Integrin Activity 7Β , and 3Β , 2 Β Chemoattractant-Stimulated Differential Regulation Of

Differential Regulation of Chemoattractant-Stimulated β2, β3, and β7 Integrin Activity

This information is current as Chanchal Sadhu, Boris Masinovsky and Donald E. Staunton of September 26, 2021. J Immunol 1998; 160:5622-5628; ; http://www.jimmunol.org/content/160/11/5622 Downloaded from References This article cites 24 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/160/11/5622.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1998 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ␤ ␤ Differential Regulation of Chemoattractant-Stimulated 2, 3, ␤ and 7 Integrin Activity

Chanchal Sadhu,1 Boris Masinovsky, and Donald E. Staunton

Leukocyte adhesion to endothelium and extravasation are dynamic processes that require activation of integrins. Chemoattrac- tants such as IL-8 and FMLP are potent activators of leukocyte integrins. To compare the chemoattractant-stimulated activation ␣ ␤ ␣ ␤ ␣ ␤ of three integrins, 4 7, L 2, and V 3, in the same cellular context, we expressed an IL-8 receptor (IL-8RA) and FMLP receptor ␣ ␤ ␣ ␤ (FPR) in the lymphoid cell line JY. Chemoattractants induced a rapid increase in L 2- and V 3-dependent JY adhesion within ␣ ␤ 5 min, and it was sustained for 30 min. In contrast, stimulation of 4 7-dependent adhesion was transient, returning to basal levels by 30 min. The activation profiles of the integrins were similar regardless of whether IL-8 or FMLP was used for induction. We ␣ ␤ also demonstrate that 4 7-dependent adhesion was uniquely responsive to the F actin-disrupting agent cytochalasin D and the ␣ ␤ ␣ ␤ Downloaded from protein kinase C (PKC) inhibitor chelerythrin. While V 3- and L 2-mediated cell adhesion was significantly reduced by ␣ ␤ ␣ ␤ cytochalasin D, 4 7-mediated adhesion was enhanced. Chelerythrin inhibited both the IL-8 and PMA activation of L 2 and ␣ ␤ ␣ ␤ V 3. In contrast, inducible 4 7 activity was unaffected, and basal activity was increased. These findings demonstrate that the ␣ ␤ ␣ ␤ ␣ ␤ mechanism of 4 7 regulation by chemoattractants is different from that of L 2 and V 3 and that it appears to involve distinct cytoskeletal and PKC dependencies. In addition, PKC activity may be a positive or negative regulator of integrin-dependent adhesion. The Journal of Immunology, 1998, 160: 5622–5628. http://www.jimmunol.org/ xtravasation of circulating leukocytes through the endo- that protein kinase C (PKC2) and phosphatidyl inositol-3 kinase thelium into tissues is a multistep process involving pri- contribute to the signals leading to integrin activation (10). Work E mary and secondary adhesion (1–4). Primary adhesion by Campbell et al. (11) and Weber et al. (8) suggests a complex involves an interaction with a rapid association and dissociation effect by chemoattractants on integrin activation and chemotaxis. rate that mediates initial contact and rolling of leukocytes on en- The T cell line Jurkat, expressing the receptors for IL-8, MIP-1␣, ␣ ␤ ␣ ␤ dothelium under flow conditions. Selectins and the integrins 4 1 C5a, or FMLP, showed a strong but transient activation of 4 1- ␣ ␤ and 4 7 can mediate primary adhesion (1, 5). During secondary dependent adhesion in the presence of a high concentration of the adhesion, leukocytes adhere firmly to the endothelium and undergo appropriate agonist (11). However, suppression of chemotaxis was ␤ ␣ by guest on September 26, 2021 a change in cell shape. Binding of leukocyte 2 (CD18) and 4 observed at higher than optimum agonist concentrations, indicat- integrins to endothelial cell ICAMs and VCAM-1 can mediate ing distinct regulation of adhesion and chemotaxis. Stimulation of secondary adhesion (1, 3). human eosinophils with RANTES, MCP-3, or C5a produced a ␣ ␤ Firm adhesion of leukocytes to the endothelium is dependent on rapid and transient activation of 4 1 but prolonged activation of ␣ ␤ ␤ signaling that leads to integrin activation, which is manifested in M 2 (8). These results suggest a differential regulation of the 1 ␤ increased avidity for ligands. Leukocyte integrins can be activated and 2 integrins. via different receptor types. For example, engagement of T cell Here, we report different signaling and cytoskeletal require- surface molecules such as TCR and L-selectin has been shown to ments for chemoattractant-stimulated activation of three integrins ␣ ␤ activate binding of the leukointegrin L 2 (CD11a/CD18, LFA-1) in the background of one cell type. We have generated lymphoid to ICAM-1 (6, 7). In addition, soluble mediators such as chemoat- cell lines to address the mechanism of integrin activation subse- tractants including chemokines have been reported to induce in- quent to stimulation by agonists of G protein-coupled receptors. creased integrin-dependent leukocyte adhesion. Classical chemoat- Thus, we have studied IL-8- and FMLP-mediated activation of ␣ ␤ ␣ ␤ ␣ ␤ tractants such as FMLP and C5a have been shown to activate 4 7, v 3, and L 2 in a B lymphoid cell line, JY, expressing CD18 integrins on eosinophils (8). Among the different chemo- IL-8 or FMLP receptors. Our results demonstrate a distinct regu- ␣ ␤ ␣ ␤ ␣ ␤ kines, IL-8 can activate integrins on neutrophils and regulate trans- lation of 4 7 integrin activity relative to that of L 2 and v 3 endothelial migration of neutrophils (9). integrins. Chemoattractants activate integrins subsequent to binding their heterotrimeric G protein-coupled receptors. However, little is Materials and Methods known of the exact downstream signaling pathway or the effects of Antibodies these inducers on individual integrins. Emerging evidence suggest The FLAG epitope-specific mAb M1 was from Eastman Kodak (Roches- ␣ ter, NY). Cells producing the L mAb, TS1/22, were from American Type ␤ Culture Collection (ATCC), Rockville, MD, and the 1 mAb, 3S3, was a ICOS Corporation, Bothell, WA 98021 gift from Dr. John Wilkins, University of Manitoba (Winnipeg, Canada). ␤ ␣ Received for publication October 30, 1997. Accepted for publication February The 2 mAb, 22F12C, and the 4 mAb, 72A1H, were generated at ICOS. ␤ ␤ 3, 1998. Fib 504.64, a rat anti-mouse 7 mAb (12) that also binds to human 7, was The costs of publication of this article were defrayed in part by the payment of page obtained from ATCC. All of the Abs were purified according to standard charges. This article must therefore be hereby marked advertisement in accordance procedures. with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Chanchal Sadhu, ICOS Corp., 22021 20th Avenue SE, Bothell, WA 98021. 2 Abbreviations used in this paper: PKC, protein kinase C; FPR, FMLP receptor.

Generation of human lymphoid cell lines expressing IL-8 or family. Based on their pertussis toxin sensitivity, it is believed that FMLP receptor both of these receptors transduce a signal through the Gi class of The human IL-8RA (IL-8 receptor subtype A) (13) sequence was amplified G proteins (15, 16). These receptors were tagged with the FLAG from genomic DNA. The FMLP receptor (FPR) cDNA was a gift from Dr. epitope at their N termini and were expressed in the human B ␣ ␤ ␣ ␤ Richard Ye (The Scripps Research Institute, La Jolla, CA). HindIII and lymphoid cell line JY. JY cells express the integrins L 2, v 3, XbaI restriction endonuclease sites were added by PCR to the 5Ј and 3Ј ␣ ␤ and 4 7 (Fig. 1), all of which may be stimulated by PMA (data ends of the IL-8RA and the FPR cDNA clones. For IL-8RA, the following Ј Ј not shown). The untransfected cells did not respond to the che- oligonucleotide primers were used: 5 (HindIII), 5 ATGCAAGCTT ␣ ␤ ␣ ␤ ␣ ␤ TCAAAT ATTACAGATCCA 3Ј; and 3Ј (XbaI), 5ЈATGCTCTAGATTT moattractants, as there was no increased L 2-, V 3-, or 4 7- TCAGAGGTTGGAAGAG AC 3Ј. Sequences of the oligonucleotide prim- dependent binding observed in the presence of either FMLP or ers used for the FPR cDNA are: 5Ј (HindIII), 5ЈATGCAAGCTTGAG IL-8 (data not presented). After transfection and G418 selection, ACAAATTCCTCTCTC 