophils and, in addition, produced conjugates that almost always consisted of one basophil bound to one VCAM–CHO cell. After about 15 min of mixing, cells were fixed by adding 400 µL of 2% paraformaldehyde. Mixing continued for 2 min, and the proportion of dually fluorescent conjugates formed between basophils and VCAM–CHO cells was measured in a FACS Caliber flow cytometer.

Every experiment included samples with added ethylenediaminetetraacetate (EDTA), which reduces VLA-4 to its least active conformation, and with added mAb 8A2 plus Mn2+, which brings VLA-4 to its maximally active conformation [1 ]. The percent of basophils forming conjugates was derived by dividing the number of conjugates (dual color events) by the number of conjugates plus free basophils. In some experiments, the percent conjugate formation in the presence of EDTA was subtracted and values were then divided by the maximum conjugate formation occurring with 8A2/Mn++, yielding a value for conjugate formation as percent of maximum.

Histamine release
Suspensions of Percoll-enriched, negatively selected basophils were washed with Hanks’ buffered saline solution without Ca2+ or Mg2+ (HBSS-), suspended to 0.5–1.1 x 106 basophils/mL and 100-µL aliquots incubated at 37°C in prewarmed HBSS+ containing either no addition (spontaneous secretion) or the addition of anti-IgE and other activating agents (stimulated secretion). Reactions were terminated by dilution in ice-cold PBS and centrifugation, and histamine in cell pellets and supernatants was measured using radioimmunoassay (Alpco, Windham, NH) as described [16 , 28 ]. Total histamine was measured in supernatants obtained by freeze-thawing cell aliquots in PBS/EDTA followed by centrifugation at 10,000 x g for 5 min to remove debris.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The VLA-4-mediated adhesion of human basophils to VCAM-1 is up-regulated by Fc{varepsilon}RI cross-linking
A typical time course of the adhesive interactions that occur in a conjugate assay between highly purified, fluorescence-labeled normal basophils and fluorescence-labeled VCAM-CHO cells is shown in Figure 1 . When cells are fixed before mixing, none of the basophils form conjugates with VCAM–CHO cells. Unstimulated basophils show a slow increase in the formation of conjugates with VCAM–CHO cells that reaches a maximum in <15 min. In contrast, basophils activated by the addition of the anti-Fc{varepsilon}RI {alpha} mAb, 22E7, show a rapid increase in conjugate formation with VCAM–CHO cells that again reaches a maximum within 15–20 min. In this experiment, approximately 6% of unstimulated cells and 18% of stimulated cells were in conjugates after 15 min of mixing.



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  		Figure 1. Time course of conjugate formation between normal human basophils and VCAM–CHO cells. Highly purified basophils were incubated on a hematology mixer with VCAM–CHO cells with no addition (no activation) or in the presence of mAb 22E7 (3.0 µg/mL). The basophil:VCAM-CHO ratio was 1:3.5. Analysis of conjugate formation by flow cytometry was performed at 5-min intervals for each condition. Data show the percentage of basophils in conjugates. Results are the average of duplicate measurements in a single experiment.

 
The results in Figure 2 establish that conjugate formation is mediated by VLA-4 and demonstrate the upper and lower limits of the assay. In Figure 2A , approximately 14% of resting and 28% of anti-IgE-activated cells formed conjugates with VCAM–CHO cells during a 15-min mixing assay. In contrast, only 4% and 2%, respectively, of basophils formed conjugates with VCAM–CHO cells when the cells were co-incubated in the presence of the VLA-4 blocking antibody, HP2/1, the mAb 4B9 to human VCAM-1, and when EDTA was included in the assay mixture to minimize the binding activity of VLA-4. In this experiment, approximately 53% of basophils formed conjugates with VCAM–CHO cells when the VLA-4-activating mAb, 8A2, was included, with the VLA-4-activating cation, Mn2+, in the incubation mixture.



