# Inflammatory neutrophils secrete annexin 1 during experimentally induced colitis in rats

Christine Coméra, D. Brousset, Nathalie Vergnolle, Lionel Bueno

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Christine Coméra, D. Brousset, Nathalie Vergnolle, Lionel Bueno. Inflammatory neutrophils secrete annexin 1 during experimentally induced colitis in rats. Digestive Diseases and Sciences, 1999, 44 (7), pp.1448-1457. hal-02688232

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# Inflammatory Neutrophils Secrete Annexin 1 During Experimentally Induced Colitis in Rats

CHRISTINE COMÉRA, PhD, PIERRE BROUSSET, MD, PhD, JEAN MORÉ, PhD, NATHALIE VERGNOLLE, PhD, and LIONEL BUÉNO, PhD

Immunoblotting and immunohistochemical analysis were performed to identify the cells expressing and secreting annexin 1 during experimental rat colitis induced by trinitrobenzene sulfonic acid. Annexin 1 expression was increased during the inflammation. Likewise, annexin 1 secretion was induced in inflamed colons at one, three, six, and nine days after trinitro-benzene sulfonic acid treatment but was not detected in colons from controls and rats at 12 days. Immunohistochemistry showed that the rise in annexin 1 expression resulted from the infiltration of two types of leukocytes highly positive for annexin 1: neutrophils (the most abundant) and macrophages. At day 1 after treatment, neutrophils of the inflammatory site, in mucosa and submucosa, are the only cells expressing annexin 1. Immunoblotting showed that they secreted annexin 1 whereas neutrophils from blood or tunica muscularis did not. This indicates that, during this colitis, annexin 1 can be secreted by neutrophils located in the inflammatory site.

**KEY WORDS:** annexin 1; neutrophils; secretion; colitis; inflammation.

Annexin 1 (lipocortin 1) belongs to a family of homologous calcium- and phospholipid-binding proteins (reviewed in 1). It was suggested to be an endogenous antiinflammatory agent that could mediate glucocorticoid action (2-4). Indeed, annexin 1 has extracellular antiinflammatory properties that mimic several of the glucocorticoid effects both *in vivo* and *in vitro*: it reduces the inflammatory reaction and inhibits eicosanoid production and leukocyte migration

Manuscript received August 24, 1998; accepted December 14, 1998.

From the Departement de Pharmacologie et Toxicologie, Institut National de la Recherche Agronomique, 180 chemin de Tournefeuille, BP3, 31931 Toulouse cedex 9, France; and Service d'Anatomie Pathologique, Hôpital Purpan, Place du Dr Baylac, 31059 Toulouse cedex, France.

N. Vergnolle was supported by a grant from the Ministère de l'Enseignement Supérieur et de la Recherche.

Address for reprint requests: Christine Coméra, Department of Pharmacology, Institut National de la Recherche Agronomique, 180 Chemin de Tournefeuille, BP3, 31931 Toulouse cedex 9, France.

(1-8). According to its extracellular action, annexin 1 was shown to be efficiently sorting through a novel secretory mechanism that seems similar to that of other proteins such as interleukin-1β (9-20). However, it is not clear when annexin 1 secretion occurs *in vivo* with regard to inflammatory reactions. An endogenous action of extracellular annexin 1 was observed *in vivo* in several studies. For instance, passive immunization of rats with anti-annexin 1 antibodies increases the extent of the inflammatory reaction and neutrophilic migration to the inflammatory site (6-8, 21, 22). It is noteworthy that one of these studies shows that these antibodies inhibit neutrophil migration when administrated systematically by intravenous injection but not when they were directly injected into the inflammatory site (8). Thus, in this inflammatory model of air-pouch in mice, it is the annexin 1 secreted in blood that is active in reducing neutrophilic migration but not the one, if any, secreted in the
inflammatory site. In a previous study, we established that annexin 1 is specifically secreted during the cellular stage of an experimental colitis in rats that was induced by the injection of TNBS into the proximal colon (20). This demonstrates secretion of annexin 1 inside an inflammatory site. Indeed, annexin 1 release is too abundant to be restricted to a secretion into blood microvessels and might actually take place in the overall inflammatory site.

