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Engagement of ICAM-3 Activates Polymorphonuclear Leukocytes: Aggregation Without Degranulation or ß₂ Integrin Recruitment¹

Michael J. Feldhaus^2,*, Julie M. Kessel, Guy A. Zimmerman and Thomas M. McIntyre^3,*,

Departments of ^* Pathology, Medicine, and Pediatrics, University of Utah, Salt Lake City, UT 84112

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
ICAM-3 is a preferred counterreceptor for the leukocyte _Lß₂ integrin. It activates T cells through outside-in signaling, but polymorphonuclear leukocytes (PMN) are reported to be refractory to ICAM-3 stimulation. We found that engagement of ICAM-3 by a mAb (CAL3.10), which binds in the region where _Lß₂ integrin binds, activates PMN homotypic aggregation and adhesion to surfaces. These functional changes were due to ICAM-3 outside-in signaling because aggregation and adhesion were ß₂ integrin-dependent, tyrosine kinase and protein kinase C activities were activated, and there was a reorganization of the cytoskeleton. This reorganization and kinase activity was required for ICAM-3-, but not FMLP-, induced aggregation. This is not an Fc-mediated event as an appropriate anti-ICAM-3 F(ab')₂ fragment still induced aggregation. Another anti-ICAM-3 Ab (HP2/19), which activates T cells, did not activate PMN. Strikingly, anti-ICAM-3 did not induce degranulation or cause an increase in surface ß₂ integrin expression, so adhesion and aggregation were due solely to the activation of the constitutively expressed ß₂ integrins. Aggregation in response to ICAM-3, but not FMLP, was compromised at lower cell densities, showing that ß₂ integrin recruitment enhances aggregation under suboptimal conditions. We conclude that engagement of ICAM-3 stimulates PMN as well as T cells, but that the appropriate epitope varies between these two cells. ICAM-3 outside-in signaling reorganizes the cytoskeleton without causing degranulation, induces serine and tyrosine kinase activation, and activates existing surface ß₂ integrins to a proadhesive state.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Adhesion molecules and their counterreceptors control the immune and inflammatory responses through their specificity and strength of intercellular and matrix interactions. In addition to the specificity imparted by the temporal and spatial display of receptors and their binding partners, the formation of appropriate ligand pairs for some receptors can transmit information about the environment into the cell. This outside-in signaling is now known to occur after appropriate ligation of members of the integrin family of adhesion molecules (1, 2). The subunit composition of the integrin heterodimers determines their targets, which for the abundant ß₁ and ß₃ integrin family includes members of the ICAM family expressed by inflammatory and immune cells. The members of this immunoglobulin domain superfamily are highly glycosylated type I transmembrane proteins. ICAM-1 (CD54) and ICAM-3 (CD50) contain five extracellular Ig-like domains (numbered sequentially from the distal end), whereas ICAM-2 contains just two of these domains (3, 4, 5, 6, 7). The first two domains of the ICAMs are homologous (8, 9). All three ICAMs are counterreceptors for the ß₂ integrin _Lß₂ (CD11a/CD18) (5, 6, 7), and _Lß₂ binding has been mapped to this distal region of ICAM-1 (4) and ICAM-3 (10).

The ligation of ICAM-3, like ICAM-1 and ICAM-2 (11), at appropriate sites on the molecule results in outside-in signaling in T cells that stimulates gene expression and alters adhesive functions, which acts as a costimulus for lymphocyte activation (12, 13, 14, 15). ICAM-3 is expressed mainly by hematopoetic cells and is well represented on the polymorphonuclear leukocyte (PMN)⁴ surface (5, 16). However, the significance of ICAM-3 expression by PMN, when compared to its potential role in lymphocyte function, is unclear. One approach to explore this issue has been to use mAbs, some of which may mimic the ligation of ICAM by the cell-associated _Lß₂ integrin. When performed with PMN, such studies have shown that anti-ICAM-3 treatment and subsequent cross-linking of the bound Ab caused an increase in intracellular Ca²⁺, increases in the level of protein tyrosine phosphorylation, and activation of the protein tyrosine kinases lck and fyn (17). These intracellular events may cause a functional change in PMN, as found with lymphocytes, as certain anti-ICAM-3 Abs stimulate homotypic PMN aggregation (12, 18). Conversely, a new study (19) shows that engagement of ICAM-3 by Abs inhibits PMN function.

We examined the effect of engagement of ICAM-3 on isolated human PMN using Abs presented in solution and found that one of two anti-ICAM-3 Abs that activate T cells (10, 12) also induced ß₂ integrin-dependent aggregation of the neutrophils. Engagement of ICAM-3 also induced tyrosine phosphorylation of multiple proteins and induced PMN aggregation that required protein kinase C and tyrosine kinase activation. We also found that cytoskeletal reorganization was required for aggregation of neutrophils. Thus, ICAM-3 transmits stimulatory outside-in signals in PMNs. Since engagement of ICAM-3 did not cause degranulation or an increase in surface ß₂ integrin expression, aggregation required only activation of the integrins constitutively present on the plasma membrane. Since the stimulatory Ab epitope maps to domain 1 of ICAM-3 where the ß₂ integrin binds (12), the prevalence of surface ICAM-3 on PMN suggests that this mechanism of activation may affect PMN function following ligation by its physiologic binding partners.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials

Staphylococcus aureus sphingomyelinase C (SmC), herbimycin A, genestein, lysophosphatidylcholine, and FITC-phalloidin were obtained from Sigma (St. Louis, MO). HBSS was from BioWhitaker (Walkersville, MD), and human serum albumin was a product of Baxter Healthcare (Glendale, CA). Chelerythrine was obtained from Calbiochem (La Jolla, CA). mAb 60.3 and mAb 60.1 were a gift from Dr. Patrick Beatty (University of Utah), and anti-ICAM-3 mAbs, CAL3.10 (BBA29), and CAL3.34 (BBA28) were obtained from R&D Systems (Minneapolis, MN). mAb ICR1.1 and F(ab')₂ of mAb ICR1.1 was a gracious gift from Joel Hayflick (ICOS Corporation, Bothell, WA). Anti-ERK1 Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All secondary Abs were obtained from Biosource International (Camarillo, CA). HP2/19, FITC-conjugated anti-CD18, FITC-conjugated anti-L-selectin, and rabbit polyclonal antiphosphotyrosine were from Immunotech (Marseille, France). Phycoerythrin-conjugated anti-CD11b was obtained from Becton Dickinson (Bedford, MA). ECL Western blotting reagents were obtained from Amersham (Arlington Heights, IL). Indo-1 AM was obtained from Molecular Probes (Eugene, OR).

Cells and stimulated adhesion

Neutrophils (PMN) were freshly isolated from human blood as described (20) and diluted to 5 x 10⁵/ml in HBSS containing 0.5% human serum albumin (HBSSA). For adhesion assays, 4-well plates from Nunclone (Roskilde, Denmark) were first coated with 0.2% gelatin in nanopure water at 37°C for 3 h and then blocked for 1 h at 37°C with HBSSA. These plates were washed twice with HBSSA before the addition of PMN (0.25 ml). Agonist at the reported concentrations was then added in 25-µl aliquots. The plates were incubated at 37°C for 5 min before nonadherent PMNs were removed by aspiration, and loosely adherent cells were depleted by two gentle 500 µl HBSSA washes. The adherent cells were immediately quantified in three random fields using a custom video imaging system connected to a microscope with 1x objective that images 20% of the well. Each sample was examined in duplicate, and the total of six readings were used to generate standard error. When the effect of cytoskeletal disruption on PMN function was to be examined, PMN at 5 x 10⁵/ml were pretreated with either 10 µg/ml cytochalasin D or with 0.5 U/ml S. aureas SMC for 30 min at 37°C before assay.

PMN aggregation and Ca²⁺ flux measurements

PMNs at 5 x 10⁶/ml were loaded in the dark with 1 µM Indo-1 AM (Molecular Probes) in HBSSA for 30 min at 37°C. Cells were washed with HBSSA before assaying intracellular Ca²⁺ levels as an emission ratio at 405/485 nm. Aggregation was measured by loss of light dispersion by suspensions of continuously stirred PMN maintained at 37°C. To determine the effects of various inhibitors on these functions, PMN were pretreated with SMC at 37°C for 30 min, with 10 µM of the protein kinase C inhibitors H7 and chelerythrine for 10 min, or with the tyrosine kinase inhibitors herbimycin A or genestein for 10 min at 10 µM. Pretreatment of PMN with the various Abs in solution is individually specified in the figure legends.

Flow cytometry

Surface expression of adhesion molecules was quantified by analyzing 10,000 cells by flow cytometry, and the data are presented as a histogram. For these experiments, 1 x 10⁶ PMN were treated as described in the figure legends, pelleted by centrifugation, and then resuspended in ice-cold PBS containing 0.1% Na azide and 10% goat serum. After 10 min, these cells were recovered by centrifugation and then resuspended for 1 h at 4°C in CAL3.10 (anti-ICAM-3), CAL3.34 (anti-ICAM-3), or both Abs at 10 µg/ml in the same azide and goat serum-modified PBS. Cells were washed three times with PBS with 1% goat serum before resuspension in FITC-conjugated goat anti-mouse in the same buffer. Cells were then fixed in 0.5% formaldehyde overnight at 4°C before analysis by flow cytometry. In some experiments, PMN were analyzed for L-selectin or CD18 surface expression after anti-ICAM-3 treatment in the aggregometer. These PMN were immediately added to 1% ice cold paraformaldehyde in PBS and incubated for 30 min. These cells were then washed with PBS by centrifugation and resuspended in 100 µl of PBS containing 10% goat serum and a directly conjugated Ab raised against one of these surface Ags. The F-actin content was determined by using a fluorescent filamentous actin (F-actin)-specific compound, FITC-phalloidin. For this, PMN (1 x 10⁶/ml) were added to an equal volume of 8% formaldehyde, 100 µg/ml lysophosphatidylcholine, and 2 µg/ml FITC-phalloidin in PBS. The cells were incubated in this mixture for 4 h on ice, washed with PBS, and resuspended in 1 ml PBS before analysis with a Becton Dickinson FACScan.

Western blotting of phosphotyrosine PMN proteins

PMNs, after the specified treatments, were pelleted by centrifugation and their proteins solubilized with boiling Laemmli sample buffer containing 1% 2-ME, 2 mM DTT, 2 mM orthovanadate, 10 µg/ml aprotinin, 100 µM leupeptin, and 1 mM PMSF in DMSO. Proteins were electrophoresed in a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon P; Millipore, Bedford, MA) (21). The membrane was blocked overnight with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 10% human serum albumin. Primary and horseradish peroxidase-conjugated secondary Abs were diluted in 10% nonimmune cognate serum, and staining was detected by enhanced chemilluminescence with subsequent exposure to Kodak (Rochester, NY) X-OMAT film.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Anti-ICAM-3 induces ß₂ integrin-dependent PMN aggregation

We found that engagement of ICAM-3 by a mAb that binds in domain 1 at or near the _Lß₂ integrin binding site (12) induced the aggregation of freshly isolated human PMN (Fig. 1A). Engagement of ICAM-3 was a strong stimulus for this response as its magnitude was equivalent to the positive control, FMLP (Fig. 1A). An isotype-matched mAb to an irrelevant leukocyte surface Ag failed to induce aggregation (not shown), so this event is specific for ICAM-3 ligation. We determined whether the increase in light transmission caused by anti-ICAM-3 was truly activation-dependent aggregation or simple clustering of PMN by the bivalent, but monomeric, Ab. To distinguish between these mechanisms, we inhibited the function of the ß₂ integrins that are required for activation-induced aggregation. We found that pretreating the PMN with the blocking anti-ß₂ Ab 60.3 completely inhibited the anti-ICAM-3-induced adhesion and severely attenuated adhesion induced by the positive control FMLP. As will be developed below, ICAM-3 ligation induces leukocyte aggregation by selective stimulation of leukocyte functions, including ß₂ integrin activation.

