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The Journal of Immunology, 2001, 166: 6294-6300.
Copyright © 2001 by The American Association of Immunologists

A Potential Role for Annexin 1 as a Physiologic Mediator of Glucocorticoid-Induced L-Selectin Shedding from Myeloid Cells1

Holly J. Strausbaugh and Steven D. Rosen2

Department of Anatomy, Program in Immunology and Cardiovascular Research Institute, University of California, San Francisco, CA 94143


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids can dampen inflammatory responses by inhibiting neutrophil recruitment to tissue sites. The detailed mechanism by which glucocorticoids exert this affect on neutrophils is unknown. L-selectin is a leukocyte cell surface receptor that is implicated in several steps of neutrophil recruitment. Recently, several studies have shown that systemic treatment of animals and humans with glucocorticoids induces decreased L-selectin expression on neutrophils, suggesting one mechanism by which inflammation may be negatively regulated. However, when neutrophils are treated in vitro with glucocorticoids, no effect on L-selectin expression is observed. Thus, the existence of an additional mediator is plausible. In this study, we investigate whether annexin 1 (ANX1), a recognized second messenger of glucocorticoids, could be such a mediator. We show that ANX1 induces a dose- and time-dependent decrease in L-selectin expression on both peripheral blood neutrophils and monocytes but has no effect on lymphocytes. The loss of L-selectin from neutrophils is due to shedding that is mediated by a cell surface metalloprotease ("sheddase"). Using cell shape and a {beta}2 integrin activation epitope, we show that the ANX1-induced shedding of L-selectin appears to occur without overt cell activation. These data may provide the basis for further understanding of mechanisms involved in the down-regulation of inflammatory responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoids curtail the inflammatory response through a number of different mechanisms including down-regulation of cytokine gene expression (1, 2, 3, 4), blocking phospholipase A2 activity (5), and inhibiting neutrophil trafficking (6, 7). Neutrophil accumulation during inflammation is a critical component of the inflammatory response. Individuals who cannot recruit neutrophils (e.g., leukocyte adhesion deficiency patients) develop chronic, severe infections and show severely impaired wound healing capability (8). Neutrophil migration to tissue sites involves a cascade of adhesion and signaling events. The early stages (i.e., tethering and rolling) of this recruitment process are mediated in part by the leukocyte adhesion molecule, L-selectin (reviewed in Ref. 9).

Recent work suggests that glucocorticoid-induced inhibition of neutrophil accumulation is mediated by an effect on the initial steps of the process (10). Therefore, it has been suggested that glucocorticoid-induced shedding of L-selectin may be one mechanism by which glucocorticoids inhibit neutrophil accumulation in vivo. Several studies show that administration of glucocorticoids to humans and animals induces L-selectin shedding on peripheral blood neutrophils (11, 12, 13, 14). In addition, our previous work has shown that inhibition of L-selectin-dependent plasma extravasation is blocked by both adrenalectomy and a glucocorticoid synthesis inhibitor (15, 16). However, when neutrophils are treated in vitro with doses of glucocorticoids that produce L-selectin shedding in vivo, shedding is not observed (17, 18, 19). This discrepancy between in vivo and in vitro results suggests that glucocorticoids do not directly affect neutrophil L-selectin expression but instead act indirectly via a heretofore unknown mediator.

Annexin 1 (ANX1)3 (annexin A1, lipocortin), a 37-kDa phospholipid- and calcium-binding protein, is a good candidate for this mediator. Glucocorticoids induce ANX1 synthesis and secretion (20, 21, 22). Moreover, exogenously provided ANX1 inhibits neutrophil accumulation at inflammatory sites (23, 24, 25), and systemic treatment with anti-ANX1 antisera blocks glucocorticoid-induced inhibition of both neutrophil and monocyte recruitment (25). Therefore, we wondered whether one of the activities of ANX1 is to induce L-selectin shedding from neutrophils. In this study, we have tested this possibility.