Several reports show an increase in the expression of annexin 1 in several damaged tissues but did not study if the protein was secreted (23-28). The cells that might be responsible for this increased expression are brain astrocytes, macrophages, or skin keratinocytes. The latter two were also reported to secrete annexin 1 *in vitro* under specific conditions (9-11, 15, 18, 19). For example, the secretion of annexin 1 from cultured cells of the macrophage lineage were shown to be glucocorticoid inducible (9-11, 15, 19) and seem dependent upon cell differentiation or activation by other factors (29). This suggests that activated macrophages could secrete annexin 1 during inflammatory processes. A recent study shows that, in blood, activated neutrophils can also release annexin 1 that inhibits neutrophil migration into the inflammatory site (21).

The aim of this work was to identify in an inflammatory site the cells that are responsible for elevated local annexin 1 expression and secretion in the model of TNBS-induced colitis in rats. The study was performed using both western-blot analysis and immunohistochemistry.

## MATERIALS AND METHODS

**Animal Preparation and Colitis Induction.** Colitis induction on the proximal colon was adapted from the model of rectocolitis previously described (20, 30) using Wistar rats weighing 225-300 g. After an overnight fast, rats were anesthetized, laparotomized, and 0.3 ml of TNBS solution (30 mg in 50% ethanol) was injected into the lumen of proximal colon. Rats were killed by cervical dislocation 1, 3, 6, 9 or 12 days later (four rats for each time point). Control animals received 0.3 ml of saline (0.9% NaCl) and were killed 6 days later. Proximal colons were rapidly collected, rinsed with saline, and cut into equivalent pieces to study annexin expression and secretion and to perform histological studies. In some experiments, rat blood was collected by cardiac puncture.

**Study of Annexin Expression.** Freshly isolated proximal colons were homogenized using a Polytron for 30 sec at 0°C and sonicated for 30 sec at 0°C in a lysis buffer (LB) containing 25 mM Tris HCl, pH 7.4, 5 mM EDTA, 0.25% (v/v) Nonidet P-40, 1 µg/ml pepstatin, 15 µg/ml benzamidine, 1 µg/ml antipain, 1 µg/ml leupeptin, 1000 IU/ml

aprotinine, and 4 mM phenylmethylsulfonyl fluoride. Homogenates were equilibrated in electrophoresis sample buffer [SB: 60 mM Tris HCl, pH 6.8, 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 2% (w/v) sodium dodecyl sulfate (SDS), 0.01% (w/v) bromophenol blue], boiled for 10 min, and stored at -80°C.

**Study of Annexin Secretion.** Tissues were cut into small pieces and washed four times for 20 min in ice-cold phosphate-buffered saline (PBS: 150 mM NaCl, 2.5 mM KCl, 10 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4), three times in the presence of 2 mM EGTA and once without EGTA. Thereafter, tissue incubation was performed in a culture medium (RPMI 1640, Eurobio, Les Ulis, France, plus 1 mM glutamine, 0.02 mM EGTA, 20 mM HEPES, pH 7.4) in two stages. First, colons were incubated for 30 min at 37°C to allow protein secretion. Second, 2 mM EGTA was added, and the samples were rapidly cooled to 0°C and further incubated on ice for a 15 min period, allowing extracellular annexin 1 to be solubilized in the media. After that, tissues were separated from supernatants and both were used to evaluate the percentage of cell lysis by measurement of lactate dehydrogenase activity and to detect annexin 1 by immunoblotting (20).