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  		Figure 2. Basophil–VCAM–CHO conjugate formation is mediated by VLA-4. Purified basophils were incubated on a hematology mixer with VCAM-CHO cells in a ratio of 1:3.5 basophils:CHO cells. In (A), incubation was for 15 min with no addition (resting), with anti-IgE (1.0 µg/mL), with the VLA-4 blocking antibody, HP2/1 (anti-VLA-4; 5.0 µg/mL), with the VCAM-1 blocking antibody, 4B9 (anti-VCAM-1; 5.0 µg/mL), with EDTA (5 mM), and with a combination of 8A2 (1:1000 from ascites) plus Mn2+ (2 mM). Cells were then fixed with 2% paraformaldehyde, and conjugate formation was analyzed by flow cytometry. Data show the percentage of basophils in conjugates. Results are the average of 2 separate experiments, each performed in duplicate. Error bars show the SEM. (* denotes t test significance, p < 0.05 level). In (B), incubation was for 15 min with no addition (resting), with 22E7 (3.0 µg/mL) or with 8A2 plus Mn2+. After 15 min, mAb HP2/1 was added to half the incubation mixtures and incubation was continued for another 15 min before fixation and analysis.

 
In Figure 2B , the initial incubation was again for 15 min with no activation or with mAbs 22E7 and 8A2. The VLA-4 blocking antibody, HP2/1, was then added to half of the samples and mixing was continued for another 15 min before the conjugate formation was analyzed. The results show that HP2/1 is almost as effective at disrupting preformed basophil–VCAM–CHO conjugates as it is at preventing their formation. This result is expected if conjugate formation principally reflects the activity of the VLA-4–VCAM interaction and not a secondary adhesive event that might conceivably follow the initial encounter of basophils with VCAM–CHO cells.

In multiple assays, 5–15% of resting basophils formed conjugates with VCAM–CHO cells, 15–40% of basophils formed conjugates after Fc{varepsilon}RI cross-linking with either mAb 22E7 (as in Figure 1 ) or anti-IgE (as in Figure 2 ), and 40–55% of cells formed conjugates in the presence of mAb 8A2 plus Mn2+. (Note: This variation was for different donors on different days. Duplicate samples within a given day’s experiment consistently showed variations of only a few percent in conjugate formation.) Although both basal and stimulated adhesion varied between experiments and between donors, the difference between resting and activated cells was almost always between two- and threefold. Conjugate formation in the presence of EDTA was generally less than 2%.

Together, these data establish that the conjugate assay provides a measure of the VLA-4-mediated adhesive activity of human basophils. They show that Fc{varepsilon}RI cross-linking up-regulates the VLA-4 adhesive activity of normal basophils. They indicate that basophil VLA-4 is neither in its least active (EDTA-induced) state in resting cells nor in its most active (8A2 plus Mn2+-induced) state in activated cells.

Src, Syk, and PI 3-kinase inhibitors all block the up-regulation of basophil VLA-4-mediated adhesion activity induced by Fc{varepsilon}RI cross-linking
Fc{varepsilon}RI signaling to secretion activates a tyrosine kinase cascade that begins with the activation of Lyn and proceeds to Syk activation and to the Syk-dependent activation of downstream enzymes, including PI 3-kinase, PLC{gamma} isoforms, and others (reviewed in refs 14 15 ). We used selective inhibitors of these signaling molecules, PP1 for Lyn and other Src kinase family members [20 , 21 ], ER27319 for Syk activation secondary to Fc{varepsilon}RI cross-linking [22 ], and wortmannin for PI 3-kinase [29 ], to help dissect the signaling pathway linking Fc{varepsilon}RI cross-linking to stimulated adhesion. ER27319 was selected over the alternative Syk-selective inhibitor, piceatannol [30 ], because the fluorescence of piceatannol interferes with the adhesion assay. All three inhibitors were used at the minimal concentrations that consistently block anti-IgE-induced histamine release under the conditions of our adhesion assay (Fig. 3 ).



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  		Figure 3. Effects of Src, Syk, and PI 3-kinase inhibitors on Fc{varepsilon}RI-mediated histamine release. Purified basophils were washed and incubated with the inhibitors indicated for 15 min at 37°C. Anti-IgE (1.0 µg/mL) was added and incubation continued for another 30 min at 37°C. Histamine levels were measured in the incubation supernatants. The experiment was performed in duplicate. Error bars show the SEM.