Adhesion of PMNs to one another or to a surface by activated ß₂ integrins is mediated through several steps; ß₂ integrin heterodimers constitutively expressed on the surface are activated to an adhesive state, additional heterodimers are recruited to the surface from intracellular stores, and the newly translocated heterodimers are then activated to the adhesive state (22). We examined the relative contributions of the constitutively expressed ß₂ integrins and the newly recruited ß₂ integrins after anti-ICAM-3 engagement or FMLP stimulation by pretreating PMN with anti-ß₂ integrin Ab to block the function of constitutively expressed molecules. We washed the cells free of excess Ab and then stimulated them in the absence of blocking Ab. We found (Fig. 1A) that aggregation induced by ICAM-3 engagement was completely blocked by this protocol, while FMLP-induced aggregation was attenuated but not completely blocked. In conjunction with the data showing that ICAM-3 ligation does not increase ß₂ integrin surface expression (shown below), we conclude that outside-in signaling via ICAM-3 activates aggregation only through ß₂ integrins constitutively expressed on the cell surface. In contrast, the pre-existing surface integrins account for only a portion of the adhesion induced by FMLP.

Differences in ß₂ integrin surface density may critically affect aggregation at lower cell concentrations. To examine this possibility, we varied the concentration of PMN exposed to anti-ICAM-3 and found that the rate of PMN aggregation was greatly affected by this parameter (Fig. 1B). Both the initial rate and the extent of aggregation induced by ICAM-3 were decreased as the concentration of PMN in the assay was decreased. This result was not solely due to an inherent limitation in aggregation at lower cell densities as the addition of FMLP rapidly induced aggregation even at low cell concentrations (Fig. 1B). It appears that the amount of ß₂ integrin constitutively expressed on the surface of PMN can limit their ability to aggregate, and that ß₂ integrin recruited from the intracellular pool augments the ability of the cells to bind one another at low cell abundance.

We next examined a second ß₂ integrin-mediated function, adhesion of stimulated PMN to a surface coated with immobilized gelatin (23). We treated PMN with FMLP or with anti-ICAM-3 for 5 min in wells coated with gelatin before removing loosely adherent and unbound PMN. We found that engagement of ICAM-3 induced PMN adherence to this surface, and this adhesion was completely inhibited by a blocking anti-ß₂ mAb (Fig. 1C). Thus adhesion was again due to the activation of ß₂ integrin function following ICAM-3 engagement.

ICAM-3 engagement does not induce complete PMN activation

Agonists that induce ß₂ integrin-dependent adhesion and aggregation typically recruit more ß₂ integrin to the cell surface from intracellular stores. The experiment above suggested that outside-in signaling via ICAM-3 might vary from this paradigm. We examined this possibility by flow cytometry of PMN stimulated with anti-ICAM-3 or FMLP. We stained these cells with FITC-conjugated anti-ß₂ integrin mAb and found (Fig. 2A) that FMLP-treated PMN displayed more ß₂ integrin on their exterior surface after stimulation, but that anti-ICAM-3-treated PMN did not. This unusual result is consistent with the experiments shown above (Fig. 1, A and B) and suggests that there was a failure to mobilize the specialized ß₂ integrin containing granules following anti-ICAM-3 treatment.

We found other differences between ICAM-3 and FMLP-exposed cells. PMN constitutively express L-selectin on their surface, and this surface expression is inversely correlated with ß₂ expression; agonist activation augments integrin heterodimer expression, and in parallel causes L-selectin to be proteolytically cleaved and released (24). We found that in contrast to the rapid and near complete shedding of L-selectin by FMLP-stimulated cells (Fig. 2B), that anti-ICAM-3 treatment failed to induce this response. One event that might account for both observations would be a failure to mobilize intracellular vesicles. Therefore, we determined whether the bulk of PMN granules were secreted following ICAM-3 ligation by examining cell granularity by the amount of side scatter detected during flow cytometric analysis. We found that FMLP, the positive control, caused a shift in this parameter, but that the anti-ICAM-3 Ab did not (Fig. 2C). We conclude that outside-in signaling via ICAM-3 induces pathways that activate the ß₂ integrins that are constitutively expressed on the cell surface. However, it does not lead to degranulation, ß₂ heterodimer recruitment, or changes in the surface expression of L-selectin.