Most of the secretion experiments were performed using full-thickness sections of the proximal colon. However, in order to determine where annexin 1 is secreted, these experiments were also performed using colonic strips of either mucosa plus submucosa or tunica muscularis as well as isolated blood neutrophils. The strips were isolated from proximal colons of two rats one day after TNBS-treatment and were carefully separated from each other by gently scraping with a glass microscope slide. The blood of these same rats was also collected by cardiac puncture in the presence of heparin, an equal volume of 0.9% NaCl was added, and the blood was subjected to neutrophil isolation. One third volume of 2% dextran 500 in 0.9% NaCl was added to 1 volume of the diluted blood, gently mixed, and allowed to sediment at room temperature for 45 min. The leukocyte-rich plasma was transferred to polypropylene tubes and centrifuged for 10 min at 100 g at room temperature. Pelleted cells were resuspended in 2 ml 0.9% NH₄Cl to lyse residual red cells and incubated on ice for 10 min. To the solution was then added 20 ml of PBS, and the solution was centrifuged for 10 min at 100 g. Leukocytes were resuspended in 15 ml PBS, carefully layered on 11 ml of Ficoll-Hypaque sodium (Pharmacia Biotech, Saint Quentin-Yvelines, France), and centrifuged at 400 g for 30 min at room temperature. Neutrophils were washed once in PBS, resuspended in 2 ml of culture medium, and incubated for 30 min at 37°C. Then, 2 mM EGTA was added and the neutrophils incubated for 2 min and rapidly centrifuged for 20 sec at 20 000 g. Supernatants and cell pellets were separated and used to measure the level of cell lysis and to detect annexin 1 by immunoblotting as described above. Hematoxylin-eosin staining of the smears of isolated polymorphonuclear cells permitted us to determine that more than 95% of the cells were polymorphonuclear, establishing the purity of the cell preparation.

**Detection of Annexins by Sodium-Dodecyl-Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Immunoblotting.** Proteins from the samples equilibrated in SB were separated by SDS-PAGE and electrophoretically
transferred onto nitrocellulose. Blots were subjected to immunoblotting using a monospecific and affinity purified rabbit polyclonal antibodies directed to annexin 1 and kindly provided by F. Russo-Marie (INSERM U332, Paris, France).

**Histology and Immunohistochemistry.** For histological studies, transversal sections of the proximal colons were fixed in 3.7% neutral buffered formalin, embedded in paraffin, cut at a thickness of 5 µm, and stained by the hematoxylin-eosin procedure. For histochemistry and immunohistochemistry, tissue sections were fixed using the previously described ModAMEX procedure (31), embedded in Paraplast X-TRA (Sigma), and cut at a thickness of 4 µm transversal sections.

Immunohistochemistry was performed as described by Delsol et al (31), using the affinity purified polyclonal antibodies against annexin 1 that were used for western blotting (1/2000), biotinylated multi-link swine anti-rabbit antibodies (diluted 1/100, Dako SA, Trappes, France), and the streptavidin-biotinylated complex coupled to alkaline phosphatase (Dako). Sites of bound alkaline phosphatase were revealed colorimetrically by a 10-min incubation with a solution containing 40 mM Tris HCl, pH 8.7, 0.17 M NaCl, 60 mM 2-amino 2-methyl 1-3 propanediol, 2 mM levamisole, 1.2 mM naphthol AS-BI phosphate, and 0.3 mM hexazotized new fuchsin. After that, the sections were washed in water, counterstained with hematoxylin and mounted in glycerin jelly. For black and white photographs, immunoperoxidase labeling of annexin 1 was performed using a streptovidin-biotinylated complex coupled to peroxidase (Dako) and a colorimetric solution containing 0.06% diaminobenzidine and 0.012% $H_2O_2$. The sections were then counterstained with hemalum, dehydrated, equilibrated in toluene, and mounted in Depex medium (BDH, Poole, England). The specificity of annexin 1 recognition by these antibodies was assessed because of a similar pattern of staining using other antibodies directed to annexin 1: rabbit monospecific polyclonal antibodies (1/200, a gift from L.A. Pradel, CNRS, Paris, France) and monoclonal antibodies (1/100, Zymed, San Francisco, California). Moreover, antibody preincubation with purified annexin I (kindly provided by L.A. Pradel) strongly reduced this staining. Negative controls were always performed in the absence of anti-annexin 1 antibodies.

The leukocytes present in the proximal colons were characterized by histological examination and histochemistry. Leukocytes were identified by both their morphological characteristics and their positive staining for acid phosphatase activity (32) in macrophages and for alkaline phosphatase in eosinophils and neutrophils. The specificity of these different stainings was verified by their inhibition in the presence of 0.1 M NaF for acid phosphatase and in the presence of 2 mM levamisole for alkaline phosphatase. Eosinophils were differentiated from neutrophils by their specific staining using Vital New Red (or Direct Red 75, CI 25380, Aldrich/Sigma, Saint-Quentin Fallavier, France) according to Duffy et al (33).