3Ј; and 3Ј (XbaI), 5ЈATGCTCTAG ATCACTT cells were sorted using a mAb that binds to the FLAG epitope. TGCCTGTAACGCCAC 3Ј. The PCR-amplified products were verified by DNA sequencing. The mammalian expression vector, pcDNA3 (Invitro- Following two rounds of sorting, cell lines expressing high levels gen, San Diego, CA) was modified by inserting the bovine prolactin signal of FPR (JY-fp) and IL-8RA (JY-8) were established (Fig. 1). Cell sequence and the FLAG epitope (Eastman Kodak) 3 prime to the CMV surface expression of both the IL-8 and FMLP receptors remained promoter. The HindIII-XbaI site-adapted cDNAs were then ligated to the stable for months without subsequent G418 selection. corresponding sites of the pcDNA3-FLAG expression vector. In the re- sulting construct, the IL-8RA and FPR cDNAs were in-frame to the pro- IL-8- or FMLP-induced activation of integrins on JY lactin signal sequence and the FLAG epitope. transfectants The FLAG-tagged receptors were expressed in the human B lympho- Downloaded from blastoid cell line, JY, obtained from ATCC. For each electorporation, 1 ϫ We tested JY-8 and JY-fp for their ability to activate integrins 107 cells were centrifuged, washed in ice-cold PBS, and resuspended in 0.5 ␤ upon treatment with IL-8 or FMLP. Of the four known 2 (CD18) ml of PBS. Thirty micrograms of the expression construct DNA was added ␣ ␤ to the cell suspension and incubated on ice for 10 min. Electroporation was integrins, JY cells express only L 2, which binds to ICAM-1. As done at 250 V, 960 ␮Fd capacitance, using a Bio-Rad (Hercules, CA) shown in Figure 2, IL-8 stimulated JY-8 adhesion to ICAM-1 in a electroporator. After incubating the electroporated cells on ice for 10 min, concentration-dependent manner. The maximum response was ob- they were transferred to the growth medium for 24 h. The transfected cells served at about 60 ng/ml of IL-8 (Fig. 2A). This concentration of were then selected using G418 at 1 mg/ml in medium. Expression of the IL-8 also resulted in maximal stimulation of ␣ ␤ - and ␣ ␤ -de- http://www.jimmunol.org/ receptors on the surface of the transfected cells was monitored using the 4 7 V 3 anti-FLAG Ab, M1 (Eastman Kodak). About 10% of the G418-resistant pendent adhesion (data now shown). At this concentration, there ␣ ␤ cells expressed the FLAG epitope. The cell population was stained with the was a two- to threefold increase in L 2-mediated adhesion to M1 Ab and sorted for high levels of receptor-expressing cells. After two ICAM-1. This response was similar in magnitude to PMA-stimu- rounds of sorting, 95% of the cells expressed the FLAG epitope. Functional lated JY-8 adhesion to ICAM-1 (Fig. 2A). Adhesion of JY-8 to expression of the receptors was confirmed by cell adhesion and chemotac- ␣ tic assays in the presence of IL-8 and FMLP (see below). These cells were ICAM-1 was completely and specifically blocked by the L ␤ designated JY-8 and JY-fp. (CD11a) mAb and the 2 (CD18) mAb (data not shown). JY cells transfected with the expression vector (JY-vector) did not demon- Adhesion assay

strate IL-8-stimulated binding (Fig. 2A). by guest on September 26, 2021 Adhesion assays were performed in 96-well Easy Wash plates (Corning JY-fp binding to ICAM-1 was specifically stimulated by FMLP, Glass, Corning, NY) using a modified procedure (14). Each well was with maximum binding observed at ϳ600 nM (Fig. 2B). At this coated with 50 ␮l of ICAM-1/Fc (10 ␮g/ml), VCAM-1/Fc (5 ␮g/ml), or concentration of FMLP, there was an approximately threefold in- vitronectin (0.5 ␮g/ml) in 50 mM bicarbonate buffer (pH 9.6). Some wells ␤ crease in binding of JY-fp to ICAM-1. FMLP did not stimulate were coated with a 2 mAb (22F12C, ICOS), to quantitate the maximum number of input cells binding, (taken as 100%) or glycophorin, to deter- adhesion of JY-vector cells (Fig. 2B). Thus, JY-fp and JY-8 ex- mine background binding. Plates were blocked with 1% BSA in PBS for press functional chemoattractant receptors. 