ICAM-3 signaling is epitope-specific

The distal domain 1 of ICAM-3 is the binding site for _Lß₂ integrin and contains the epitope, the "A region," for the anti-ICAM-3 that we used to stimulate PMN adhesion and aggregation (12). We determined whether other Abs that map to this region also stimulate outside-in signaling. We found that HP2/19 (IgG2a), which maps in this same A region where _Lß₂ integrin binds (10), did not stimulate PMN adhesion (Fig. 3A). In contrast, a second anti-ICAM-3 Ab that is isotype matched to HP2/19 (ICR1.1, an IgG2a) did induce PMN homotypic aggregation. We also examined homotypic aggregation in response to a third anti-ICAM-3 Ab, CAL3.34, that binds in domain 4 and partially blocks _Lß₂ integrin binding to ICAM-3 (12). We found that this ICAM-3 Ab did not stimulate aggregation (Fig. 3A). In fact, it proved to be an inhibitor of ICAM-3 signaling as it completely blocked aggregation in response to the stimulatory CAL3.10 anti-ICAM-3 Ab (Fig. 3A). The effect of CAL3.34 was not plieotropic as PMN exposed to it still aggregated in response to either FMLP or PMA. We considered the possibility that the suppression of aggregation by pretreatment with mAb CAL3.34 was due to mutual exclusion of the two ICAM-3 Ab. However, this hypothesis does not account for the inhibition as these two Abs showed additive binding by flow cytometric quantitation (Fig. 3B). Instead, we postulate that the conformation of ICAM-3 is altered by the binding of certain ligands and that CAL3.34 interferes with this alteration. We also found the aggregation response is not due to Fc-mediated signaling or cross-linking of Fc receptors as a F(ab')₂ of mAb ICR1.1 also induced PMN aggregation in a time- and concentration-dependent manner (Fig. 3C). This experiment and the experiments in Fig. 3A clearly show that the anti-ICAM-3-induced aggregation response of PMN is specific to a particular epitope in domain 1 of ICAM-3 and is not dependent on Fc-mediated signaling.

ICAM-3 signaling depends on cytoskeletal reorganization

We postulated that ICAM-3, like other ICAMs, might interact with the cytoskeleton and that this interaction could be needed to transmit stimulatory signals. We determined whether the activating Ab CAL3.10 induced a change in total cellular F-actin content by staining the cytoskeleton with FITC-phalloidin, an F-actin-specific compound. PMN treated with FMLP as a positive control demonstrated a rapid increase in F-actin content that was maintained over the next 5 min. (Fig. 4A). Engagement of ICAM-3 also induced a transient increase in F-actin content (Fig. 4B). ICAM-3 engagement therefore modulates intracellular cytoskeletal structure in a way similar to FMLP.

We asked if this mobilization of actin monomers is vital for the intracellular signal emanating from ICAM-3 by first treating the cells with cytochalasin D to disrupt the cytoskeletal organization before addition of CAL3.10. We observed that treating PMN with cytochalasin D or SMC⁵ to disrupt cytoskeletal organization completely suppressed the aggregation caused by the activating anti-ICAM-3 Ab (Fig. 5). Disruption of cytoskeletal rearrangement by either of these two agents did not fundamentally alter PMN function as these PMN still aggregated in response to FMLP. Thus signaling from the extracellular domain of ICAM-3 depends on the cytoskeletal alterations that are blocked by cytochalasin D, and this differs from the receptor-mediated signaling through the FMLP receptor.

View larger version (10K): [in this window] [in a new window]   FIGURE 5. Disruption of cytoskeletal rearrangement inhibits anti-ICAM-3 induced aggregation. PMN were pretreated with S. aureus SMC (1 U/ml) for 30 min, or cytochalasin D (5 µM) for 15 min at 37°C before being transferred to an aggregometer cuvette. After a stable baseline had been achieved, FMLP or anti-ICAM-3 (10 µg/ml) was added and aggregation recorded by the increase in light transmission. The aggregation assay was repeated six times and a representative example is shown. To ensure changes in transmittance reflected aggregation, an aliquot was removed for staining and microscopy.

To elucidate the nature of the outside-in signals induced by engagement of ICAM-3, we first determined whether it involved altered intracellular Ca²⁺ levels. We found that, unlike FMLP stimulation, neither the activating anti-ICAM-3 Ab nor PMA produced a change in this parameter (Fig. 6).

View larger version (6K): [in this window] [in a new window]   FIGURE 6. Activating anti-ICAM-3 Ab does not induce a calcium flux. PMN were loaded with Indo-1 AM (0.5 mM) for 10 min at room temperature. Excess reagent was removed by centrifuging the PMN twice in HBSSA and resuspending cells in this buffer (1 x 107 PMN/ml). Fluorescent emissions at 405 and 485 were monitored, and once a stable ratio was obtained, FMLP or anti-ICAM-3 (10 µg/ml) or PMA (10-8 M) was added and emission ratios followed for 6 min. An aliquot of the PMN suspension was recovered for microscopy to ensure that agonist-induced aggregation had occurred. The light transmittance/time tracing is a representative example of four other separate experiments. Indo-1 itself does not affect aggregation by any of the agonists used (not shown).

We determined whether protein kinase inhibitors would block anti-ICAM-3-induced aggregation. We found that PMN treated with the protein kinase C inhibitor chelerythrine (or the less specific kinase inhibitor H7; not shown) blocked aggregation induced by exposure to anti-ICAM-3 mAb. These two inhibitors also blocked aggregation induced by the agonist PMA (Fig. 7). We also exposed PMN to the general tyrosine kinase inhibitors herbimycin A and genistein and found that these effectively blocked anti-ICAM-3-induced aggregation. However, the subsequent addition of FMLP to these samples resulted in aggregation that was unaffected by the individual inhibitors and was only slightly attenuated when the two tyrosine kinase inhibitors were used together. Thus, ICAM-3 engagement is completely dependent on tyrosine kinases that are inhibited by herbimycin A and genistein, a property not shared by FMLP.

View larger version (17K): [in this window] [in a new window]   FIGURE 7. Protein kinase C and tyrosine kinase activities are essential for anti-ICAM-3-induced PMN aggregation. Human PMN were treated with chelerythrine (1 µM), herbimycin A (10 µM), and genistein (80 µM) for 20 min. The response to anti-ICAM-3, FMLP, or PMA was determined by aggregometry, and visually verified at the end of the incubations as before. Each inhibitor was examined on three separate days using different donors’ PMN.