**Image Analysis.** Microscopic images of stained sections were detected by a JVC 3-CCD video camera and transduced to an image grabbing program (Neotech, Eastleigh, UK) and an Optilab-pro image analysis software package (Graftek, Mirmande, France) running on a Power Macin-

<figure><img src="image_2.png" /><figcaption>**Fig 1.** Western-blot analysis of colonic annexin 1 expression and secretion. Animals were killed at 1, 3, 6, 9 and 12 days after TNBS induction of colitis. Control animals are referred as 0 (day 0 after TNBS treatment). (A) Expression of annexin 1 in whole tissues; (B) annexin 1 secretion in supernatants after incubation in culture medium. Equivalent samples were loaded for homogenates (15 µg of total proteins) and supernatants. At 9 days after TNBS treatment, the colonic sample (a) did not secrete annexin 1 whereas sample (b) secreted annexin 1.</figcaption></figure>

tosh 7100/80 computer. All sections were investigated at constant voltage of the light source. At least three fields were captured. The cells that expressed either annexin 1, alkaline phosphatase, acid phosphatase, or that were Vital New Red positive were pink stained and could be detected as the area where the intensity of the green component was greater than that of the other components (blue or red).

RESULTS

**Annexin 1 Expression and Secretion.** In parallel to the histological study, annexin 1 expression and secretion by colonic specimens were investigated by immunoblotting, which permits relating cell protein expressing to the occurrence of annexin 1 secretion. As shown in Figure 1A, annexin 1 was overexpressed in the proximal colon of rats treated by TNBS (Figure 1A). Maximal annexin 1 expression was concomitant with high tissue myeloperoxidase (MPO) activity (not shown) and with the presence of two forms of annexin 1 of 33 and 38 kDa. The 38-kDa annexin 1 was previously shown to be the native protein and the 33-kDa form to be the product of an extracellular proteolysis of secreted annexin 1 (13, 14, 20). All colonic specimens from days 1, 3, and 6 after TNBS-treatment and one colon out of four at day 9 expressed these two forms of annexin 1 in their homogenates (Figure 1A). All these colons, and only these, also secreted the protein *in vitro*, when incubated in culture medium (Figure 1B). The presence of these two annexin 1 forms in the whole colonic homogenates is in fact due to an extracellular location of the
protein that should have been secreted *in vivo*. In contrast, the other colons (originating from control rats, from three of four rats at 9 days after TNBS-treatment, and from rats at 12 days) expressed only the native 38-kDa form of annexin 1 and did not secrete it *in vitro*. For all animals, the percentage of cell lysis was always <6.5% (mean 5.12% ± 0.86, N = 20) and was not correlated to annexin 1 release. This shows that the observed *in vitro* secretion of annexin 1 was not an nonspecific release due to cell death.

**Histological Study.** Microscopic examination of the proximal colons of control rats revealed a characteristic organization of normal tissue with mucosa, submucosa and tunica muscularis and did not show any inflammation (Figure 2A). In contrast, at days 1, 3, and 6 after TNBS-administration, a uniform colitis had developed over ≥2 cm in the proximal colon. These tissues showed complete necrosis of the epithelium and colonic glands, with mild to moderate signs of ulceration, edema, hyperemia, and crypt abscesses (Figure 2B-D). At day 1 after colitis induction, an important inflammatory infiltrate was revealed in the mucosa and submucosa (Figure 2B). Leukocytes infiltrated mainly the lamina propria, surrounding dead epithelial cells, but also the submucosa. By day 3 or 6, the mucosa was completely atrophied and mucus glands had almost completely disappeared (Figure 2C and D). The submucosa was hypertrophied and showed significant infiltration of leukocytes, especially concentrated beneath the dead tissues. The internal muscularis was also thicker and partially disorganized. It showed mild leukocyte infiltration except in localized areas where muscular necrosis was associated with greater leukocytic infiltration. By day 9 after TNBS treatment, the appearance of the colon was close to that observed at days 3 or 6, but the necrosed mucosa has been partially or totally eliminated from the tissue (Figure 2E). Areas of active epithelial regeneration were visible as early as 6 days after TNBS treatment, but were larger at 9 and 12 days. By day 9, the only proximal colon secreting annexin 1 showed less extensive epithelial regeneration than the three nonsecreting colons. At day 12, tissues had recovered an almost normal organization with a uniformly regenerated mucosa, but colonic diameters remained larger than in control tissues (Figure 2F). Moderate leukocytic infiltration was revealed in submucosa.