 
The results in the first four columns of Figure 4A 4B 4C show that 15 min of preincubation with all three inhibitors before initiating the conjugate assay blocks the increased adhesion of normal basophils to VCAM–CHO cells that is normally induced by Fc{varepsilon}RI cross-linking. PP1 (Figure 4A) , but not ER27319 or wortmannin (Figure 4B 4C) , also consistently reduces the adhesion of unstimulated normal basophils to VCAM–CHO cells.



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  		Figure 4. Effects of Src, Syk, and PI 3-kinase inhibitors on basal and stimulated basophil VLA-4-mediated adhesion. Purified IgE-primed basophils were incubated for 15 min at 37°C with and without inhibitors (10 µM PP1 in A, 30 µM ER27319 in B, 10 nM wortmannin in C), then transferred to a hematology mixer with VCAM–CHO cells plus stimuli. Stimuli were none (resting cells), 22E7 (3.0 µg/mL), fMet peptide (100 nM), A23187 (5.0 µg/mL), PMA (25 nM), EDTA (5 mM), and 8A2 (1:1000 from ascites) plus Mn2+ (2 mM). After 15 min, cells were fixed with 2% paraformaldehyde and conjugate formation was analyzed by flow cytometry. Data show the percentage of basophils in conjugates. Results are the average of 2 separate experiments for each inhibitor, each performed in duplicate. Error bars show the SEM. (* denotes t test significance, P < 0.05 level.)

 
Effects of Src, Syk, and PI 3-kinase inhibitors on the up-regulation of basophil VLA-4-mediated adhesion activity induced by fMET peptide, Ca2+ ionophore, and PMA
The implication that PP1 may block both basal and Fc{varepsilon}RI-stimulated adhesion, whereas ER27319 and wortmannin may block only Fc{varepsilon}RI-stimulated adhesion, was explored further by introducing a series of independent activating agents: fMET peptide, which activates a G protein-coupled receptor pathway; A23187, which activates signaling pathways downstream of Ca2+ mobilization; and PMA, which activates signaling pathways downstream of protein kinase C activation. These results are also shown in Figure 4 . All three stimuli increased the VLA-4-mediated adhesion of normal basophils to VCAM–CHO cells. Neither ER27319 (Figure 4B) nor wortmannin (Figure 4C) inhibited the up-regulation of VLA-4-mediated adhesion induced by fMET peptide, A23187, or PMA. These results suggest that neither Syk nor PI 3-kinase lies on the pathways linking these activators to VLA-4-mediated adhesion. PP1-treated cells (Fig. 4A) also responded to fMET peptide, A23187, or PMA with an increase in VLA-4-mediated adhesion. However, the maximum adhesion induced by these stimuli was always less in PP1-treated cells than in cells treated with ER27319 or wortmannin. These results suggest that PP1 blocks a component of VLA-4 adhesive activity that is common to all adhesion activators working from inside the cell. Not even PP1 reduced the up-regulation of VLA-4-mediated adhesion induced by mAb 8A2 plus Mn+ (Fig. 4A) . Thus, activators that work from outside the cell are insensitive to the Src inhibitor.

PP1-treated normal basophils and nonreleaser basophils share a similar adhesion phenotype
Figure 5A shows averaged results of adhesion assays from more than 10 separate experiments, each performed in duplicate, with resting and Fc{varepsilon}RI-stimulated normal and nonreleaser basophils isolated from different donors on different occasions. The figure confirms that Fc{varepsilon}RI cross-linking causes no increase in VLA-4-mediated adhesion in nonreleaser basophils. Moreover, it establishes through replicate analyses that basal adhesion is reduced in the nonreleaser basophils as it is in PP1-treated normal basophils.



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  		Figure 5. The adhesion phenotype of nonreleaser basophils. Purified releaser and nonreleaser basophils were incubated on a hematology mixer for 15 min with VCAM–CHO cells in the presence or absence of activating agents. In (A), cells were incubated with no addition (resting) or with anti-IgE (1.0 µg/mL) or 22E7 (3.0 µg/mL), both of which activate by cross-linking Fc{varepsilon}RI and therefore depicted by "XL." Results are from at least 10 separate experiments with resting and Fc{varepsilon}RI-stimulated normal and nonreleaser basophils isolated from different donors on different occasions. In (B), cells were incubated with no addition (resting) or with anti-IgE (1.0 µg/mL), fMET peptide (100 nM), ionomycin (1 µM), or PMA (25 mM). Results are the average of 2 separate experiments, each performed in duplicate. EDTA and 8A2/Mn2+ controls were included in each experiment and used to determine percent of maximum conjugate formation. Error bars show the SEM. (* denotes t test significance, P < 0.05 level.)