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

Purified bovine lung ANX1 was purchased from Biodesign International (Saco, ME). Ro31-9790 was a gift from W. H. Johnson of Roche Discovery Welwyn (Welwyn Garden City, U.K.). A mAb to L-selectin (DREG 56-FITC) was purchased from Beckman Coulter (Fullerton, CA). FITC-labeled mAbs to human CD14, CD16, CD3, and CD11b, control Abs (mouse IgG1-FITC and mouse IgG2a-FITC), and the secondary Ab, FITC-labeled goat anti-mouse IgG, were purchased from Caltag (Burlingame, CA). The mAb24 was a gift from Dr. Nancy Hogg (Imperial Cancer Research Fund, London, U.K.). FMLP, PMA, N-t-Boc-Met-Leu-Phe (Boc), dexamethasone, and Sepharose (S-400) beads were purchased from Sigma (St. Louis, MO). Cal-Lyse solution was purchased from Caltag.

Flow cytometry

Venous blood was collected in anticoagulant-containing tubes from human volunteers. Blood was treated with various agents (described below) and incubated at 37°C for specified amounts of time. Before Ab staining, the blood was placed on ice for 10 min. Whole blood was then stained with specific Abs (DREG 56-FITC, anti-CD11b-FITC, or mAb24). For the mAb24 experiments, after the primary incubation, cells were treated with FITC-labeled goat anti-mouse IgG. Background was defined using FITC-labeled isotype-matched control mouse IgGs or with secondary Ab alone (mAb24). After Ab incubations, blood was washed with staining buffer (PBS with 1% FCS or BSA and 0.1% NaN3), leukocytes were fixed and prepared for lysis with Cal-Lyse solution, and erythrocytes were lysed with deionized water. Fluorescence was analyzed by flow cytometry. Leukocyte classes were defined based on forward and side scatter pattern parameters and by using Ab staining to cell-type specific markers (CD3 for T cells, CD14 for monocytes, and CD16 for neutrophils).

Dexamethasone and ANX1 dose-response experiments

Whole blood was incubated for 30 min at 37°C with specified concentrations of ANX1 or dexamethasone. Control blood was incubated with identical amounts of ANX1 vehicle (40 mM Tris (pH7.5), 0.15 mM NaCl, 1 mM DTT) or dexamethasone vehicle (1% ethanol). For time-course experiments, ANX1 was incubated for specified lengths of time with 5 µg/ml ANX1 or an equal concentration of vehicle.

Sheddase inhibitor experiments

Whole blood was incubated for 15 min at 37°C with Ro31-9790 (50 µm) or with vehicle (1% DMSO). ANX1 (5 µg/ml) or PMA (100 nM) were then added and incubated for an additional 30 min.

Boc-inhibitor experiments

Whole blood was preincubated with Boc (20 µM) or vehicle (0.2% DMSO) for 10 min at room temperature. ANX1 (5 µg/ml), FMLP (50 nM), ANX1 vehicle, or FMLP vehicle (0.005% DMSO) were then added and incubated for an additional 30 min at 37°C.

Preclearing of ANX1 with Sepharose beads

Sepharose beads were washed extensively with PBS. ANX1 (1 µg) was added to 100 µl of PBS containing 10 µl packed Sepharose beads. A control solution of ANX1 was not treated with beads. All incubations took place in BSA (3%)-coated tubes. After 90 min on ice, the supernatants were subjected to SDS-PAGE on 10% polyacrylamide gels and silver staining (26). The control ANX1 solution, the Sepharose-exposed supernatant, and a vehicle control were then tested for their ability to induce L-selectin shedding as described above. We found that Sepharose, whether it was unconjugated or bound to protein A or protein G (data not shown), was able to completely clear ANX1. Therefore, we pooled these data when analyzing the shedding activity of precleared supernatants (Fig. 2GoC).