1 h at room temperature. Wells were then rinsed and 200 ␮l of adhesion ␣ ␤ ␣ ␤ ␣ ␤ buffer (RPMI ϩ 0.2% human serum albumin) was added with or without Activation profiles of L 2, v 3, and 4 7 by IL-8 and FMLP PMA (20 ng/ml), IL-8, or FMLP. Cells (100 ␮lof1ϫ 106/ml) were then We determined and compared the magnitude and rate of chemoat- added to each well, and plates were incubated at 37°C in 5% CO2 for the indicated time. Cells were allowed to settle on the plate for 25 min, and tractant-mediated activation of three integrins expressed in JY-8 ␣ ␤ ␣ ␤ then PMA (20 ng/ml) or IL-8 or FMLP was added. In some assays, cells and JY-fp. JY cells express the integrins 4 7 and V 3 (Fig. 1), were mixed with cytochalasin D and added to the wells such that the final which bind to VCAM-1 and vitronectin, respectively (17, 18). JY ␮ concentration of cytochalasin D was 10 g/ml. For chelerythrin treatment, cells may express a very low level of ␤ integrin (Fig. 1, second cells were preincubated with the inhibitor at 37°C for 10 min and then 1 added to wells. Adherent cells were fixed with the addition of 50 ␮lofa row, middle panel), however, binding to VCAM-1 was blocked by ␤ ␤ 10% glutaraldehyde solution and stained with 0.5% crystal violet (Sigma, 7-blocking mAb, and not by 1-blocking mAb (data not shown). St. Louis, MO) solution. The plates were washed in several changes of Thus, we compared binding of the JY-8 and JY-fp cells to water. After washing, 70% ethanol was added, and adherent cells were ICAM-1, VCAM-1, and vitronectin as an indication of the activa- quantitated by determining absorbance at 570 nm using a SPECTRAmax ␣ ␤ ␣ ␤ ␣ ␤ 250 microplate spectrophotometer system (Molecular Devices, Sunnyvale, tion profile of L 2, 4 7, and V 3. CA). Percentage of cell binding was determined using the formula: In the absence of stimulation, there was a slight increase in JY-8 and JY-fp binding to ICAM-1 within the first 5 min. Thereafter, ͑ A570 binding to ICAM-1 or VCAM-1 this level of binding did not change significantly for up to 30 min or VN͒ Ϫ A ͑binding to BSA͒ 570 ϫ (Fig. 3, A and D). In the presence of the appropriate agonists, ͑ ͒ 100. A570 binding to CD18 mAb however, both the JY-8 and the JY-fp cells showed two- to threefold enhanced binding to ICAM-1 within 5 min. Thus, there is a Results ␣ ␤ rapid activation of L 2 induced by the binding of either agonist Establishment of IL-8 and FMLP receptor-expressing cell lines to their corresponding receptors. The initial two- to threefold in- For a detailed analysis of integrin activation by chemoattractants, crease in binding was sustained for at least 30 min. ␣ ␤ ␣ ␤ we have established cell lines stably expressing either of two che- The activation profile of V 3 was similar to that of L 2 (Fig. moattractant receptors. The formyl peptide receptor, FPR, is rep- 3, C and F). There was a rapid increase in JY-8 and JY-fp cell resentative of the classical chemoattractant receptor, and the other, binding to vitronectin within 5 min of adding either IL-8 or FMLP, IL-8RA (CCR1), is a member of the ␣ chemokine (CXC) receptor as compared with the binding in the absence of either agonist. The 5624 DIFFERENTIAL REGULATION OF INTEGRIN ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. Expression of chemoattractant receptors and integrins on JY transfectants. JY cells were transfected with either IL-8RA (JY-8) or FPR (JY-fp) expression constructs, selected with G418, and sorted using the anti-FLAG mAb, M1. Cell surface expression (shaded areas) of the indicated ␣ ␤ ␣ ␤ ␣ ␤ molecule was determined by staining with the following mAb: IL-8RA (M1); L (TS1/22); 2 (22F12C); FPR (M1); V 3 (mAb 1976; V 5 (mAb 1961); ␣ ␤ ␤ 4 (72A1H); 1 (3S3); 7 (Fib 504.64). Unshaded curves show staining with isotype-matched irrelevant mAb. Expression of integrins in JY-8 cells are shown. The pattern of integrin expression in JY-fp cells (not shown) was identical to that of the JY-8 cells. The level of FPR expression by the JY-fp cells is shown in the bottom right panel.