We determined what effect ICAM-3 engagement had on the level of protein phosphotyrosine in these cells and found that CAL3.10 caused a rapid increase in the phosphotyrosyl content of a number of proteins (Fig. 8). The increase was evident by 1 min of stimulation and continued to increase over the next few minutes. Particularly prominent changes occurred in proteins migrating as approximately 180-, 150-, 100-, 80-, and 40-kDa proteins. The increase in total phosphotyrosyl residues induced by anti-ICAM-3 was greater than that induced by FMLP or PMA. We determined whether the 40-kDa phosphoprotein might be ERK MAP kinase by reprobing the gel with anti-ERK1/2 Ab. This showed that there was near equal loading of the lanes, and that FMLP and PMA caused a mobility shift associated with enhanced phosphorylation of the ERK mitogen-activated protein (MAP) kinases (Fig. 8). However, ICAM-3 ligation did not induce a change in mobility, so we conclude these ERKs are not major downstream targets of ICAM-3 signaling in PMNs. We determined whether stimulation of tyrosine kinase activity through ICAM-3 engagement occurred just when epitopes that cause cell activation were ligated, or whether this occurred whenever ICAM-3 was ligated. We found that the nonstimulatory Ab HP2/19 (-IT) failed to induce an increase in protein phosphorylation (Fig. 8), suggesting that phosphorylation correlates with function. These results show ICAM-3 ligation leads to intracellular signaling that results in a phosphoprotein profile much like that induced by the classical agonists FMLP and PMA, even though no obvious phosphoprotein correlated with ICAM-3 induced adhesion.

View larger version (137K): [in this window] [in a new window]   FIGURE 8. ICAM-3 ligation induces phosphotyrosyl accumulation. PMN were treated with anti-ICAM-3 Abs, FMLP, or PMA for the stated times while stirring an aggregometer to measure functional changes. At the stated times aliquots were removed, and PMN were recovered by centrifugation and resuspended in boiling SDS-PAGE reducing buffer. The samples were boiled for 10 min, electrophoresed on a 10% SDS-PAGE, and transferred to PVDF membranes, and phosphotyrosine residues were detected with rabbit polyclonal antiphosphotyrosine and ECL detection system as described in Materials and Methods. The phosphotyrosine blots were repeated three times with samples prepared from three different donors. The phosphotyrosine blots were stripped and reprobed to detect ERK1 and ERK2. This experiment was repeated twice. -D (CAL3.10); -IT(CAL3.34).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

ICAM-3 is a counterligand for the ß₂ integrin, _Lß₂, and for the newly recognized a_dß₂ integrin (25) that is expressed by all myeloid cells (5, 6). Although ICAM-3 is most abundant on monocytes and lymphocytes, it is well represented on PMN where it exceeds the density of ICAM-1 by 6-fold (5). In addition to its role in cell adhesive interactions, it has been investigated as a signal transducer that conveys information from the extracellular environment into the cell. These studies, for the most part, have employed T cells and cell lines where treatment with certain anti-domain 1 Abs, classified as binding to an A epitope, will stimulate (12, 15) or costimulate (12, 14, 26) T cell function. The A epitope is an important functional definition as this includes the region of the molecule where _Lß₂ binds (12, 26). This outside-in signaling can be enhanced when T cell ICAM-3 is clustered by cross-linking the Ab or presenting it as a polyvalent Ab-coated surface. Cross-linking causes a Ca²⁺ influx and activation of tyrosine kinase activity (17) that is needed for ICAM-3-induced aggregation (13, 15). This finding appears to be a fundamental distinction between signaling mechanisms in T cells and PMN, as we found no such requirement for crosslinking of ICAM-3 on PMN to promote outside-in signaling.

The role of ICAM-3 engagement in modulating PMN function, in contrast to a documented role in T cell function, has not been established, and the few published experiments have given conflicting results (12, 18, 19, 27). In fact, a recent report used the HP2/19 Ab, which we found to be nonstimulatory, to suggest that ICAM-3 is a negative modulator of PMN function in a different type of assay (19). Here we show that appropriate ligation of PMN ICAM-3 is stimulatory. Thus our study shows that ICAM-3 ligation generates stimulating signals in PMN as it does in T cells and monocytes (28). However, we find that the epitope that can induce such changes differs when ICAM-3 is expressed on these two cells: the HP2/19 Ab that activates T cells failed to induce PMN aggregation, as reported (19, 27). We also demonstrated that the isotype of the aggregation-inducing Abs is not important as CAL3.10 (IgG1) and ICR1.1 (IgG2a) both induce aggregation. This is relevant as the Fc receptors that interact with these two classes of Ab are different. Accordingly, we found that a F(ab')₂ fragment of the anti-ICAM-3 Ab ICR1.1 also induces PMN aggregation (Fig. 3C).

We found that ligation of ICAM-3 by CAL3.10 caused a rapid increase in the phosphotyrosine content of numerous proteins, a change not found when the cells were treated with the nonstimulatory HP2/19 anti-ICAM-3 Ab. Modest changes in tyrosyl phosphorylation have been found when T cells coactivated with the anti-ICAM-3 Ab HP2/19 (15), an effect that may depend on the recruitment of the tyrosine kinase lck and fyn to ICAM-3 after clustering of ICAM-3 with a second Ab (17). ICAM-3 is physically associated with tyrosine kinase activity in PMN (29). We found two tyrosine kinase inhibitors, herbimycin and genistein, blocked aggregation induced by anti-ICAM-3 (Fig. 7 ), so one or more tyrosine kinase are essential for ICAM-3 induction of functional changes in PMN. Most of the increase in protein phosphotyrosine content, however, is not relevant to ICAM-3-induced aggregation since most of these proteins were also phosphorylated in response to FMLP where aggregation did not depend on tyrosine kinase activity. While the phosphoproteins needed for a functional change are apparently minor entities, the importance of these data are that they show that ICAM-3 ligation initiates a true outside-in signal. That is, ICAM-3 ligation induces phosphorylation of many of the same proteins that are downstream of the FMLP receptor.