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  		FIGURE 2. ANX1 effects are specific. A, SDS-PAGE analysis of ANX1. Silver stain (500 ng/lane) of ANX1 (lane 2) or an equivalent volume of vehicle (lane 1). The doublet at 37 kDa is consistent with the reported molecular mass of ANX1 (lane 2). Both lanes show a band at 66 kDa that corresponds to BSA added as a stabilizer. B, ANX1 was incubated with Sepharose beads. The starting solution (lane 1) and the precleared solution (lane 2) were analyzed by SDS-PAGE with silver staining. In the upper portion of the gel (data not shown), the BSA band was not depleted by Sepharose gel preincubation. C, ANX1 (3.3 µg/ml) and the precleared solution of ANX1 were tested for their ability to induce L-selectin shedding from neutrophils. Precleared ANX1 (SUP) did not induce L-selectin shedding compared with a corresponding volume of ANX1 (F = 88.8, p < 0.001, ANX1 vs control and ANX1 vs precleared ANX1; precleared ANX1 vs control, NS). Data are expressed as percentage of control. The control consisted of a paired vehicle-treated or vehicle-precleared sample. L-selectin levels on ANX1-treated or ANX1-precleared cells are expressed as a percentage of these controls. Data represent the mean and SE for 6–10 experiments.

 
Statistics

Student’s t test was used to compare two groups. One-way ANOVA was used to compare three or more treatment groups. Student Newman-Keuls post hoc test was then used to determine which groups were significantly different from each other.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In vitro treatment with dexamethasone does not affect L-selectin expression

We treated peripheral blood with dexamethasone and measured L-selectin expression on peripheral blood leukocytes. Based on our studies of plasma glucocorticoids in a rat model in which L-selectin was shed from neutrophils (27), we tested an extensive dose range of dexamethasone (2–200 µg/ml). Similar to findings of others (17, 18), dexamethasone had no effect on L-selectin expression on neutrophils (data not shown). Monocytes and lymphocytes were also unaffected (data not shown).

ANX1 decreases L-selectin expression in a dose- and time-dependent manner

To determine whether ANX1 affects L-selectin expression, we exposed whole blood to varying concentrations of ANX1 and measured L-selectin expression on peripheral blood leukocytes by flow cytometry. As shown in Fig. 1Go, A–C, ANX1 induced a dose-dependent decrease in L-selectin expression on peripheral blood neutrophils. L-selectin expression was significantly decreased at concentrations as low as 1 µg/ml ANX1. The ED50 for the ANX1 effect was 2.5 µg/ml. ANX1 also decreased L-selectin expression on monocytes (Fig. 1GoD). Monocytes were more responsive to ANX1 than neutrophils. Treatment of monocytes with 0.5 µg/ml of ANX1, a concentration that had no effect on neutrophils, induced L-selectin shedding from >80% of the cells. In contrast, ANX1 did not affect L-selectin expression on lymphocytes, even at 10 µg/ml, a concentration that produced a maximal effect on neutrophils. To determine the time course of the ANX1-induced effects, we incubated peripheral blood with ANX1 for varying lengths of time. Significant loss of L-selectin from neutrophils was observed by 15 min of incubation. Treatment for 30 min was required to achieve complete shedding (Fig. 1GoE).



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  		FIGURE 1. ANX1 decreases L-selectin expression in a dose-dependent manner. Blood was treated with varying concentrations of ANX1, and L-selectin levels on neutrophils were measured. A representative experiment (top) is shown depicting neutrophils positive for L-selectin after vehicle treatment (A) and after ANX1 (5 µg/ml) treatment (B). The average and SE of four to eight experiments is shown in (C). ANX1 induced a dose-dependent decrease in L-selectin expression (F = 25.0, p < 0.01) with a maximal effect achieved at 5 µg/ml. ANX1 also decreased L-selectin expression on monocytes (D) at 0.5 µg/ml (t = 33.8, p < 0.001) and at a range of other concentrations (data not shown). ANX1 had no effect on lymphocyte L-selectin expression (D) at 10 µg/ml (t = 0.7; NS) or at any other concentration tested (data not shown). The time course of ANX1 effects are shown in (E). Peripheral blood was incubated with ANX1 (5 µg/ml) for indicated lengths of time, and L-selectin was measured on neutrophils. ANX1-induced shedding was half-maximal at 15 min and required between 30 and 60 min to achieve maximum shedding (F = 40.5, p < 0.001). Data represent the mean and SE of three experiments. For all experiments (C–E), the control consisted of a paired vehicle-treated sample. L-selectin levels on ANX1-treated cells are expressed as a percentage of this control. The ANX1 effects shown in all panels were observed both as a reduction in the percentage of cells expressing L-selectin and a decrease in mean fluorescence intensity (data not shown).