The nature of the infiltrated leukocytes was determined by histochemistry and quantified using image analysis. Neutrophils were identified by their characteristic structure with lobulated nuclei. Moreover,

they were positive for alkaline phosphatase but not Vital New Red staining (not shown). Proximal colons of rats killed one day after TNBS treatment revealed an inflammatory infiltrate originating from the submucosa and containing more than 95% of neutrophils. At days 3, 6, and 9, neutrophils remained the major infiltrated leukocytes, representing more than 83% of total leukocytes by day 3 and more than 65% by day 6 or 9. However, the presence of eosinophils and macrophages was determined by their morphologic characteristics, acid phosphatase activity for macrophages, and staining using Vital New Red for eosinophils. At days 6 and 9, fibroblasts were also abundant in the expanded submucosa. No granulomas were observed in this study.

**Immunohistochemistry.** The specificity of annexin 1 immunohistochemistry was established by similar staining with all polyclonal or monoclonal antibodies used. Moreover, a strong reduction in staining was observed by antibody preincubation with purified annexin 1 (not shown). In all tissue sections studied, the cells that were strongly positive to annexin 1 were leukocytes (Figure 3). In saline-treated colons there were few isolated neutrophils and macrophages present, mostly in the lamina propria but also in the submucosa and the tunica muscularis. At days 1, 3, 6, and 9 after TNBS-treatment, the annexin 1-positive cells were neutrophils and macrophages that infiltrated the inflamed tissues; they also were present at 12 days, in reduced numbers, in the newly formed mucosa and submucosa. Figure 4 shows photographs and an image analysis representation of the pattern of annexin 1 expression in control and inflamed colons at days 1, 3 and 9. Annexin 1 immunoreactivity clearly overlaid that of leukocytic distribution. Cells that were enriched in annexin 1 were concentrated in the submucosa and the mucosa at day 1 and in the inflamed submucosa at days 3 and 6. They were especially concentrated beneath the necrosed tissue and more dispersed in the muscularis. Other cells were immunoreactive to annexin 1 as smooth muscle and vascular endothelial cells but with a fainter labeling than for leukocytes. Their detection needed longer incubation times with the alkaline phosphatase substrate. Furthermore, they stained with similar intensity in colons from saline-treated or TNBS-treated rats.

**Annexin 1 Secretion by Activated Neutrophils.** At day 1 after TNBS treatment, neutrophils were the only cells that were present in the mucosa; epithelial and glandular cells were all dead because of the TNBS treatment. Neutrophils were also very abun-
<figure><img src="image_3.png" /><figcaption>**Fig 2.** Light micrographs of proximal colonic histological changes after TNBS administration. Hematoxylin and eosin staining. (A) Control tissues from saline treated rats; no signs of inflammation were visible. Damage at 1 (B), 3 (C), 6 (D), 9 (E), and 12 (F) days after induction of the colitis. Original magnification, ×10 for A–F. Bar = 10 µm.</figcaption></figure>

In the isolated part of proximal colon containing mucosa and submucosa, annexin 1 was almost totally expressed by neutrophils. Therefore, we investigated if these cells were responsible for annexin 1 secretion. For that, mucosa and

submucosa as well as the tunica muscularis of two independent colonic specimens were collected at day 1 after TNBS administration and were separately washed and incubated in culture medium. In parallel, the blood neutrophils from these rats were isolated
<figure><img src="image_4.png" /><figcaption>**Fig 3.** Immunohistochemical study of annexin 1 expression in neutrophils of colons at day 1 after TNB administration. (A) Hematoxylin/eosin staining of cell nucleus identifying polynuclear neutrophils; (B) annexin 1 immunoreactivity of neutrophils; (C) immunostaining of annexin 1 in mucosa; (D) negative control of the immunostaining in mucosa. Original magnification, ×100 for A and B and ×40 for C and D. Bar = 1 µm.</figcaption></figure>

and similarly incubated to determine if they also
secreted annexin 1. As shown in Figure 5, and accord-
ing to the immunohistological study, annexin 1 ex-
pression was higher in the mucosa and submucosa
than in the muscularis. Annexin 1 was secreted by the
mucosal part of colon, but not by the tunica muscu-
laris or the isolated blood neutrophils. Indeed, for the

