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Aberrant inflammation and resistance to glucocorticoids in annexin 1^-/- mouse¹

ROBERT HANNON, JAMIE D. CROXTALL, STEVE J. GETTING, FIORITA ROVIEZZO^*, SIMON YONA, MARK J. PAUL-CLARK, FELICITY N. E. GAVINS, MAURO PERRETTI, JOHN F. MORRIS, JULIA. C. BUCKINGHAM and RODERICK J. FLOWER²

Department of Biochemical Pharmacology, William Harvey Research Institute, Queen Mary, University of London, Charterhouse Square, EC1M 6BQ, UK;
^* Universita Degli Studi Di Napoli Dipartimento di Farmacologia Sperimentale Federico II, Napoli, Italy;
Department of Human Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK; and
Division of Neuroscience and Psychological Medicine, Imperial College School of Medicine, Hammersmith Hospital Campus, London, W12 0NN, UK

²Correspondence: Department of Biochemical Pharmacology, William Harvey Research Institute, Queen Mary, University of London, Charterhouse Square, EC1M 6BQ, UK. E-mail: r.j.flower{at}qmul.ac.uk

SPECIFIC AIMS

The 37 kDa protein annexin 1 (Anx-1; lipocortin 1) has been implicated in the regulation of phagocytosis, cell signaling, and proliferation and postulated to be a mediator of glucocorticoid action in inflammation. To test this hypothesis, we have generated for the first time an Anx-1 null mouse line and compared its sensitivity to inflammatory stimuli and its response to glucocorticoids with wild-type controls. A dual-purpose targeting vector designed to simultaneously inactivate the gene and report on the activity of the Anx-1 promoter allowed us to measure gene expression.

PRINCIPAL FINDINGS

1. Generation of Anx-1^-/- mice
Anx-1^-/- animals were viable and appeared healthy. There was no obvious difference between any of the (sex-matched) groups Anx-1^-/- Anx-1^+/- and Anx-1^+/+ littermate control mice in terms of gross physical appearance, behavior, or weight. Anx-1^-/- animals of both sexes bred normally.

2. Anx-1 and related protein expression in Anx-1^-/- mice
There was strong Anx-1 gene expression (as assessed by lac-Z staining) in stomach, lung, spleen, ovary, and uterus whereas kidney, thymus, and heart exhibited intermediate levels; some expression was detected in thyroid, pancreas, and testes. At the protein level, Anx-1 was absent from Anx-1^-/- mice and reduced in Anx-1^+/-animals. Tissues taken from Anx-1^-/- mice exhibited an altered (mainly up-regulated) expression of other annexins (principally annexins 2, 5, and 6) and in some cases (lung and thymus) by a strong (2- to 100-fold) up-regulation of COX-2 and cPLA_2.

3. Inflammation in Anx-1^-/- mice
Mouse paw carrageenin-induced edema follows a characteristic biphasic pattern with a PMN-dependent phase peaking at 4–6 h and resolving at 24 h, and a lymphocyte/macrophage/eosinophil-dependent phase (48–96 h). In an age- and weight-matched experiment, Anx-1^-/- mice displayed enhanced sensitivity to this inflammatory stimulus (Fig. 1 A). Dexamethasone [10 µg/kg x3, intraperitoneal (i.p.)] strongly inhibited the first phase of edema (mainly PMN dependent) in Anx-1^+/+ mice but was completely without effect in Anx-1^-/- mice (Fig. 1B ). In contrast, there was no change in glucocorticoid sensitivity of the second phase (Fig. 1C ).