Ligation of T cell ICAM-3 by appropriate anti-ICAM-3 Abs results in the redistribution of both _Lß₂ integrin and ICAM-3 to areas of cell-cell contact (26), a similar redistribution of phosphotyrosyl proteins (15), and a redistribution of actin to form a uropod that is enriched with ICAM-3 (14). We show that appropriate ligation of ICAM-3 on PMN also leads to a reorganization of the cytoskeleton, although for PMN this did not require clustering of the anti-ICAM-3 Ab with a second Ab, nor did it require a costimulus. Inhibition of this transient increase in F-actin content by cytochalasin D or SMC treatment showed that these changes in the cytoskeleton were needed for ligation of ICAM-3 to induce adhesion and aggregation. This finding shows that signaling subsequent to ICAM-3 engagement, rather than the actual development of adhesive forces, is dependent on the actin cytoskeleton rearrangement.

Activation of PMN via ICAM-3 ligation resulted in rapid PMN aggregation and adhesion to an immobilized ligand. Unusually, this depended on just the ß₂ integrins that are constitutively expressed on the surface of PMN without augmentation from intracellular stores. This lack of recruitment and activation of supplementary integrins from the intracellular pool allowed us to show that this recruitment amplifies aggregation at low cell density. Conversely, this also shows that activation of just the constitutive surface ß₂ integrin is adequate to support both aggregation at higher cell densities and adhesion to a surface where multiple bonds can form. Basally expressed and newly recruited ß₂ integrins are not equivalent, as newly recruited ß₂ integrins are separately activated (22); and, it is the constitutively expressed ß₂ integrins that cluster in the uropod, while newly expressed ß₂ integrins remain broadly distributed over the cell surface (30). ICAM-3 ligation, like chilling (31), shows that just the basally expressed ß₂ integrin can, after appropriate ligation, support adhesion. However, as might be anticipated, recruitment of the intracellular pool of ß₂ integrins to the cell surface enhances adhesive interactions under suboptimal conditions.

View larger version (21K): [in this window] [in a new window]   FIGURE 1. ICAM-3 ligation activates ß2 integrins. A, PMN aggregation. Human PMN (5 x 106 in 0.5 ml HBSSA) were exposed to 10 µg/ml anti-ICAM-3 (CAL3.10) and/or FMLP (10-7 M) and aggregation was recorded as an increase in light transmittance while stirring in an aggregometer tube. The effects of blocking the ß2 integrin CD18 were determined in two ways: PMN were pretreated with 20 µg/ml of blocking anti-CD18 Ab (mAb 60.3) and then excess Ab was removed by twice centrifuging the PMN and resuspending them in HBSSA. Alternatively, mAb 60.3 was added 10 min prior to the initiation of the assay, and the inhibitory Ab was then also present during the aggregation assay. PMN aggregation was verified visually by removing a 10 µl aliquot at the end of the incubation for fixation, staining, and microscopy. The experiments that utilized the blocking mAb 60.3 were repeated three times with this Ab and twice more with a different CD18 blocking Ab. Other aggregation experiments were repeated at least six times. In all cases a representative aggregometer tracing is shown. B, The rate of anti-ICAM-3-induced aggregation is affected by PMN density. PMN were resuspended at the stated cell density in HBSSA. Aggregation was initiated by addition of 5 µg/ml of anti-ICAM-3 Ab and aggregation over 5 min was followed at 37°C. In some samples FMLP (10-7 M) was added after 3 min, and the rate of aggregation was monitored for a subsequent 2 min. This experiment is representative of two others. C, Anti-ICAM-3 induces CD18-dependent adhesion. PMN were layered over tissue culture wells precoated with gelatin and then exposed to either FMLP (10-7 M) or anti-ICAM-3 for 5 min. Nonadherent and loosely adherent cells were then removed, and the number of tightly bound PMN was determined by video imaging. Three fields of view per well of duplicate wells were used to calculate the standard error. Some PMN were exposed to anti-CD18 mAb 60.3 (10 µg/ml) for 10 min prior to addition to the adhesion assay. This experiment was repeated with near identical results on two other days using different donors.