maximum level of induced binding in the presence of the agonists was not sustained. Since both JY-8 and JY-fp demonstrated a tran- ␣ ␤ was two- to threefold. Since the activation of v 3 was sustained, sient induction of binding to VCAM-1 relative to ICAM-1 and the ratio of induced binding to uninduced binding did not change vitronectin, this suggests a unique and integrin-proximal modula- ␣ ␤ significantly for as long as 30 min. tion of the 4 7 activity. The activation profile of ␣ ␤ in JY-8 and JY-fp differed from 4 7 ␣ ␤ that of ␣ ␤ and ␣ ␤ . As shown in Figure 3B, there was in- Differential effect of cytochalasin D on activation of L 2-, L 2 V 3 ␣ ␤ ␣ ␤ creased binding of the JY-8 cells to VCAM-1 within the first 5 min 4 7-, and V 3-mediated activation of cell adhesion ␣ ␤ after adding IL-8, indicating a rapid activation of 4 7. Both the Cytochalasin D, which prevents actin polymerization (19), blocks ␣ ␤ ␣ ␤ response time and fold increase was similar to that of L 2 and PMA-induced L 2-mediated aggregation of JY cells, suggesting a ␣ ␤ ␣ ␤ ␣ ␤ V 3. However, the induced 4 7-mediated binding in response to role for F actin in the regulation of L 2 activity (20). We tested IL-8 was not sustained. After the initial 5 min, a steady decrease in whether IL-8- or PMA-induced activation of integrins on JY-8 re- binding was observed. By 30 min, the extent of cell adhesion to quires an intact cytoskeleton. Because FMLP- and IL-8-stimulated VCAM-1 in the presence of IL-8 was essentially the same as in the responses were identical, with perhaps both receptors interacting with absence of IL-8. This resulted in a decreased ratio of binding to the same G␣i, only IL-8 was used in these and subsequent assays. VCAM-1 in the presence of IL-8 vs in the absence of IL-8. The Cytochalasin D treatment significantly reduced both IL-8- and PMA- observed decrease in the ratio between 5 and 30 min is in contrast induced cell binding to ICAM-1 (Fig. 4A). A similar pattern of cy- ␣ ␤ to the profile of binding to ICAM-1 and vitronectin, indicating a tochalasin D inhibition was observed for V 3-dependent adhesion to ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ differential temporal regulation of 4 7. vitronectin (Fig. 4C). In contrast to that of L 2 and V 3, 4 7- Chemoattractant-stimulated binding of JY-fp cells to VCAM-1 mediated JY-8 binding was markedly enchanced in the presence of was similar to that of JY-8. As shown in Figure 3E, FMLP caused the same concentration of cytochalasin D (Fig. 4B). A greater than ␣ ␤ a rapid increase in the JY-fp cell binding to VCAM-1 within 5 min. threefold enhancement of 4 7-mediated JY-8 cell binding to With longer periods of incubation, the agonist-stimulated binding VCAM-1 was observed in the presence of cytochalasin D alone. IL-8 The Journal of Immunology 5625 Downloaded from http://www.jimmunol.org/

FIGURE 2. Dose-response of agonist-induced cell adhesion to ICAM-1. A, IL-8 concentration dependence of JY-8 (open bars) and JY- vector (solid bars) adhesion; B, FMLP concentration dependence of JY-fp

(open bars) and JY-vector (solid bars) adhesion. Error bars show the range by guest on September 26, 2021 of triplicate samples. One representative example of several experiments is shown. Percentage of cell binding was determined by crystal violet staining, quantiﬁcation of A570, and normalized according to the formula presented in Materials and Methods. or PMA further increased JY-8 cell adhesion to VCAM-1 in the pres- ␣ ␤ ␣ ␤ ␣ ␤ ence of cytochalasin D. Thus, 4 7 differs from L 2 and V 3 in its requirement of F actin.