latter two, no release of annexin 1 was detected even
after extensive exposure of the fluorographs. In the
two secretory experiments performed, cell lysis was
always low (0.7 and 1.9% for blood neutrophils, 5.4
and 4.5% for mucosa plus submucosa and 6.3 and
6.5% for tunica muscularis) and was not correlated
with annexin 1 secretion.
<figure><img src="image_5.png" /><figcaption>**Fig 4.** Annexin 1 immunoreactivity: (1) image analysis at 1 (a), 3 (b) and 9 (c) days after TNBS treatment, in proximal colons secreting annexin 1: (A) colonic lumen, (B) necrosed tissues, (C) mucosa, (D) submucosa, (E) internal muscularis. Annexin 1 immunoreactivity is represented in black. (2) Photographs of the colonic immunoperoxidase labeling of annexin 1 from control animals (d) and at day 1 (e, f) after TNBS treatment. Samples (d) and (e) were incubated with anti-annexin 1 antibodies and sample (f) is the negative control of immunostaining (not incubated with anti-annexin 1 antibodies). In control colons, a few leukocytes were the only annexin 1-positive cells. During colitis annexin 1 labeling is greatly increased and is expressed by the infiltrated leukocytes, which were mostly present in the mucosa and the submucosa. At days 3 and 9, annexin 1 immunoreactivity was more significant in some areas of the internal muscularis, which corresponded to leukocyte-rich areas of muscular necrosis.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig 5.** Western-blot analysis of colonic annexin 1 secretion in blood neutrophils (sample 1), colonic mucosa plus submucosa (sample 2), and tunica muscularis (sample 3) of animals treated for 1 day by intracolonic injection of TNBS. Samples were incubated separately in culture medium. The presence of annexin 1 was then detected in 15 µg of total proteins of the tissue homogenates (H) and supernatants (S). The supernatant volumes loaded represent five times those of the corresponding homogenates. This figure is representative of two separate experiments.</figcaption></figure>

DISCUSSION

In this study using TNBS-induced colitis in rats, we have attempted to characterize the inflammation histologically and to identify the cells that are responsible for most of the increased annexin 1 expression and secretion. The study indicates that neutrophils play a leading part in the cellular stage of this inflammatory reaction in the proximal colon. Indeed, they are the major leukocytic cells that infiltrated the tissue and are responsible for most of the increased annexin 1 expression and secretion.

Histological examination shows that TNBS induces severe colitis, with extensive necrosis of the epithelium and mucus glands. This is associated with annexin 1 secretion and with significant leukocyte infiltration from days 1 to 6 after treatment, in accordance with the previously reported high MPO activities of these tissues (20). Around day 9, the disappearance of necrosed mucosa and tissue regeneration coincides with a significant decrease of leukocytic infiltration and MPO activity and with the cessation of annexin 1 secretion (only one colon of four is still secreting annexin 1 at day 9). At day 12, the leukocytic infiltrates are very reduced, consisting of scattered cells in the newly formed mucosa and submucosa. This rapid regeneration contrasts with the usually observed persistence of inflammatory parameters (damage score and MPO activity) reported for the initial model of TNBS induced colitis involving distal colons and performed with Sprague-Dawley rats (30). This discrepancy could be due to differences in the rat lines used or to different reactivities to TNBS/ethanol between distal and proximal colons.

In this study, the histochemical characterization and the quantification of infiltrated leukocytes in TNBS-treated proximal colons from Wistar rats permitted

us to emphasize differences between the response in our model and in the one using inflammation of the distal colon of Sprague-Dawley rats (30). Indeed, in the proximal colon, neutrophils remain the major cells present in the infiltrates during the entire cellular stage of the inflammation. Macrophages and eosinophils have also infiltrated the inflammatory site but remained in a minority, representing, respectively, 9.5% (±8.8 SD) and 10.8% (±9.0 SD) by days 3 to 9. Moreover, we did not observe any granulomas in histological sections of proximal colon. Indeed, the TNBS-induced inflammation of proximal colon of Wistar rats has the characteristics of an acute reaction and does not show the chronicity reported by Morris et al (30).

