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*BACITRACIN
The Journal of Immunology, 1998, 161: 6323-6329.
Copyright © 1998 by The American Association of Immunologists

The Selective Inhibition of ß1 and ß7 Integrin-Mediated Lymphocyte Adhesion by Bacitracin1

Yanglong Mou{dagger},*,{ddagger}, Heyu Ni{dagger},* and John A. Wilkins2,{dagger},*,{ddagger},§

* The Rheumatic Diseases Research Laboratory and Departments of {dagger} Immunology, {ddagger} Medical Microbiology, and § Medicine, University of Manitoba, Winnipeg, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment. The regulation of such adhesion often involves changes in the functional state of the integrins rather than alterations in their expression levels. Although the functional basis for such transitions is unknown, a possible role for disulfide exchange might be postulated based on the observations that integrin function can be activated by bifunctional reducing agents or by Abs that react with areas adjacent to predicted long-range disulfide bonds in integrins. Recently, it has been reported that enzymes that catalyze disulfide exchanges such as protein disulfide isomerase (PDI) are present on the surface of lymphoid cells, raising the possibility that such enzymes might be involved in the control of lymphocyte adhesion. A number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of T cells. The results did not support role for PDI in the regulation of integrin function, as the inhibitors somatostatin A, tocinoic acid, dithiobisnitrobenzoic acid, and anti-PDI mAb did not interfere with adherence. However, one of the PDI inhibitors, bacitracin, selectively interfered with the ß1 integrin-mediated adherence of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and with {alpha}4ß7-dependent adherence to fibronectin and to VCAM-1. In contrast, {alpha}vß3- and {alpha}Lß2-mediated adherence were not inhibited. Thus, it appears that bacitracin may be a selective inhibitor of ß1 and ß7 integrin functions by an as yet unknown mechanism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lymphoid cells display marked variations in their requirements to interact with other cells and with extracellular matrix components of their microenvironment. These interactions are essential for the appropriate recruitment to and retention of cells at sites of immunologic responsiveness (1). Thus, it is important for lymphocytes to acquire adhesive potential at the appropriate tissue sites. Agents such as Ags, mitogenic stimuli, chemokines, and cytokines have been shown to induce lymphocyte adhesion, thus providing a basis for the selection of the appropriate cells from the circulating lymphocyte pool (2, 3, 4).

Integrins represent one of the major adhesive systems employed by lymphoid cells for extravasation, migration, and adherence to the extracellular matrix. Integrins are expressed on the surface of leukocytes in a latent form that can be activated by a variety of stimuli (1, 2, 3, 4). Often the acquisition of adhesion competency is not associated with changes in the levels of integrin expression, implying that the surface-expressed molecules undergo conformational changes (5). Although there is considerable evidence from a variety of biochemical and immunologic approaches to support such a contention, the molecular basis for the transition to an active state is unknown (6).

A number of stimuli, including divalent cations such as Mn2+ and Mg2+, have been shown to activate integrin function (7, 8, 9). In many cases, it has been possible to demonstrate that the integrin complex acquires a conformation that resembles that of a ligand-occupied structure (10, 11, 12), suggesting that these agents may promote binding by stabilizing an integrin conformation that facilitates ligand-receptor interactions (6). Collectively, these observations raise the possibility that physiologic mechanisms might also generate such a conformational transition.

The ß1, ß2, and ß3 integrins can be activated by bifunctional reducing agents such as DTT (13, 14, 15, 16). There are structural constraints on the distance separating the sulfydryl groups of the reducing agent, implying that there may be a minimal distance that must be spanned to allow for simultaneous -SH exchange on the target molecule (15). We have demonstrated that the induction of ß1 adhesion by DTT is associated with conformational changes in the integrins, suggesting that the acquisition of adhesion competence may be the result of the direct actions of the reducing agent on the integrin (13).