Role of PKC in IL-8-stimulated integrin activation Members of the PKC family of enzymes can stimulate integrin- dependent adhesion, since the PKC agonist, PMA, stimulates integrin activity. Calphostin C, a potent inhibitor of diacylglycerol and Ca2ϩ-dependent PKC isoforms (21), can block PMA-induced ␣ ␤ ␣ ␤ 4 1 and M 2 integrin activation (16). However, calphostin C did ␣ ␤ ␣ ␤ not inhibit IL-8- or FMLP-stimulated 4 1- and M 2-dependent adhesion (16). We used chelerythrin (22), a catalytic domain antagonist of different PKC isoforms, to determine whether different PKC family

␣ ␤ FIGURE 3. IL-8 or FMLP induced prolonged activation of L 2 and ␣ ␤ ␣ ␤ V 3 and transient activation of 4 7. Shown are the time courses of agonist induced cell adhesion to ICAM-1 (10 ␮g/ml), VCAM-1 (5 ␮g/ml), and vitronectin (0.5 ␮g/ml). A, B, C, IL-8; D, E, F, FMLP. Gray bar indicates with agonist; open bar, without agonist. Final concentration of IL-8 was 100 ng/ml and that of FMLP was 500 nM. After cells settled, agonists were added and the plate was incubated at 37°C. Incubation was stopped by the addition of glutaraldehyde (ﬁnal concentration of 1.5%) at the indicated times. Percentage of cell adhesion was determined as described in Figure 2 legend. Shown is one representative of three experiments. 5626 DIFFERENTIAL REGULATION OF INTEGRIN ACTIVATION

␣ ␤ FIGURE 5. Inhibition of L 2-mediated cell adhesion by the PKC inhibitor chelerythrin. JY-8 cells were pretreated with chelerythrin for 10 min

at 37°C and allowed to settle in wells coated with ICAM-1. Percentage of Downloaded from cell binding at the end of 5 min (with no inducer ᮀ, IL-8 ᮀ,orPMAf) was measured as described in the legends to Figures 2 and 4. Final concentration of chelerythrin in each well is indicated. Duration and concentration of chelerythrin during the experiment did not affect cell viability as measured by trypan blue staining. One representative of three experiments is shown. http://www.jimmunol.org/ IL-8- and PMA-stimulated cell adhesion. These results suggest a ␣ ␤ role for PKC in IL-8- mediated stimulation of L 2. ␣ ␤ ␣ ␤ We further compared the effect of chelerythrin on L 2, 4 7, ␣ ␤ ␮ and V 3 activity. Because at 12.5 M chelerythrin blocked both chemoattractant- and PMA-stimulated adhesion of JY-8 to ICAM-1 (Fig. 5), it was used in additional experiments. Chel- erythrin was not used at higher concentrations at which additional PKC isoforms or kinases might be inhibited. As seen in Figure 6, A and C, at this concentration chelerythrin markedly inhibited both by guest on September 26, 2021 ␣ ␤ ␣ ␤ IL-8- and PMA-stimulated L 2 and v 3 activity. In contrast, chelerythrin did not inhibit IL-8-stimulated JY-8 adhesion and only marginally inhibited PMA-stimulated adhesion (Fig. 6B)to VCAM-1 (Fig. 6B). Furthermore, chelerythrin treatment increased basal level binding to VCAM-1 by approximately twofold and IL-8-induced binding by about 20%. These results indicate that ␣ ␤ ␣ ␤ ␣ ␤ 4 7 differs from L 2 and V 3 in PKC dependency. Discussion We expressed functional chemoattractant receptors in the human B lymphoid cell line, JY, to compare activation as well as cytoskel- ␣ ␤ ␣ ␤ etal and PKC dependencies of three integrins, L 2, V 3, and FIGURE 4. Differential cytoskeleton dependency for integrin-depen- ␣ ␤ 4 7, in a common cellular context. These studies resulted in three dent cell adhesion. JY-8 cells were treated with cytochalasin D (solid bars) key observations. First, stimulation of JY transfectants with the or DMSO (open bars) and allowed to bind to ICAM-1 (A)-, VCAM-1 (B)-, appropriate chemoattractant resulted in a rapid activation of all or vitronectin (VN) (C)-coated surfaces. Cells were incubated for an additional 5 min in the presence of no agonist, IL-8, or PMA. Percentage of three integrins. Second, although their initial activation profiles cell adhesion was determined as described in the Figure 2 legend. Shown were similar, a significant difference was observed in the temporal is one representative of