View larger version (20K): [in this window] [in a new window]   FIGURE 2. Anti-ICAM-3 does not alter surface expression of adhesion molecules. A, ß2 integrin. PMN were treated with anti-ICAM-3 (10 µg/ml) or FMLP for 5 min while stirring in an aggregometer to confirm both agonists induced homotypic adhesion as in Fig. 1. After 5 min, the PMN were removed from the assay and immediately fixed in 1% paraformaldehyde in PBS for 20 min on ice. The sample was washed twice in PBS containing 10% goat serum and the resuspended in 10% goat serum/PBS containing 10 µg FITC-conjugated anti-CD18 mAb (60.3). After 30 min, these PMN were washed once and resuspended in PBS before flow cytometric analysis. The abscissa is the number of cells at a specific level of fluorescence that is indicated by the ordinate. This histogram is representative of five other experiments. B, L-selectin. PMN were treated with anti-ICAM-3, fixed, and washed as in A before they were resuspended in buffer containing FITC-conjugated anti-L-selectin (DREG200). After 30 min, the samples were washed, resuspended in PBS, and cellular fluorescence analyzed by flow cytometry. C, Degranulation. PMN were treated with either FMLP or anti-ICAM-3 while stirring in an aggregometer at 37°C. After 5 min, PMN granularity was determined by side scatter during flow analysis. The histogram is representative of five other similar experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. Activation of ICAM-3 is domain and epitope-specific. A, PMN were exposed to FMLP (10-7 M), PMA (10-8 M), or anti-ICAM-3 domain 1 Abs CAL3.10 (an IgG1 used at 5 µg/ml) or HP2/19 (an IgG2a used at 20 µg/ml), or ICR1.1 (an IgG2a used at 10 µg/ml) as shown. Where stated PMN were also exposed to 20 µg/ml of the domain 4 anti-ICAM-3 Ab CAL3.34. The aggregation assays were done three times using different donors’ PMN, and a representative example is shown. B, Domain 1 and domain 4 anti-ICAM-3 binding are not mutually exclusive. PMN were treated with domain 1 CAL3.10 or additionally with domain 4 CAL3.4 at 20 µg/ml for 5 min, recovered from the assay, fixed with paraformaldehyde, and stained in 10% goat serum/PBS with FITC-conjugated goat anti-mouse Ab and analyzed by flow cytometry as in Fig. 2. The histograms are representative of two other experiments. The variation shown for the fluorescence intensity of cells stained with anti-ICAM-3 domain 1 and 4 is the geometric mean of 10,000 cells. C, Anti-ICAM-3-mediated aggregation is not Fc-receptor mediated. PMN were exposed to a ICR1.1 F(ab')2 at 10 µg/ml at 37°C for 5 min. The aggregation assays were done three times and a representative example is shown. At the end of the 5-min assay, an aliquot was removed and aggregation verified by microscopy.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. ICAM-3 ligation increases F-actin accumulation in PMN. PMN were exposed to FMLP (A) or anti-ICAM-3 (B) for the stated times. These PMN were then placed in ice-cold fixative containing FITC-conjugated phalloidin for 4 h as described in Materials and Methods. The PMN were then washed by centrifugation in ice-cold PBS and resuspended in PBS prior to flow analysis. The histogram is representative of two other experiments.

	Acknowledgments

	Footnotes

 
¹ This work was supported by National Institutes of Health Grants HL 50513 and HL 44525, and by National Cancer Institute Grant CC56 SP30 CA 42014 support of core facilities.

² Current address: Ventana Genetics Inc., Salt Lake City, UT 84108.

³ Address correspondence and reprint requests to Dr. Thomas Marty McIntyre, University of Utah, CVRTI, 95 S 2000 E, Salt Lake City, UT 84112-5000. E-mail address:

⁴ Abbreviations used in this paper: PMN, polymorphonuclear leukocyte(s); HBSSA, HBSS containing 0.5% human serum albumin; SMC, sphingomyelinase C; ERK, extracellularly regulated kinase.

⁵ M. J. Feldhaus, G. A. Zimmerman, S. M. Prescott, and T. M. McIntyre. Ceramide or cytochalasin D block neutrophil ß₂ integrin clustering and adhesion to surfaces without blocking aggregation. Submitted for publication.