 
We reported before that nonreleaser basophils seemed to show a more modest increase than normal basophils in VLA-4 adhesive activity in response to fMET peptide, ionomycin, and PMA. In contrast, their increase in VLA-4 adhesive activity to 8A2 plus Mn2+ was not different from normal. The results shown in Figure 5B confirm that the VLA-4 adhesive responses of an expanded panel of nonreleaser are smaller than those of normal basophils to this series of stimuli.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We showed recently that Fc{varepsilon}RI cross-linking increases the adhesive activity of human basophil VLA-4 toward its counter-receptor, VCAM-1. The adhesive activity of VLA-4 is up-regulated without an increase in the expression levels of this integrin. Increased VLA-4 activity is not induced by Fc{varepsilon}RI cross-linking in Syk-deficient nonreleaser basophils, a result that suggests it requires the integrity of the tyrosine kinase-coupled signaling pathway that has already been shown to link Fc{varepsilon}RI cross-linking to other responses, including secretion, ruffling, and cytokine production in human basophils [16 , 17 ].

In our study, we first confirm that our conjugate assay indeed measures the interaction of VLA-4 on basophils with its counter-receptor VCAM-1 on CHO cells and that the interaction is up-regulated after Fc{varepsilon}RI cross-linking with both anti-IgE and mAb 22E7. Because our resting and activated samples consistently yield a proportion of basophils in conjugates with VCAM–CHO cells that falls between the proportion of basophils in conjugates in EDTA-treated and in 8A2/Mn2+-treated samples, we infer that basophil VLA-4 is in neither its least active (EDTA-induced) affinity state in resting cells nor in its most active (8A2 plus Mn2+-induced) affinity state in activated cells. We recognize that events other than the affinity of VLA-4 for VCAM-1 for example, cell shape changes, the secondary activation of other adhesion molecules, or receptor clustering may contribute to the formation of basophil-VCAM–CHO conjugates. Evidence that cell shape changes probably do not complicate our assay was obtained from experiments in which a series of other stimuli, some capable of inducing cell shape changes in leukocytes (fMET peptide and PMA) and some causing little or no shape change (ionomycin and A23187) [31 , 32 ], all supported increases in VLA-4-mediated basophil adhesion to VCAM–CHO cells. The secondary activation of other adhesion molecules seems unlikely based on evidence that the VLA-4-blocking mAb HP2/1 not only prevents conjugate formation but causes a prompt disassembly of preformed basophil–VCAM–CHO conjugates. It is still possible that the different VLA-4 adhesive activities of resting and stimulated basophils measured in our conjugate assay may result in part from differences in integrin clustering that might modulate the avidity rather than the affinity of the integrin. We recently developed an immunoelectron microscopic technique to observe the distribution of proteins in native membrane sheets torn from basophils and mast cells [33 ]. Integrin mapping studies are in progress to determine if any of our stimuli induce VLA-4 clustering on basophil membranes.

We established that the Fc{varepsilon}RI-mediated up-regulation of VLA-4-mediated basophil adhesion is abolished in the conjugate assay by PP1, ER27319, and wortmannin and thus is most likely dependent on the sequential activation of Lyn (inhibited, with other Src kinases, by PP1), Syk (interaction with Fc{varepsilon}RI blocked by ER27319), and PI-3 kinase (selectively inhibited by wortmannin). In contrast, the adhesion activation induced in the same cells with fMET peptide, which stimulates a G protein-coupled signaling pathway, and with ionophores and phorbol ester, which bypass both tyrosine kinase- and G protein-coupled receptors altogether, is insensitive to ER27319 and wortmannin and is reduced, but not eliminated, by PP1. These data lead to some straightforward conclusions. First, because the Fc{varepsilon}RI/Syk interaction and PI 3-kinase inhibitors, ER27319 and wortmannin, abolish only the Fc{varepsilon}RI-mediated activation of VLA-4-mediated basophil adhesion, we propose that Syk and PI 3-kinase are uniquely involved in the pathway linking Fc{varepsilon}RI cross-linking to adhesion up-regulation. Second, because the Src inhibitor, PP1, abolishes the Fc{varepsilon}RI-mediated activation of VLA-4 adhesion, we infer that a Src kinase, presumably Lyn, is critical for the Fc{varepsilon}RI-induced up-regulation of VLA-4 adhesion.