three experiments. behavior of the integrins. IL-8 or FMLP induced prolonged stim- ␣ ␤ ␣ ␤ ulation of L 2 and V 3-dependent adhesion, whereas under sim- ␣ ␤ ilar conditions, 4 7-dependent adhesion was transient. After 5 ␣ ␤ min of IL-8 or FMLP stimulation, 4 7-mediated cell adhesion to members are involved in IL-8-mediated integrin activation in VCAM-1 begins to decline, and by 30 min it returns to the un- ␣ ␤ ␣ ␤ JY-8. Although chelerythrin may have broad specificity, its IC50 stimulated level. Third, the 4 7 integrin differed from L 2 and ␣ ␤ for different PKC isoforms can vary. Initially, we determined the V 3 in its sensitivity to both cytochalasin D and chelerythrin ␣ ␤ ␣ ␤ ␣ ␤ effect of a range of concentrations of chelerythrin on L 2-depen- treatment. While L 2- and V 3-dependent adhesion was signif- Ͻ ␮ ␣ ␤ dent JY-8 adhesion to ICAM-1 (Fig. 5). At concentrations 6 M, icantly reduced by cytochalasin D, 4 7 function was enhanced chelerythrin did not inhibit IL-8- or PMA-stimulated binding. At 6 under identical conditions. Chelerythrin significantly blocked ␮ ␣ ␤ ␣ ␤ M, there was no inhibition of PMA-stimulated binding, although L 2 and V 3 activity, whereas, at the same concentration, it ␮ ␣ ␤ IL-8-stimulated binding was blocked 50%, while at 12.5 Mor enhanced 4 7 activity. These data demonstrate differential inte- higher concentrations, chelerythrin substantially blocked both grin regulation in JY cells. The Journal of Immunology 5627

␣ ␤ manner, whereas M 2 activity demonstrated a prolonged increase in the presence of several chemoattractants (8). In accordance with ␣ ␤ these results, transient activation of 4 1 in Jurkat cell transfectants expressing the IL-8RA has been reported (11). A transient ␣ ␤ up-regulation of the activity of 4 7 in mouse pre-B cell transfec- ␤ tants expressing 7 and FPR has also been reported (11). Our ␣ ␤ results with JY cells that express endogenous 4 7 further extends this observation. Collectively, these studies demonstrate that the ␣ ␣ ␤ ␣ ␤ 4 integrins 4 1 and 4 7 are transiently activated by several chemokines/chemoattractants in a variety of cell types. Data presented here provide evidence that two chemoattractants can stim- ␣ ␤ ␣ ␤ ulate prolonged up-regulation of the activity of L 2 and V 3 in ␣ ␤ ␣ ␤ the same cell that demonstrates transient 4 7 activity. Thus, 4 7 may possess a different integrin-proximal mechanism of regulation. To begin to determine the mechanism for this difference, we addressed the role of F actin involvement. Our results with cytochalasin D demonstrate a requirement of intact actin filaments in JY cells for both IL-8- and PMA-stimu- ␣ ␤ ␣ ␤ Downloaded from lated L 2 and V 3-dependent adhesion. Consistent with this finding, cytochalasin B has been reported to abolish PMA-stimu- ␣ ␤ lated, L 2-mediated aggregation of JY cells (20). At similar concentrations of cytochalasin D as that used in our assays, Kucink et ␣ ␤ al. (23) also reported inhibition of L 2 activity, although at lower concentrations cytochalasin D showed a stimulatory effect. Both our data and others (20) suggest that an intact actin cytoskeleton is http://www.jimmunol.org/ ␣ ␤ essential for L 2-mediated adhesion. In addition, using CHO ␣ ␣ ␤ ␤ cells expressing a chimeric integrin, IIb L/ 3 2, which contains ␣ ␤ the cytoplasmic tails of L and 2 chains, a similar inhibitory effect of cytochalasin on adhesion to fibrinogen was observed, although it did not alter the high affinity status of the integrin thus implying ␣ ␤ a requirement for an L 2 cytoplasmic tail-cytoskeleton interaction for adhesion (24). These results contrasts with that of the ␤ ␣ ␤ activity of another 2 integrin, M 2 (Mac-1), in eosinophils. Che- by guest on September 26, 2021 ␣ ␤ moattractant-stimulated increase in the adhesiveness of M 2 in eosinophils was unaffected by cytochalasin treatment (8). Thus, ␣ ␤ ␣ ␤ ␤ even though both L 2 and M 2 share a common -chain, they demonstrate distinct regulation. This difference may therefore be a function of their ␣-chains