We show a high annexin 1 immunoreactivity in neutrophils and macrophages. In agreement, previous studies reported that annexin 1 has a discrete localization in a restricted number of cells including specialized epithelia, epidermic keratinocytes, brain astrocytes, endothelial cells from umbilical cord, chromaffin cells, germinal cells, monocytes, as well as neutrophils and macrophages displaying the strongest immunoreactivity (18, 24-28, 34-38). Moreover, in this study and according to Fava et al (35), smooth muscle and vascular endothelial cells were also identified as immunoreactive to annexin 1 but with a much fainter labeling than for leukocytes. In fact, the detection of these two types of cells as annexin 1-positive needed longer incubation times with the alkaline phosphatase substrate (not shown). Furthermore, they were stained with similar intensity in colons from saline-treated and from TNBS-treated rats. Thus, the increase of annexin 1 expression during this experimental colitis cannot be related to any rise of annexin 1 expression by resident colonic cells. In fact, the elevated expression of annexin 1 is due to the significant infiltration of leukocytes (neutrophils and macrophages) that are highly enriched in annexin 1. Other reports show that the activation of local cells during inflammatory processes raises their expression of annexin 1: in keratinocytes during psoriasis (25), in reactive astrocytes during brain injuries (26, 27), and in synovial macrophages during rheumatoid arthritis (28). Even if not apparent in this study, the level of annexin 1 expression by leukocytes could also be raised when the cells were activated in the inflammatory site. Another study reported that annexin 1 expression of biopsies from Crohn's disease patients was lower or equivalent to control levels depending on the severity of the damage, but was not increased by the inflammation (39). It is actually difficult to
compare these results with ours. Indeed, we studied whole colonic tissue whereas the biopsies used in this other study where representative of only a small part of the human tissue that may not contain as many inflammatory cells as observed in our model.

At one day after TNBS treatment, neutrophils are very abundant in the mucosa and submucosa and are almost the only annexin 1-enriched cells. The mucosal part of the colon secretes annexin 1, establishing that neutrophils are responsible for the majority of, if not all, annexin 1 secretion during this colitis. In contrast, no annexin 1 secretion from the tunica muscularis was detectable, suggesting that the cells that express the protein in this tissue do not secrete it (smooth muscle cells expressing moderate amount of annexin 1 and few neutrophils highly enriched in annexin 1). We cannot exclude that the failure to detect annexin 1 secretion from the tunica muscularis is related to the sensitivity of the immunoblotting assay. However, this experiment shows that the major contributors for annexin 1 secretion in inflamed colons are neutrophils, which had invaded the inflammatory site in the mucosa plus submucosa. In addition, blood neutrophils do not show any detectable annexin 1 secretion. This strongly suggests that annexin 1 secretion by neutrophils is linked to the activation of these cells in the inflammatory site. By days 3, 6, and 9 after colitis induction, the cells are responsible for annexin 1 secretion should be primarily neutrophils, but also macrophages that can represent up to 15% of total leukocytes in secreting tissues. It has been proposed that macrophage activation during an inflammatory reaction raises annexin 1 expression and secretion and their induction by glucocorticoids (29). Our results suggest that neutrophil activation during inflammation also induces active annexin 1 secretion. Perretti et al (21) showed that during an inflammatory process, blood neutrophils can secrete annexin 1, which inhibits their migration through the endothelial barrier. The significant amount of annexin 1 secretion from TNBS-inflamed colons (up to 25% of total annexin 1 was secreted in 30 min (20) can not only be attributed to the few leukocytes that are present in blood vessels but to all the leukocytes of the inflammatory site.

We conclude that in this experimental colitis model the infiltration of neutrophils and macrophages was important from 1 to 9 days after TNBS treatment and was responsible for increased annexin 1 expression. This study also establishes that, at least in this experimental colitis model, the involvement of local cells in annexin 1 secretion is nonexistent or negligible,

whereas neutrophils and probably macrophages that have migrated into the inflammatory site actively secrete it. The biological function of annexin 1 secretion during colitis remains to be studied. Based on its antiinflammatory properties, we can suggest that secreted annexin 1 inhibits neutrophilic migration and also, for the leukocytes that have reached the inflammatory site, the local production of inflammatory lipidic mediators. Both actions should consequently reduce the extent of inflammation.

## ACKNOWLEDGMENTS

The authors thank Josy Bigot and Martine Deuillhe for excellent technical assistance.

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