During these studies, we observed a small but consistent inhibition by PP1, but not by the other inhibitors, of the basal VLA-4 adhesive activity of normal human basophils. PP1 also slightly inhibited the VLA-4 adhesion activation induced with fMET peptide, ionophores, and phorbol ester. The likeliest explanation is that a PP1-sensitive Src family member determines the basal activity of basophil VLA-4-mediated adhesion and that incubation with adhesion-activating agents does not restore the basal component of VLA-4 activity. This small effect might have seemed unremarkable except that parallel studies in the laboratory had given the impression that nonreleaser basophils also show a loss of basal as well as Fc{varepsilon}RI-mediated VLA-4-dependent adhesion to VCAM–CHO cells. The analysis of a large body of experiments comparing normal and nonreleaser basophils, reported here, confirms that untreated nonreleaser basophils and PP1-treated normal basophils indeed have defects in their basal VLA-4-dependent adhesion to VCAM-1. Adding to their similarity to PP1-treated normal basophils, nonreleaser basophils also have smaller than normal adhesive responses to fMET peptide, ionophores, and PMA.

It has been known since the pioneering studies of Masumoto and Hemler [34 ] that VLA-4 occurs in cell membranes in multiple activation states. The evidence was based primarily on the results of static or dynamic adhesion assays in which VLA-4-mediated adhesion was altered from the outside of the cells with different anti-VLA-4 Abs and different divalent cations or from the inside of the cells with different ligand–receptor complexes. The concept was strengthened recently by evidence that leukocytes contain a pool of VLA-4 that is maintained in a state that is conformationally responsive to ligand [6 ]. It was extended by the recent synthesis of radiolabeled LDV-containing ligands that can directly measure the affinity of VLA-4 for its ligands [5 , 35 ]. Although these promising reagents have not yet demonstrated rapid enough kinetics for dynamic studies of changes in VLA-4 adhesion, they clearly reveal the ability of VLA-4 to occur in multiple activation states on T cells and so, by extrapolation, on basophils.

Our results suggest that basal VLA-4 adhesion may be regulated by Src family members in human basophils and that this regulation may be impaired in nonreleaser basophils. Lyn is the principal Src kinase of human basophils, and our previous work demonstrated only inconsistent changes in levels of Lyn among five nonreleaser donors [16 , 17 ]. However Lavens-Phillips and MacGlashan recently reproduced the lack of Syk in basophils from several more nonreleaser donors [36 ]. They also reported that neither of the two donors they studied had detectable basophil Lyn. It is thus possible that nonreleaser basophils have a second defect, expressed variably as changes in Lyn expression or function, that contributes to their impaired basal VLA-4 adhesion. Alternatively, the putative Src kinase–regulating basal adhesion might not be Lyn but a related Src family member functioning at or near the integrin to maintain VLA-4 in a specific affinity state. Supporting this, Lyn, Src, and Yes have all been detected in mast cells [37 ], and there is abundant evidence that Src family members acting at or near integrins can modulate the integrin-mediated adhesive properties of many cell types [38 39 40 ]. In either case, the impairment of a VLA-4-modulating Src kinase is likely to reduce the recruitment of nonreleaser basophils from the blood to sites of tissue inflammation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by NIH grants P50 HL56384 and RO1 GM49814. We thank Drs. Jarko Kochan, Richard Larson, and John Harlan for generous gifts of Abs; Dr. Juan Rivera for providing ER27319; Dr. David Leavesley for providing VCAM-CHO cells; and Drs. Larry Sklar, Richard Larson, and Bruce Edwards for discussion. We also thank the UNM Clinical Research Center for assistance in coordinating blood donors and for phlebotomy and the UNM Cancer Research and Treatment Center for facilities and skilled personnel for flow cytometry.

Received April 1, 2001; revised July 3, 2001; accepted July 9, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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