It has recently been reported that the enzyme protein disulfide isomerase is located on the surfaces of a variety of cell types, including lymphocytes (17, 18, 19, 20, 21). The distribution of this enzyme was previously thought to be restricted to the endoplasmic reticulum, where it plays an essential role in the exchange of disulfide bonds and in the proper folding of newly synthesized protein (20). Surface-associated thiol disulfide transferase activity has been shown to be necessary for diphtheria toxin activation and for the infection of lymphoid cells with HIV (22, 23). Activation of platelet adhesion has also been shown to lead to a marked increase in PDI expression on the surface of these cells. It was therefore questioned whether cell surface PDI3 might play a role in integrin activation, possibly by facilitating intramolecular disulfide exchanges. As an approach to examining this possibility, the effects of a number of inhibitors of PDI function were examined for their effects on integrin-mediated adherence of lymphoid cells to a number of ligands.

The present studies demonstrate that an inhibitor of PDI activity, bacitracin, inhibits ß1 integrin-mediated adhesion of lymphoid cells to collagen, fibronectin, laminin, and VCAM-1, and ß7 to fibronectin and VCAM-1. Bacitracin interferes with binding of soluble fibronectin, implying that the inhibition is due to a direct effect on integrin-ligand interactions. These effects appear to be selective, as ß2 and ß3 integrin-dependent adherence was not inhibited by bacitracin. The fact that these effects were not observed with other inhibitors of PDI activity suggests that the mechanism of bacitracin action does not involve interference with surface thiol-reductase activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

Unless otherwise indicated, all chemicals were purchased from Sigma (St. Louis, MO). Media and FBS were obtained from Life Technologies (Gaithersburg, MD). Purified human plasma fibronectin and FITC goat anti-mouse IgG were obtained from Chemicon (Temecula, CA).

Antibodies

The Abs to ß1, JB1A (24), B3B11, N29 (25), and 3S3 (26) have been described previously. Anti-PDI, RL 77 (27), was purchased from Affinity Bioreagents (Golden, CO). The hybridoma was subsequently obtained through Dr. Charlotte Kaetzel (University of Kentucky). A rat anti-mouse ß7 integrin chain that cross-reacts with human ß7 integrin, FIB 504 was purchased from PharMingen (San Diego, CA). Anti-ß2 and anti-{alpha}vß3, LM-609, were purchased from Chemicon.

Cells and culture

PBMCs were isolated from normal healthy volunteers on Ficoll-Hypaque and cultured for 72 h in RPMI 1640 containing 10% FBS and 10 µg/ml of phytohemagglutinin-P (PHA-P). Human rIL-2 (25 U/ml) was added to the culture, and the cells were maintained by dilution every 2 to 3 days in fresh media containing IL-2 until day 12, after which time they were used for analysis.

IL-2-dependent T cells were washed twice in RPMI and resuspended in media containing bacitracin (0.1–10 mM), tocinoic acid (0.5 mM), or anti-PDI (800 µg/ml) for 30 min before activation for adherence. Cells were stimulated with PMA (50 ng/ml), or anti-ß1 (10 µg/ml) or cross-linked anti-CD3 (150 ng/ml) and added at 2 x 105 cells/well to microtiter wells coated with fibronectin (1 µg/well) (28). The plates were incubated at 37°C for 10 min. The wells were then filled with 0.15 M saline, sealed with an adhesive plate sealer, inverted, and centrifuged at 70 x g for 5 min. The supernatant was aspirated, and the adherent cells were stained with crystal violet for at least 30 min with 0.5% crystal violet in a 30% solution of methanol in water. The plates were washed with tap water to remove unbound dye. The cell-bound dye was dissolved in methanol, and the absorbance at 550 nm was determined. In all assays, the adherence to BSA (OD < 0.1) was subtracted from the values obtained for the fibronectin-coated wells. Unless indicated otherwise, all assays were performed at least three times in pentuplicate.

The human lymphoid cell lines Jurkat and RPMI 8866 were examined for adhesion, as described above. In some cases, the adherence to fibronectin fragments, 120 or 40 kDa (Life Technologies), was determined by substituting the purified fragments for fibronectin in the coating of the wells.