View larger version (26K): [in this window] [in a new window]   Figure 1. Failure of dexamethasone to inhibit the first phase of carrageenin paw edema in Anx-1-/- mouse. A) Age- and sex-matched experiment demonstrating that edema follows the same time course in wild-type and null animals and that Anx-1-/- mice display an enhanced sensitivity to this irritant. Black columns, Anx-1+/+; open columns, Anx-1-/- mice. Data analyzed by Student’s t test, n = 6 per group. **P < 0.01 relative to Anx-1+/+. B) Failure of dexamethasone to inhibit the PMN-dependent phase of carrageenin paw edema in Anx-1-/- mice. Black columns, Anx-1+/+ mice treated with vehicle; cross-hatched columns, treated with 10 µg dexamethasone i.p. at 0, 2, 6, and 24 h. Open columns, Anx-1-/- mice treated with vehicle; dotted columns, treated with dexamethasone. Data analyzed relative to vehicle-treated controls by Student’s t test, n = 17–18 per group; *P < 0.05; **P < 0.01. C) Maintenance of dexamethasone action on the second phase of paw edema in Anx-1-/- mice. Column codes as above.

In zymosan-induced peritonitis experiments with matched groups, we noted a substantial increase in PMN migration at all points in the Anx-1^-/- mice relative to their Anx-1^+/+ counterparts; at 4 and 6 h, the response in the Anx-1^-/+ animals was intermediate (Fig. 2 A). At 2 h, IL-1ß levels were higher in females than males (299.8±90.1 ng/mL vs. 142.6±29.6 ng/mL respectively: P=0.01) but were significantly increased in Anx-1 null animals in both sexes (Fig. 2B ). Dexamethasone (0.1 mg/kg) produced 49.2% inhibition of PMN emigration in Anx^+/+ mice but progressively less in Anx^+/- (36.1%) and Anx^-/- mice (22.3%) (Fig. 2C ).

View larger version (25K): [in this window] [in a new window]   Figure 2. Leukocyte dynamics in Zymosan peritonitis. A) Leukocyte emigration is enhanced in peritonitis in Anx-1+/- (shaded columns) and Anx-1-/- (open columns) compared to Anx-1+/+ (black columns) mice. Composite figure of three separate experiments at 2, 4, and 24 h, data analyzed by unpaired Student’s t test, n = 7–10 per group. *P < 0.05; ***P < 0.001. B) Increased IL-1ß generation during (2 h) zymosan peritonitis in male (light shading) and female (dark shading) Anx-1-/- mice. Data analyzed by unpaired Student’s t test, n = 4–6 per group. **P < 0.01, relative to appropriate Anx-1+/+ controls. C) Dexamethasone (0.1 mg/kg) is less effective as an inhibitor of cell migration in Anx-1+/- and Anx-1-/- mice. Composite of three separate experiments using female mice, n = 6–15 per group, expressed as a % inhibition of migration relative to vehicle-treated mice using two-tailed unpaired Student’s t test. *P < 0.05; **P < 0.01. D) Leukopenia observed in female Anx-1-/- mice during the course of zymosan peritonitis. Data shown are 4 h after injection of zymosan. Total and differential cell count are shown (PMN, polymorphonuclear cells; Mono, monocytes; Lymph, lymphocytes). Black columns, Anx-1+/+; gray columns, Anx-1+/-; open columns, Anx-1-/- mice. Data analyzed by 1-way ANOVA using Dunnett’s correction for multiple comparisons with control, n = 4–9. **P < 0.01.

4. Gender differences in inflammatory response in Anx-1^-/- mice
We found that after 4 h zymosan peritonitis, there was a profound alteration in the disposition of peripheral blood cells in female, though not in male, Anx-1^-/- animals and that this was mainly accounted for by a dramatic fall in circulating PMN (Fig. 2D ).

5. Leukocyte behavior and adhesion molecule expression in Anx-1^-/- mice
CD11b expression on PMN and monocytes from Anx-1^-/- mice (either sex) were significantly reduced (-32% and -28.5% respectively) compared with Anx-1^+/+ controls whereas CD62L expression was significantly enhanced (+27.8% and +30.0%, respectively). In contrast, CD11b expression on PMN and monocytes taken from Anx-1^-/- mice was significantly more sensitive to activation by FMLP (1–10 µM) or PAF (0.3–3.0 µM) than were their Anx-1^+/+ counterparts.