 
L-selectin effects are specific to ANX1

We next wanted to verify that the observed effects of the ANX1 preparation were due to this protein and not to a contaminant. To determine the purity of the preparation, we subjected it to SDS-PAGE with silver staining for protein detection. When we loaded 500 ng of protein, the only protein band we observed in addition to BSA (added as a stabilizer) was a doublet at 37 kDa, which is consistent with the reported molecular mass of ANX1 (Fig. 2GoA). Because the detection limit of silver staining is 2–5 ng of protein per band (28), the purity of the ANX1 preparation was at least 99% based on protein. To further establish that the effects we observed were due to ANX1, we took advantage of our observation that ANX1 is able to bind to unconjugated Sepharose. When we exposed the ANX1 solution to Sepharose, the supernatant could no longer reduce L-selectin expression on neutrophils (Fig. 2GoC) in correspondence with the depletion of the ANX1 band (Fig. 2GoB).

ANX1 induces L-selectin shedding

L-selectin shedding is induced by a number of stimuli (29, 30, 31). This shedding is thought to be mediated by a metalloprotease on the leukocyte surface, referred to as the "sheddase" (32, 33). To investigate whether the ANX1-induced decrease in L-selectin expression was a result of L-selectin shedding, we determined whether pretreatment with the sheddase inhibitor, Ro31-9790, would block the effect. Ro31-9790 is a hydroxamic acid-based inhibitor of zinc-dependent metalloproteinases that has been shown to block L-selectin shedding by inhibiting a cell surface enzyme referred to as the sheddase (32). As shown in Fig. 3Go, the ability of ANX1 to induce the loss of L-selectin from neutrophils was completely blocked when this inhibitor was added. As expected from previous results, Ro31-9790 also completely prevented PMA-induced shedding. We interpret these findings to indicate that ANX1 induces L-selectin shedding from the neutrophil surface.



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  		FIGURE 3. ANX1 induces L-selectin shedding. Neutrophils were treated with Ro31-9790 (50 µM; sheddase inhibitor) and exposed to ANX1 (5 µg/ml) or PMA (100 nM). L-selectin was then measured on the treated cells. The effect of ANX1 on neutrophil L-selectin expression was completely blocked when cells were pretreated with the sheddase inhibitor (F = 184.8, p < 0.001; ANX1 vs ANX1 plus Ro31-9790, p < 0.001). Ro31-9790 also completely blocked PMA-induced shedding (F = 3084, p < 0.001; PMA vs PMA plus Ro31-9790, p < 0.001). No effect on L-selectin expression was observed when cells were preincubated with Ro31-9790 vehicle (1% DMSO; F = 184.8, p < 0.001; ANX1 vs ANX1 plus DMSO, NS). For all experiments the control consisted of a paired vehicle-treated sample. L-selectin levels on ANX1-treated and PMA-treated cells are expressed as a percentage of these controls. The effects shown were observed both as a reduction in the percentage of cells expressing L-selectin and a decrease in mean fluorescence intensity (data not shown). Data represent the mean and SE for two (PMA) and four (ANX1) experiments.

 
ANX1 does not induce L-selectin shedding through an interaction with the FMLP receptor

Recently it has been suggested that ANX1 may exert some of its effects on neutrophils by binding to and acting through the FMLP receptor (34). To determine whether ANX1-L-selectin shedding is mediated by this mechanism, we pretreated neutrophils with the FMLP receptor antagonist, Boc, and determined the effects of this treatment on the ANX1 activity. Blocking the FMLP receptor had no effect on ANX1-induced shedding (Fig. 4Go). However, Boc effectively blocked the FMLP receptor in these experiments, because pretreatment of neutrophils with this antagonist completely abolished FMLP-induced L-selectin shedding.