Ab blocking of cell adhesion was achieved by preincubating the cells with the indicated Abs for 30 min before the addition to the cell-binding assays.

Homotypic aggregation assays

JY cells were treated with 50 ng/ml PMA with or without 3.5 mM bacitracin for 3 h and microscopically examined for homotypic aggregation. Ab inhibition studies were performed by incubating the cells with 10 µg/ml of blocking anti-ß2.

Flow cytometry analysis

Cells were preincubated with the indicated stimuli at room temperature and then incubated with the indicated Ab (5 µg/ml) for 30 min at 37°C. The cells were washed twice with PBS and incubated for 60 min at 4°C with a FITC-labeled goat anti-mouse Ig (Chemicon). Fluorescence analysis was performed with a BD FACScaliber.

The direct binding of soluble fibronectin was monitored by incubating control or bacitracin-treated (3.5 mM) cells with 50 µg of biotinylated plasma fibronectin for 30 min at 37°C. The cells were washed three times with PBS and incubated with FITC-conjugated avidin at 4°C for 30 min. The cells were washed and examined for fluorescence by FACS, as described above.

Purification of ß1 integrin

Integrin was isolated from human placenta, used as previously described (13). Briefly, 300 g of washed placenta was homogenized in 300 ml of 50 mM n-octylglucopyranoside in 25 mM Tris, pH 7.6, 150 mM NaCl, 2 mM CaCl2, and 1 mM PMSF. The homogenate was centrifuged 10,000 x g for 1 h at 4°C, after which the supernatant was collected and passed sequentially through an OVA Sepharose 4B and a JB1 Sepharose 4B column at a rate of 1 ml/min. The column was washed sequentially with 20-column volumes each of 1) 0.1% Nonidet P-40 in 25 mM Tris, pH 7.6, 150 mM NaCl, and 2 mM CaCl2, and 2) 0.1% Nonidet P-40 in 0.01 M sodium acetate buffer, pH 4.5. The ß1 integrin was eluted from the JB1 column in 0.1% Nonidet P-40, 10 mM sodium acetate buffer, pH 3.6, and 3-ml fractions were collected into tubes containing 0.5 ml of 3 M Tris, pH 8.8.

The purity of the fractions was assessed by SDS-PAGE and Coomassie blue staining. The fractions containing ß1 also had a mixture of the associated {alpha}-chains. However, the {alpha}- and ß1-chains collectively represented greater than 85% of the total stained proteins. The presence of ß1 in the fractions was confirmed by Western blot with JB1A and B3B11.

Fibronectin binding to purified ß1 integrin

Purified integrin was diluted in PBS, and microtiter wells were coated for 18 h at 4°C with 100 µl (50–100 ng) of the dilute integrin preparation. The plates were washed with PBS and blocked with 2% BSA in PBS, and 1 µg of biotin-labeled fibronectin (10 µg/ml) in RPMI was added in the presence or absence of 3.5 mM bacitracin. The plates were incubated at 37°C for 3.5 h and subsequently washed five times with PBS containing 0.05% Tween-20. Alkaline phosphatase-labeled avidin was added to the wells, and the plates were then incubated at 37°C for 30 min. The plates were washed five times and substrate was added. The plates were incubated at 37°C for 30 min, and the OD was determined at 405 nm. The experiment was performed twice in replicates of five each time.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of PDI inhibitors on ß1-mediated T lymphocyte adherence

Several inhibitors on PDI activity were examined for their effects on the adhesion of IL-2-dependent T cells to fibronectin. We and others have demonstrated previously that the adherence of these cells to fibronectin is mediated by {alpha}4ß1 and {alpha}5ß1 integrins (29). Bacitracin, tocinoic acid, and dithiobisnitrobenzoic acid (DTNB) have previously been reported to interfere with cell surface thiol-reductase activity and with PDI function (22, 23, 30, 31). Treatment with 1.5 mM bacitracin resulted in a >80% inhibition of PMA-induced adherence (Fig. 1GoA). In contrast, exposure to 5 mM DTNB, a concentration greater than those previously reported for inhibition enzyme activity, did not influence adhesion (Fig. 1GoA). Similarly, pretreatment of the cells with concentrations of tocinoic acid reported to block PDI activity did not affect the induction of adhesion (Fig. 1GoB). Another inhibitor of PDI activity, somatostatin A (0.2 mM), also had no effect on cell adhesion (data not shown).