We assessed the phagocytic activity of peritoneal lavage macrophages in the three genotypes using light microscopy. We found that zymosan (unopsonised) was taken up by 50% of Anx-1^+/+ cells after 2 h, but a significantly decreasing proportion of phagocytosing cells was seen in preparations obtained from Anx-1^+/- and Anx-1^-/- mice. To investigate the extent of this phagocytic defect, we examined ROS generation induced by IgG complexes using the Red Oxyburst^TM technique. Phagocytosis of IgG was not significantly different among any of the control groups tested. Glucocorticoids inhibited the reaction rate in Anx-1^+/+ animals by 29.55 ± 1.96% but were inactive in Anx-1^-/- animals (P<0.028, two-sided Mann Whitney test).

Using intravital microscopy in the mouse mesentery, we investigated the effects of genotype on the behavior of leukocytes in the microcirculation under unstimulated conditions. There was no significant difference in the velocity of rolling of Anx-1^+/+ or Anx-1^-/- cells under basal conditions, but we observed a significant increase in cell adhesion in the Anx-1^-/- animals relative to wild-type controls (1.0±0.27 Anx-1^+/+ vs. 2.0±0.36 Anx-1^-/- per 100 µm vessel; P=0.02, unpaired t test, n=11–17) as well as in the emigration (0.0, range 0–4 Anx-1^+/+ vs. 1.0 range 0–5 Anx-1^-/- per 100x50 µm² field: P=0.48, Mann Whitney test, n=11–17).

CONCLUSIONS AND SIGNIFICANCE

Earlier attempts to delineate the role of Anx-1 in physiopathology used acute passive immunization strategies. In models of rodent inflammation such as the zymosan-inflamed air pouch, we found that administration of neutralizing antibodies exacerbated inflammation as assessed by PMN influx, cytokine, and eicosanoid synthesis and resolution of the inflammatory response. These findings were confirmed in this study using the transgenic approach. There were marked variations in leukocyte behavior and phagocytic response between the Anx-1^+/+ and Anx-1^-/- animals as well as differing responses to inflammatory stimuli and glucocorticoid sensitivity. Because of the apparent redundancy of glucocorticoid mechanisms, it would be surprising if every effect of these drugs was suppressed in the Anx-1^-/- animals. Indeed, we have observed that recombinant Anx-1 exerts anti-inflammatory effects in some, but not all, inflammatory models.

The overall pathophysiological picture of Anx-1 gene deletion that emerges from our studies suggests a heightened sensitivity to inflammatory and other environmental stimuli. This is no doubt exacerbated in vivo by the inability of endogenous glucocorticoids to adequately control the inflammatory response because of the absence of this protein. Our observation that female (but not male) Anx-1^-/- mice respond with a strong leukopenia during an inflammatory episode may be significant and suggests a novel and potentially important link between sex hormones and the annexin system.

View larger version (25K): [in this window] [in a new window]   Figure 3. Schematic summary of the alterations in leukocyte recruitment in the Anx-1-/- mouse. Top panel: Leukocyte extravasation in wild-type mice. On activation, white blood cells (PMN leukocytes shown) roll on the postcapillary venule endothelium and adhere. This is followed in some instances by detachment and in others by emigration through the endothelial barrier. Previous data suggest a crucial role for Anx-1 in this "decision" process (?). At the site of inflammation, emigrated cells may phagocytose micro-organisms, release proteolytic enzymes, etc., before entering apoptosis. B) In Anx-1-/- mice, the process of PMN extravasation is greatly enhanced: fewer cells will detach from the vessel wall (?), as leukocytes from these animals express higher cell surface CD11b when activated. This effect is mirrored by the increase in adherent and migratory cells seen in the inflammatory and microcirculation models. Both circulating and emigrated cells seem more sensitive to inflammatory stimuli and less susceptible to glucocorticoid counter-regulation resulting in an enhanced inflammatory response. The result is a more intense inflammatory response.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0239fje; to cite this article, use FASEB J. (December 3, 2002) 10.1096/fj.02-0239fje

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