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  		FIGURE 4. ANX1-induced L-selectin shedding is not mediated through the FMLP receptor. Blood was pretreated with Boc (20 µM) and then exposed to either FMLP (50 nM) or ANX1 (5 µg/ml). L-selectin was then measured on neutrophils. FMLP-induced L-selectin shedding from neutrophils was blocked by the FMLP receptor antagonist, Boc (F = 124.4, p < 0.001; FMLP vs FMLP plus Boc, p < 0.001). Pretreatment with Boc had no effect on ANX1-induced shedding from neutrophils (F = 0.11, NS). The Boc vehicle (0.2% DMSO) had no effect on FMLP- or ANX1-induced shedding (F = 124.4, p < 0.001; FMLP plus DMSO vs FMLP, NS). For all experiments, the control consisted of a paired vehicle-treated sample. L-selectin levels on ANX1-treated cells and FMLP-treated cells are expressed as a percentage of these controls. The effects shown were observed both as a reduction in the percentage of cells expressing L-selectin and a decrease in mean fluorescence intensity (data not shown). Data represent the mean and SE of four experiments.

 
Effect of ANX1 on neutrophil activation

Up-regulation of expression of the {beta}2 integrin, Mac-1, occurs after exposure of neutrophils to activating agents such as PMA, TNF-{alpha}, leukotriene B4, and C5a fragments of complement (29). Conflicting results exist as to whether in vivo glucocorticoid treatment affects Mac-1 expression on neutrophils. Some studies show decreased CD18 expression (11), whereas others show increased expression (19) or no change (17). To determine whether ANX1 activates neutrophils, we investigated the effect of ANX1 treatment on Mac-1 expression. Because PMA is a potent activator of neutrophils, we also compared the effects of PMA to those of ANX1. As shown in Fig. 5Go, ANX1 induced a 2-fold up-regulation of Mac-1, whereas PMA treatment induced a 4-fold up-regulation. Previous studies have shown that neutrophil activators (i.e., TNF-{alpha}, C5a, and PMA) induce Mac-1 up-regulation to a similar extent (29, 35). Therefore, we wondered whether the relatively limited increase in Mac-1 expression in response to ANX1 might indicate a partially activated state of the cells. Therefore, we examined expression of a {beta}2 integrin activation epitope that is recognized by mAb24 (36). ANX1 treatment did not induce this epitope, whereas PMA induced a 4-fold increase in its expression. We further investigated the activation state of ANX1-treated neutrophils by performing an analysis of cell shape. Activated neutrophils have an altered morphology, which can be quantitatively monitored by flow cytometry using cell scatter profiles (37). This parameter for measuring cell-shape change has been shown to be very well correlated with FITC-phalloidin staining of F-actin (37). As shown in Fig. 5Go, ANX1 treatment did not alter neutrophil shape (Fig. 5Go). In contrast, PMA treatment induced a significant change in neutrophil shape.



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  		FIGURE 5. Effect of ANX1 on neutrophil activation. Blood was treated with ANX1 (5 µg/ml) or with PMA (100 nM), and neutrophils were evaluated for different activation parameters. A, A representative experiment (left) is shown depicting fluorescence intensity of neutrophils stained for Mac-1 in ANX1-treated samples (top) or PMA- treated samples (bottom). Dotted lines indicate isotype-matched control Ab staining, dashed lines indicate Mac-1 staining of vehicle-treated neutrophils, and solid lines indicate Mac-1 staining of ANX1- (top) or PMA-treated (bottom) neutrophils. The average and SE of 6–15 experiments is shown (right). ANX1 induced an ~2-fold up-regulation of Mac-1, whereas PMA treatment induced a 4-fold up-regulation (F = 22.3, p < 0.001; all comparisons, p < 0.01). For each experiment, a paired control of vehicle-treated cells was included. In all panels, (A–C), data are expressed as the percentage of change from these vehicle-treated controls. B, A representative experiment (left) is shown giving the expression of the mAb24 epitope in ANX1-treated samples (top) or PMA-treated samples (bottom). Dotted lines indicate staining with secondary Ab alone, dashed lines indicate mAb24 staining of vehicle-treated neutrophils, and solid lines indicate mAb24 staining of ANX1- (top) or PMA-treated (bottom) neutrophils. The average and SE of five experiments is shown (right). ANX1 treatment did not induce the mAb24 epitope, whereas PMA induced a >4-fold increase in its expression (F = 7.9, p < 0.01; control vs PMA and ANX1 vs PMA, p < 0.01; control vs ANX1, NS). C, A representative experiment (left) is shown depicting neutrophil forward scatter pattern (FSC) in vehicle-treated and ANX1-treated neutrophils (top left) or in vehicle-treated and PMA-treated neutrophils (bottom left). The average and SE of seven to nine experiments is shown (right). ANX1 treatment, in contrast to PMA treatment, did not alter neutrophil shape (F = 10.6, p < 0.001; control vs PMA and ANX1 vs PMA, p < 0.01; control vs ANX1, NS).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ANX1 belongs to a large family of calcium-dependent phospholipid-binding proteins known as the annexins (reviewed in Refs. 38, 39). ANX1 is primarily a cytosolic protein found in myeloid cells and a variety of other cells. ANX1 can be externalized to the cell surface of leukocytes and occurs as a soluble protein in blood plasma (40, 41) and bodily fluids (40, 42). Glucocorticoids, as well as other mediators such as IL-6, can stimulate the synthesis of ANX1 (20, 21, 22). Glucocorticoids and other stimuli also induce externalization of ANX1 (43) and increase systemic levels of the protein (44). ANX1 is released from leukocytes during the inflammatory response (21, 45), and it has recently been suggested that ANX1 should be classified as a type 2 acute phase protein (22).