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  		FIGURE 1. The effects of inhibitors of PDI and disulfide exchange on lymphocyte adherence to fibronectin. A, IL-2-dependent T cells were pretreated with the indicated concentrations of bacitracin or DTNB and assayed for adherence to immobilized fibronectin following PMA stimulation. B, Cells were untreated or pretreated with tocinoic acid (0.5 mM) or bacitracin (3.5 mM), and either stimulated with PMA or untreated and assessed for binding to fibronectin. The results for all experiments are the mean of sextuplicate assays that have been repeated at least three times.

 
As an alternative approach to determining a possible role for PDI in lymphocyte adhesion, cells were treated with concentrations of a function-blocking anti-PDI mAb, RL-77 (22). Exposure to Ab concentrations up to 800 µg/ml (the highest concentration tested) failed to influence adherence, while bacitracin caused a concentration-dependent inhibition of the adherence with a 50% inhibition at 1–2 mM (Fig. 2Go).



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  		FIGURE 2. The effects of anti-PDI on lymphocyte adherence. Cells were pretreated with the indicated concentrations of anti-PDI or bacitracin and assessed for PMA-induced binding to fibronectin in the presence of the indicated inhibitors.

 
Bacitracin effects on T cell adhesion

The above results suggested that PDI was not directly involved in the induction of ß1 integrin-mediated adhesion to fibronectin. This raised the question of the mechanism(s) of bacitracin inhibition of adherence.

Stimulation of T cell adherence by PMA or anti-CD3 treatment was inhibited by pretreatment with 3.5 mM bacitracin (Fig. 3Go). Under these conditions, the adherence of the non-PMA-treated cells was also inhibited by bacitracin, implying that the inhibition was occurring at a postactivation event. To test this possibility, cells were treated with PMA for 30 min to activate adhesion, exposed to bacitracin for 30 min, and assayed for adherence. The control cells that were activated by PMA displayed strong binding to fibronectin. This contrasted with cells activated with PMA and treated with bacitracin, in which almost complete inhibition was observed. It would appear that bacitracin inhibits adherence either by reversing adhesion competence or by interfering with the integrin function because it can block preactivated integrin function.



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  		FIGURE 3. The effects of bacitracin on PMA- or anti-CD3-activated adherence of lymphocytes. Cells were untreated or stimulated with anti-CD3 or PMA in the presence or absence of bacitracin (3.5 mM) and assessed for binding to fibronectin.

 
The activation of adhesion by PMA and stimulatory Abs has been shown to have different biochemical requirements, as only the former was staurosporine H sensitive (32). The adhesion induced by the stimulatory anti-ß1, B44, was also fully inhibited by bacitracin (Fig. 4Go). The induction of adherence by Mn2+, anti-ß1, TS2/16, or DTT was also inhibited by bacitracin (data not shown). These results suggested that the site of action was at a postactivational ß1 step.



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  		FIGURE 4. The effects of bacitracin on activated cell adhesion. Cells were activated with the indicated stimuli and then either exposed to bacitracin (3.5 mM) or untreated and assayed for binding to fibronectin.

 
The possibility that the action of bacitracin was due to a toxic effect on the cells was examined by exposing the cells to bacitracin, and removing the bacitracin from one set of cells. The spontaneous and PMA-induced adhesion was then compared with either cells that were nonbacitracin treated or with cells that had bacitracin present throughout the incubation period. Within 30 min of removal of bacitracin, the cells displayed a 40–45% recovery of adhesive potential (Fig. 5Go). These results, in conjunction with direct viability measurements, using trypan blue staining of the cells (data not shown), suggested that the bacitracin effects were at least partially reversible and they were not a result of direct cytotoxicity for the cells.