Received for publication October 14, 1997. Accepted for publication July 29, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Figdor, C. G., M. Lub, Y. van Kooyk. 1995. Ins and outs of LFA-1. Immunol. Today 16:479.[Medline]
2. Shattil, S. J., M. H. Ginsberg. 1997. Intergrin signaling in vascular biology. J. Clin. Invest. 100:1.[Medline]
3. Staunton, D. E., S. D. Marlin, C. Stratowa, M. L. Dustin, T. A. Springer. 1988. Primary structure of ICAM-1 demonstrates interaction between members of the immunoglobulin and integrin supergene families. Cell 52:925.[Medline]
4. Simmons, D., M. W. Makgoba, B. Seed. 1988. ICAM, an adhesion ligand of LFA-1, is homologous to the neural cell adhesion molecule NCAM. Nature 331:624.[Medline]
5. Vazeux, R., P. A. Hoffman, J. K. Tomita, E. S. Dickinson, R. L. Jasman, T. St. John, W. M. Gallatin. 1992. Cloning and characterization of a new intercellular adhesion molecule ICAM-R. Nature 360:485.[Medline]
6. Fawcett, J., C. L. L. Holness, L. A. Needham, H. Turley, K. C. Gatter, D. Y. Mason, D. L. Simmons. 1992. Molecular cloning of ICAM-3, a third ligand for LFA-1, constitutively expressed on resting leukocytes. Nature 360:481.[Medline]
7. de Fougerolles, A. R., T. A. Springer. 1992. Intercellular adhesion molecule 3, a third adhesion counter-receptor for lymphocyte function-associated molecule 1 on resting lymphocytes. J. Exp. Med. 175:185.[Abstract/Free Full Text]
8. Diamond, M. S., D. E. Staunton, S. D. Marlin, T. A. Springer. 1991. Binding of the integrin Mac-1 (CD11b/CD18) to the third immunoglobulin-like domain of ICAM-1 (CD54) and its regulation by glycosylation. Cell 65:961.[Medline]
9. Staunton, D. E., M. L. Dustin, H. P. Erickson, T. A. Springer. 1990. The arrangement of the immunoglobulin-like domains of ICAM-1 and the binding sites for LFA-1 and rhinovirus. Cell 61:243.[Medline]
10. Klickstein, L. B., M. R. York, A. R. de Fougerolles, T. A. Springer. 1996. Localization of the binding site on intercellular adhesion molecule-3 (ICAM-3) for lymphocyte function-associated antigen 1 (LFA-1). J. Biol. Chem. 271:23920.[Abstract/Free Full Text]
11. Damle, N. K., K. Klussman, A. Aruffo. 1992. Intercellular adhesion molecule-2: a second counter-receptor for CD11a/CD18 (leukocyte function-associated antigen-1) provides a costimulatory signal for T-cell receptor-initiated activation of human T cells. J. Immunol. 148:665.[Abstract]
12. Bossy, D., C. D. Buckley, C. L. Holness, A. J. Littler, N. Murray, I. Collins, D. L. Simmons. 1995. Epitope mapping and functional properties of anti-intercellular adhesion molecule-3 (CD50) monoclonal antibodies. Eur. J. Immunol. 25:459.[Medline]
13. Cid, M. C., J. Esparza, M. Juan, A. Miralles, J. Ordi, R. Vilella, A. Urbano-Marquez, A. Gaya, J. Vives, J. Yague. 1994. Signaling through CD50 (ICAM-3) stimulates T lymphocyte binding to human umbilical vein endothelial cells and extracellular matrix proteins via an increase in ß₁ and ß₂ integrin function. Eur. J. Immunol. 24:1377.[Medline]
14. Campanero, M. R., P. Sanchez-Mateos, M. A. del Pozo, F. Sanchez-Madrid. 1994. ICAM-3 regulates lymphocyte morphology and integrin-mediated T cell interaction with endothelial cell and extracellular matrix ligands. J. Cell Biol. 127:867.[Abstract/Free Full Text]
15. Arroyo, A. G., R. Campanero, P. Sanchez-Mateos, J. M. Zapata, M. A. Ursa, M. A. del Pozo, F. Sanchez-Madrid. 1994. Induction of tyrosine phosphorylation during ICAM-3 and LFA-1-mediated intercellular adhesion, and its regulation by the CD45 tyrosine phosphatase. J. Cell Biol. 126:1277.[Abstract/Free Full Text]
16. de Fougerolles, A. R., M. S. Diamond, T. A. Springer. 1995. Heterogenous glycosylation of ICAM-3 and lack of interaction with Mac-1 and p150,95. Eur. J. Immunol. 25:1008.[Medline]
17. Juan, M., O. Vinas, M. R. Pino-Otin, L. Places, E. Martinez-Caceres, J. J. Barcelo, A. Miralles, R. Vilella, M. A. de la Fuente, J. Vives. 1994. CD50 (intercellular adhesion molecule 3) stimulation induces calcium mobilization and tyrosine phosphorylation through p59^fyn and p56^lck in Jurkat T cell line. J. Exp. Med. 179:1747.[Abstract/Free Full Text]
18. del Pozo, M. A., R. Pulido, C. Munoz, V. Alvarez, A. Humbria, M. R. Campanero, F. Sanchez-Madrid. 1994. Regulation of ICAM-3 (CD50) membrane expression on human neutrophils through a proteolytic shedding mechanism. Eur. J. Immunol. 24:2586.[Medline]
19. Skubitz, K. M., K. D. Campbell, A. P. N. Skubitz. 1997. CD50 monoclonal antibodies inhibit neutrophil activation. J. Immunol. 159:820.[Abstract]
20. Zimmerman, G. A., T. M. McIntyre, S. M. Prescott. 1985. Thrombin stimulates the adherence of neutrophils to human endothelial cells in vitro. J. Clin. Invest. 76:2235.
21. Laemmli, U. K.. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680.[Medline]
22. Hughes, B. J., J. C. Hollers, E. Crockett-Torabi, C. W. Smith. 1992. Recruitment of CD11b/CD18 to the neutrophil surface and adherence-dependent cell locomotion. J. Clin. Invest. 90:1687.
23. Zimmerman, G. A., T. M. McIntyre. 1988. Neutrophil adherence to human endothelium in vitro occurs by CDw18 (Mo1, MAC-1/LFA-1/GP 150,95) glycoprotein-dependent and -independent mechanisms. J. Clin. Invest. 81:531.
24. Borregaard, N., L. Kjeldsen, H. Sengelov, M. S. Diamond, T. A. Springer, H. Clarke-Anderson, T. K. Kishimoto, D. F. Baint. 1994. Changes in subcellular localization and surface expression L-selectin, alkaline phosphatase, and Mac-1 in human neutrophils during stimulation with inflammatory mediators. J. Leukocyte Biol. 56:80.[Abstract]
25. Van der Vieren, M., H. L. Trong, C. L. Wood, P. F. Moore, T. St. John, D. E. Staunton, W. M. Gallatin. 1995. A novel leukointegrin, _dß₂, binds preferentially to ICAM-3. Immunity 3:683.[Medline]
26. Campanero, M. R., M. A. del Pozo, A. G. Arroyo, P. Sanchez-Mateos, T. Hernandez-Caselles, A. Craig, R. Pulido, F. Sanchez-Madrid. 1993. ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway. J. Cell Biol. 123:1007.[Abstract/Free Full Text]
27. Okuyama, M., J. Kambayashi, M. Sakon, M. Monden. 1996. LFA-1/ICAM-3 mediates neutrophil homotypic aggregation under fluid shear stress. J. Cell. Biochem. 60:550.[Medline]
28. Kessel, J. M., J. Hayflick, A. S. Weyrich, P. A. Hoffman, M. Gallatin, T. M. McIntyre, S. M. Prescott, G. A. Zimmerman. 1998. Coengagement of ICAM-3 and Fc receptors induces chemokine secretion and spreading by myeloid leukocytes. J. Immunol. 160:5579.[Abstract/Free Full Text]
29. Skubitz, K. M., K. Ahmed, K. D. Campbell, A. P. N. Skubitz. 1995. CD50 (ICAM-3) is phosphorylated on tyrosine and is associated with tyrosine kinase activity in human neutrophils. J. Immunol. 154:2888.[Abstract]
30. Francis, J. W., R. F. Todd, L. A. Boxer, H. R. Petty. 1989. Sequential expression of cell surface C3bi receptors during neutrophil locomotion. J. Cell Biol. 140:519.
31. Schleiffenbaum, B., R. Moser, M. Patarroyo, J. Fehr. 1989. The cell surface glycoprotein Mac-1 (CD11b/CD18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression. J. Immunol. 142:3537.[Abstract]

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