A variety of studies using rANX1, derivative peptides, and neutralizing Abs indicate that ANX1 has immunosuppressive activities. These activities include inhibition of phospholipase A2 (20, 46), the binding and neutralization of lipid A (47), and inhibition of neutrophil and monocyte recruitment to inflammatory sites (25). One potential mechanism for the ANX1 effects on leukocyte recruitment is through blockade of leukocyte-endothelial interactions. In vitro, ANX1 can inhibit the adhesion of a monocyte cell line to activated endothelial cells. The effect appears to be mediated by ANX1 inhibition of the interaction between VLA4 and VCAM-1 (48). Here, we propose another potential mechanism, namely that ANX1 inhibits leukocyte recruitment by affecting L-selectin expression. L-selectin has been implicated in several steps during neutrophil recruitment to inflammatory sites: tethering and rolling of the neutrophil on the endothelium (49), neutrophil-neutrophil interactions (50), activation of intracellular signaling pathways (51), and extravascular migration (52). Previous studies have shown that modulation of L-selectin levels via shedding can have a marked impact on neutrophil-endothelial interactions (16, 53, 54).

In the present study, we have shown that exogenous ANX1 induces L-selectin shedding from peripheral blood neutrophils in a dose- and time-dependent manner. It also induces shedding from monocytes but has no effect on the expression of L-selectin by lymphocytes. Our results provide a rationale for the previously reported discrepancies between in vitro and in vivo effects of glucocorticoids with respect to L-selectin shedding. As we have verified, glucocorticoids do not cause shedding from neutrophils, although they have this effect in vivo when administered to humans and animals (11, 12, 13, 14). From our results and the fact that ANX1 is a second messenger for glucocorticoids, it is plausible that systemically available ANX1 is a mediator of glucocorticoid-induced L-selectin shedding. Importantly, the ED50 for the neutrophil effect is consistent with the dose of administered ANX1 demonstrated to inhibit neutrophil recruitment in a murine air pouch model in vivo (24). Although the high levels (60 µg/ml) observed in some extracellular fluids (40) suggest that our effective dose may be in the physiological range, determination of serum ANX1 levels during various inflammatory states will make it possible to assess the physiological relevance of the ED50 reported in this study.

Other explanations have been offered to account for the ability of glucocorticoids to produce L-selectin shedding. Nakagawa et al. suggest that glucocorticoids do not affect L-selectin shedding by mature peripheral blood neutrophils but rather act by inducing shedding in bone marrow neutrophils and promoting the release of these lower expressors into the circulation (55). They observe these effects as early as 6 h after glucocorticoid treatment. Although intriguing, this hypothesis cannot account for effects of glucocorticoids observed at earlier time points. For example, Fassbender et al. reported dexamethasone-induced shedding of L-selectin 1 h after systemic treatment (19). In addition, our previous studies indicate that L-selectin is shed from circulating neutrophils within 30 min of exposure to elevated systemic levels of glucocorticoids (16).