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  		FIGURE 5. The reversibility of bacitracin effects on cell adhesion. Cells were treated with bacitracin (3.5 mM) for 30 min and the bacitracin was removed. The cells were then incubated for 30 min in fresh media and assessed for spontaneous and PMA-induced adherence to fibronectin. The adhesion was compared with control cells that had been handled in a similar fashion, but not exposed to bacitracin, or with cells that had been maintained in the presence of bacitracin.

 
The effects of bacitracin exposure on integrin expression levels were determined by comparing the staining patterns of treated cells with those of control cells. Four Abs were used for the analysis. JB1A inhibits adhesion and it detects an epitope (residues 82–87) that is normally accessible on the ß1-chain (28). N29 stimulates adhesion and it recognizes an epitope near the -NH2 terminus of the ß1-chain (13). Access to this epitope appears to be related to functional status of the integrin. The stimulatory Ab B3B11 reacts with a membrane-proximal epitope (666–668) (25). The Ab 3S3 blocks adhesion and it recognizes a discontinuous epitope (26). The level of staining of cells with these Abs was not influenced by the presence of 3.5 mM bacitracin (Fig. 6Go). These results suggested that the integrin levels and conformations were not altered by bacitracin.



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  		FIGURE 6. The effect of bacitracin on B1 integrin expression levels and conformation. Cells were incubated with the indicated Abs in the absence or presence of bacitracin (3.5 mM). The expression levels and patterns were examined by flow cytometry. The light line in each histogram represents the negative control. Note the staining patterns of each of the Abs in the presence of bacitracin are totally superimposed on their corresponding untreated counterparts.

 
The ability of cells to bind soluble fibronectin was assessed as a direct test of integrin competence. Treatment of the cells with bacitracin resulted in a complete inhibition of fibronectin binding, suggesting that the integrin function was the site of action rather than postreceptor occupancy events required for cell adherence (Fig. 7Go).



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  		FIGURE 7. The effect of bacitracin on the binding of soluble fibronectin to cell surface {alpha}5ß1. Cell were incubated with biotinylated fibronectin in the presence or absence of 3.5 mM bacitracin for 30 min and washed. The cells were then reacted with FITC-labeled avidin and examined for staining by FACS. The control binding of fibronectin is marked with the dashed lines; the binding in the presence of bacitracin is indicated by the solid dark line; and the negative control binding is indicated by the solid light line.

 
As a direct test of the above prediction, the effects of 3.5 mM bacitracin on the binding of fibronectin to purified ß1 integrin were examined. Coincubation of biotinylated fibronectin with bacitracin resulted in >80% inhibition of fibronectin binding (Fig. 8Go). In contrast, the background binding of fibronectin to BSA-coated plates was relatively unaffected by this treatment. These results suggest that bacitracin can directly interfere with fibronectin binding to purified ß1 integrin.



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  		FIGURE 8. The effect of bacitracin on ligand binding to purified fibronectin. Biotinylated fibronectin alone or the presence of 3.5 mM bacitracin was added to the microtiter wells coated with BSA or purified ß1 integrin. The level of bound fibronectin was then determined following reaction with alkaline phosphatase-conjugated avidin and substrate. The experiment was performed twice in replicates of five each time. The mean and the SE of one such experiment is given.

 
Integrin inhibition patterns of bacitracin

Jurkat cells have previously been shown to employ both {alpha}4ß1 and {alpha}5ß1 to bind to fibronectin (7). The former receptor binds to a 40-kDa chymotryptic fragment of fibronectin, while the latter recognizes a 120-kDa fibronectin fragment (33, 34). The use of fragments that contain only one of the binding sites allowed for the analysis of cell binding by one receptor type in isolation of possible contributions by occupancy of the other.

Jurkat cells were incubated with 3.5 mM bacitracin and examined for adherence to either 40- or 120-kDa fragments of fibronectin. The adherence to both fragments was markedly inhibited by this treatment (Table IGo). In both cases, the inhibition was comparable with that seen with the inhibitory anti-ß1, 3S3 (data not shown). Thus, it appears that both {alpha}4ß1- and {alpha}5ß1-mediated binding to fibronectin are inhibited by bacitracin.