or of the different cell types utilized. ␣ ␣ ␤ ␣ ␤ Integrins that share a common -chain, 4 1 and 4 7, may also differ in their cytochalasin sensitivity. We demonstrated that in JY ␣ ␤ cells 4 7-dependent basal level adhesion was enhanced by cy- ␣ ␤ tochalasin. In addition, IL-8- or PMA-induced 4 7-dependent adhesion was further augmented by cytochalasin. However, in human eosinophils the chemoattractant-stimulated increase in the ␣ ␤ activity of 4 1 was inhibited by cytochalasins, suggesting a requirement of an intact cytoskeleton (8). Thus, the cytochalasin- ␣ sensitive or -insensitive nature of 4 integrins could be attributed ␤ ␤ ␤ to the -chains ( 1 vs 7) or to differences in the cell types used in the binding studies. In addition to F-actin, we also addressed the role of PKC in integrin activation. Chelerythrin binds to the catalytic domain of PKC and is thought to inhibit all PKC isoforms (22). IL-8 and PMA stimulate translocation of PKC from the cytosol to the mem- brane in JY-8 (C. Sadhu, K. Dick, and D. E. Staunton, unpublished FIGURE 6. Differential effect of chelerythrin on integrin activation. observation). Our data show that both PMA and IL-8 stimulation ␣ ␤ ␣ ␤ JY-8 cells were pretreated with chelerythrin as described in Figure 5 legend of L 2- and V 3-dependent adhesion are chelerythrin sensitive, and allowed to bind to ICAM-1 (A), VCAM-1 (B), or vitronectin (C)inthe indicating a role for PKC in the signaling pathways employed by presence of no agonist, IL-8, or PMA as described in Figure 3. Activation both stimuli. However, under identical conditions chelerythrin had ␣ ␤ by the agonists was terminated at the end of 5 min, and bound cells were an opposite effect on activation of 4 7. While chelerythrin inhib- quantitated. Shown is one representative of three experiments. ␣ ␤ ␣ ␤ ␣ ␤ ited L 2 and V 3 activity, it enhanced the activity of 4 7. This suggests that PKC may also function as a negative regulator of ␣ ␤ Different temporal regulation of integrin activity by chemoat- 4 7-dependent adhesion. tractants has been demonstrated in leukocytes isolated from blood. In summary, we have shown that in the same cellular context, ␣ ␤ ␣ ␤ ␣ ␤ ␣ ␤ In human eosinophils, 4 1 activity was up-regulated in a transient 4 7 regulation differs from L 2 and V 3 in its 1) temporal 5628 DIFFERENTIAL REGULATION OF INTEGRIN ACTIVATION pattern of activation, 2) requirement of an intact cytoskeleton, and 10. Kolanus, W., and B. Seed. 1997. Integrins and inside-out signal transduction: 3) sensitivity to PKC inhibition. These results suggest the exis- converging signals from PKC and PIP3. Curr. Opin. Cell Biol. 9:725. 11. Campbell, J. J., S. Qin, K. B. Bacon, C. R. Mackay, and E. C. Butcher. 1996. tence of integrin-specific steps in the pathways of integrin regula- Biology of chemokine and classical chemoattractant receptors: differential re- ␣ ␤ tion. The transient nature of 4 7 adhesion and relative indepen- quirements for adhesion-triggering versus chemotactic responses in lymphoid ␣ ␤ cells. J. Cell Biol. 134:255. dence of F actin structure may reflect the distinct function of 4 7 in primary or rolling adhesion. 12. Andrew, D. P., C. Berlin, S. Honda, T. Yoshino, A. Hamann, B. Holzmann, P. J. Kilshaw, and E. C. Butcher. 1994. Distinct but overlapping epitopes are involved in alpha 4 beta 7-mediated adhesion to vascular cell adhesion mole- Acknowledgments cule-1, mucosal addressin-1, fibronectin, and lymphocyte aggregation. J. Immu- nol. 153:3847. We thank Drs. Richard Ye and Carol Raport for the FPR and IL-8RA 13. Holmes, W. E., J. Lee, W. J. Kuang, G. C. Rice, and W. I. Wood. 1991. Structure cDNAs; Dr. John Wilkins for the 3S3 mAb; Lee Hendrickson and Amy and functional expression of a human interleukin-8 receptor. Science 253:1278. Wilson for excellent technical assistance; and Alice Dersham for assistance 14. Morla, A., Z. Zhang, and E. Ruoslahti. 1994. Superfibronectin is a functionally in preparing the manuscript. 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