The mechanism by which ANX1 induces L-selectin shedding remains to be elucidated. Binding studies suggest that ANX1 receptors are present on monocytes and neutrophils but not on lymphocytes (56). However, these receptors have not been identified at the biochemical level. Recently it has been suggested that the active N-terminal peptides of ANX1 (Ac1–26 or Ac9–25) bind to and exert their biological effects through the FMLP receptor (34). However, in the present study, when we blocked the FMLP receptor with the antagonist peptide, Boc, there was no effect on ANX1-induced shedding, indicating that ANX1-induced shedding was not mediated through the FMLP receptor. It is possible that the lack of involvement of the FMLP receptor in our system results from our use of the full-length ANX1 protein rather than the N-terminal peptides. The N-terminal peptides of ANX1 and the full-length protein may induce different biological effects and may involve different mechanisms. Indeed, others have shown that the N-terminal peptide does not induce L-selectin shedding (57). The time course of ANX1-induced L-selectin shedding in our study was notably slower than FMLP or PMA. Whereas these agents induce maximum shedding within 10 min of exposure, ANX1 begins to induce shedding only after 15 min of exposure. Maximum shedding is reached after 30 min of exposure. Of note, both LPS- (29, 58) and IL-1 (29)-induced L-selectin shedding show a similar time course to that of ANX1. This longer time course may be indicative of slower kinetics of ANX1 binding to its cell surface receptor or that ANX1 acts through a distinct signaling pathway.

In this study, we also investigated whether ANX1-induced L-selectin shedding is accompanied by neutrophil activation. Although many agents that induce L-selectin shedding also activate cells (29), several treatments, including chymotrypsin exposure (59), cross-linking of Leu-13 (60), cross-linking of L-selectin itself (30, 60), ligation of CD4 (31), and exposure to C-reactive protein (61) or to phenylarsine oxide (62), induce L-selectin shedding without activating the cells. Our data indicate that ANX1 causes up-regulation of the {beta}2 integrin, Mac-1, which is often used as an indicator of cell activation. However, this Mac-1 up-regulation is markedly less than that observed with PMA treatment, suggesting a partially activated phenotype. ANX1 does not induce cell activation by two other indicators of neutrophil activation: expression of a {beta}2 integrin activation epitope and changes in cell shape. In contrast, PMA-treated cells showed strong activation by these two criteria. Taken together, these data suggest that ANX1 treatment does not produce overt activation of neutrophils.

In summary, the present study provides a mechanism by which glucocorticoids may induce L-selectin shedding from neutrophils and monocytes in vivo. The observed induction of L-selectin shedding by ANX1 may be part of an endogenous regulatory circuit that regulates the inflammatory response. We have previously described such a circuit in a model of inflammation in the rat knee joint (15, 16). We have shown that stimulation of pain fibers, designed to mimic inflammatory pain, activates the hypothalamic-pituitary-adrenal axis, resulting in glucocorticoid release and subsequent L-selectin shedding from circulating neutrophils. The consequence of this shedding is a dampening of the inflammatory response, as measured by neutrophil recruitment and plasma extravasation into the synovial space (16). Although other factors (i.e., IL-1, TNF-{alpha}, leukotriene B4, and C-reactive protein) that are up-regulated in blood during inflammation or injury (63) can induce L-selectin shedding (29, 61), ANX1 is specifically induced by glucocorticoids and thus is an excellent candidate to mediate these glucocorticoid-induced effects. Our findings may provide the basis for further understanding of physiological mechanisms for down-modulation of inflammatory reactions.


   Acknowledgments
 
We thank Annemieke van Zante and Dr. Vedang Londhe for kindly performing blood draws, Mark Singer for advice on preclearing experiments, and Dr. Joel Ernst for helpful discussions. We thank Dr. Nancy Hogg for providing the mAb24 Ab and W. H. Johnson for providing Ro31-9790.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant R37GM23547 (to S.D.R.) and a postdoctoral fellowship from the Arthritis Foundation (to H.J.S.). Back

2 Address correspondence and reprint requests to Dr. Steven D. Rosen, Department of Anatomy, University of California, 513 Parnassus Avenue, Box 0452, San Francisco, CA 94143-0452. Back

3 Abbreviations used in this paper: ANX1, annexin 1; Boc, N-t-Boc-Met-Leu-Phe. Back

Received for publication December 26, 2000. Accepted for publication March 5, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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