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  		Table I. The effects of bacitracin on integrin binding of selected ligands

 
The binding of IL-2-dependent cell lines to laminin and collagen was also blocked by bacitracin (Table IGo). These results indicated that at least {alpha}2ß1-, {alpha}4ß1-, {alpha}5ß1-, and {alpha}6ß1-mediated adherence were inhibited by bacitracin. It was noteworthy that inhibition of collagen adherence required a much higher concentration of bacitracin than was required for fibronectin and laminin.

The B cell line, RPMI 8866, displays spontaneous and PMA-inducible adherence to the 40-kDa fragment of fibronectin (35, 36). This adherence was fully inhibited by an Ab to the ß7-chain, FIB 504, but not by the anti-ß1, 3S3 (Fig. 9GoA). The RPMI 8866 adhesion to this fragment of fibronectin was fully inhibited by bacitracin, indicating that adhesion by the {alpha}4ß7 complex was also sensitive to bacitracin (Fig. 9GoA).



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  		FIGURE 9. The effects of bacitracin on RPMI 8866 adherence to the 40- and 120-kDa fragments of fibronectin. PMA-stimulated RPMI 8866 cells were treated with bacitracin (3.5 mM), anti-ß7 (FIB 504), anti-ß1 (3S3), or anti-{alpha}vß3 (LM-609) (c) and examined for binding to the 40-kDa (A) or 120-kDa (B and C) fragments of fibronectin.

 
RPMI 8866 cells can also be induced by PMA to adhere to fibronectin via {alpha}vß3. This integrin complex binds to the same region of the 120-kDa fibronectin fragment as {alpha}5ß1 (35). Pretreatment with these cells with 3.5 mM bacitracin (Fig. 9GoB) or even up to 8 mM did not inhibit their adherence to this fragment. Since this concentration fully blocked the {alpha}4ß7-dependent binding of these same cells to the 40-kDa fragment, these results further exclude cytotoxicity as the mechanism of bacitracin action.

The cell line JY undergoes PMA-induced aggregation that is inhibited by Abs to {alpha}Lß2 (37). As a test of the range of bacitracin effects, these cells were pretreated with up to 7 mM of the antibiotic and examined for their capacities to aggregate. The bacitracin treatment did not inhibit adherence; in fact, it may have enhanced the aggregation (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present studies were initiated to examine the possible role of protein disulfide isomerase-like activity in the control of cellular adhesion. The rationale for this proposal was 1) the demonstration of immunochemical and biochemical PDI-like activity on the surface of lymphoid cells (17, 38); 2) the capacity of bifunctional reducing agents to activate adhesion (15); and 3) the localization of the epitopes of a number of stimulatory Abs to the integrin ß-chains to regions adjacent to predicted long-range disulfide bonds (13, 25, 39, 40, 41). Collectively, these observations suggest that changes in disulfide bond pairing or associated regions can influence integrin function, and that the necessary enzyme activities to facilitate such reactions are present on the surface of lymphocytes.

Cell surface-associated thiol-reductase activity has been demonstrated on a number of cell types, including lymphocytes (17, 18, 19, 38). The infection of T cells by HIV has been shown to require a cell surface-associated thiol-reductase activity (23). In the case of B cells, the activity appears to have immunologic differences with conventional PDI (17). Inhibition of this activity results in a marked increase in the levels of free thiol groups on the surface of CLL, suggesting that related enzymes play a role in regulating the oxidation status of membrane proteins (38).

The results of our studies do not support a role for PDI activity in the regulation of integrin-mediated adhesion. Treatment with chemical or immunologic inhibitors of PDI did not influence adhesion. The concentrations used were in excess of the reported KI values of the various compounds, and they were equal to or exceeding those reported to block T cell membrane thiol-reductase activity. The addition of exogenous functional PDI did not modify the adhesive properties of a number of cell lines that displayed distinctive binding phenotypes (data not shown). These results suggest that PDI activity is not involved in the acquisition or maintenance of T cell adhesion competence. However, the endoplasmic reticulum-associated form of the enzyme undoubtedly plays a critical role in the proper folding of newly synthesized integrins.

The anti-adhesive effects of bacitracin are dependent upon continuous presence of the inhibitor, and the effects on integrin function are reversible. Bacitracin blocks the activity of functional integrins rather than the induction of integrin activity. The FACS results suggest that the mode of inhibition does not involve gross changes in integrin conformation or expression levels. Bacitracin has been shown to be an inhibitor of purified and cell-associated PDI (22, 23, 30, 31). However, as discussed above, it seems unlikely that this is the mode of action in the current studies. The direct inhibition of the binding of soluble fibronectin to cells suggests that the integrins may be the targets for bacitracin effects. This point was further supported by the fact that the binding of fibronectin to purified ß1 was almost fully inhibited by 3.5 mM bacitracin. It should be kept in mind that bacitracin has also been reported to inhibit a broad range of proteases, including members of the aspartic, serine, and cysteine proteases, and metalloproteinases (42). It is not possible to exclude a role for this activity in the observed anti-adhesive effects observed with whole cells. This question warrants further investigation, and biochemical studies are in progress to address this issue.

The full ranges of integrin sensitivities to bacitracin have yet to be determined. To date we have demonstrated that ß1- and ß7-mediated adherence to collagen, fibronectin (both CS-1 and CBD domains), laminin, or VCAM-1 is inhibited by bacitracin. Although the sequences of the ß1 and the ß7 subunits display considerable sequence homology, they are clearly antigenically and functionally distinct with the latter, displaying greater sequence homology with ß2 than ß1 (43). However, it may be that there are critical shared regions of homology between ß1 and the ß7 that are the targets of bacitracin action. The fact that neither ß2 nor ß3 functions are inhibited by bacitracin suggests that these effects do not relate to alterations in central processes that are shared in all adhesive interactions, but rather to aspects of the ligand interaction that are unique to at least the ß1- and ß7-chains. A direct test of this point will require the direct analysis of bacitracin binding sites on purified integrins.

The fact that {alpha}vß3 is not inhibited by bacitracin at concentrations that are 10-fold higher than those required to inhibit ß7 function on the same cell indicates that the inhibition is not due to direct cytotoxic effects of the treatment. In preliminary studies, it has been determined that cells can be grown in the presence of 5 mM bacitracin for at least 72 h without any effect on viability. The removal of the bacitracin at this point leads to a complete recovery of cell adherence and growth properties (unpublished data, J.A.W. and M. A. Miranda).

While the mechanism of bacitracin action remains to be elucidated, the selectivity of the effects suggests that this agent may be a useful probe for the analysis of integrin function. It may afford a means to identify functionally homologous regions on distinct integrin families. Also, because of its previous history as a safe therapeutic agent, bacitracin may offer a useful adjunct for the selective modification of adhesion in vivo.


   Acknowledgments
 
We thank Dr. Charlotte Kaetzel (University of Kentucky) for making the R77 hybridoma available to us, and Dr. Dianne Dottaviao (Leukosite, Boston, MA) for providing us with recombinant VCAM-1. A special thanks to Dr. Hiram Gilbert (Baylor College of Medicine) for his patient and helpful discussions and for providing the purified protein disulfide isomerase.


   Footnotes
 
1 The Medical Research Council of Canada and the Canadian Arthritis Society supported this research. H.N. and Y.M., respectively, are recipients of studentships from the Manitoba Health Research Council and the Faculty of Graduate Studies, University of Manitoba. Back

2 Address correspondence and reprint requests to Dr. John A. Wilkins, Rheumatic Diseases Research Laboratory, RR014 800 Sherbrook St., Winnipeg MB R3A 1 M4, Canada. E-mail address: Back

3 Abbreviations used in this paper: PDI, protein disulfide isomerase; DTNB, dithiobisnitrobenzoic acid. Back

Received for publication February 27, 1998. Accepted for publication August 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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