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Published online before print July 6, 2005
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* Centre of Biochemical Pharmacology, The William Harvey Research Institute, London, United Kingdom; and
Eric Byswaters Centre for Vascular Inflammation, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, United Kingdom
1Correspondence: Centre of Biochemical Pharmacology, Bart’s and The London, Queen Mary School of Medicine and Dentistry, Charterhouse Square, London, EC1M 6BQ, UK. E-mail: m.perretti{at}qmul.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: inflammation • intravital microscopy • CD11b • cell trafficking • chemotaxis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within the complex processes that comprise the innate immune response, migration of white blood cells (WBC) from the vascular lumen into the interstitial tissue is probably the most important and potentially damaging event shared by numerous inflammatory conditions [2 ]. For this reason, it is not surprising that leukocyte migration is closely controlled. Endogenous inhibitors of leukocyte recruitment include endothelial-derived nitric oxide, adenosine, short-lived lipids produced via transcellular synthesis [3 , 4 ], and the 37-kDa protein, annexin 1 (AnxA1). Originally termed lipocortin 1 and proposed to be involved in the anti-inflammatory actions of glucocorticoids [5 ], AnxA1 was cloned in 1986 and found to be a member of a large family of structurally related proteins [6 ]. Human and rodent granulocytes [predominantly neutrophils, polymorphonuclear leukocyte (PMN)] contain high levels of this protein [7 8 9 ], which is rapidly mobilized upon leukocyte adhesion to endothelial monolayers in vitro [10 ]. In pharmacological studies, exogenous application of human recombinant AnxA1 and its peptido-mimetics exerts inhibitory actions on human PMN activation [11 , 12 ] as well as on PMN recruitment in models of acute inflammation [13 , 14 ]. The latter effect has been described in several experimental settings and in response to various inflammatory stimuli, including zymosan, cytokines, and reperfusion injury [15 16 17 ].
Much less is known about the role of the endogenous protein. AnxA1 null mice exhibit a prolonged and exacerbated inflammatory response to locally applied stimuli (e.g., paw edema model) [18 ] and are partially resistant to the anti-inflammatory properties of glucocorticoids [18 , 19 ]. More recently, we demonstrated higher susceptibility of AnxA1 null mice to the systemic response promoted by lipopolysaccharide [20 ]; in the same study, we reported rapid kinetics of AnxA1 gene activation in the mesenteric microcirculation, suggesting a function for endogenous AnxA1 on microscopic events occurring within inflamed vascular beds.
Similarly, few studies have investigated cell behavior in the absence of AnxA1. Peritoneal macrophages have defected phagocytosis (related to the size of the particle added) and display altered activation profiles if taken from AnxA1 null mice [21 ]. Similarly, AnxA1 null fibroblasts are more prone to activation, releasing increased amounts of prostaglandins upon stimulation and showing partial resistance to the inhibitory effects of glucocorticoids [22 ]. In the present study, we have used AnxA1 null mice to increase our knowledge of the role of this mediator in cell activation, by combining analyses of PMN trafficking and interaction with the postcapillary venule endothelium, and selected in vitro assays of activation using blood-purified PMN.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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25 g body weight) were purchased from B&K (Hull, UK). In all cases, animals were housed at a density of five animals per cage in a room with controlled lighting (lights on from 8:00 a.m. to 8:00 p.m.), in which the temperature was maintained at 21–23°C. Animal work was performed according to UK Home Office regulations (Guidance on the Operation of Animals, Scientific Procedures, Act 1986) and complying with the directives of the European Union.
In vivo protocols
Model of peritonitis
Zymosan peritonitis was induced by injecting 1 mg zymosan A (Sigma-Aldrich, Poole, UK) in 0.5 ml phosphate-buffered saline (PBS) [23
]. Peritoneal lavage fluid aliquots were stained in Turk’s solution and used for total cell counts as well as differential counting (light microscopy values) using an Olympus B061 microscope (London, UK). Remaining lavage fluids were centrifuged at 400 g for 10 min. Pellets were resuspended in fluorescein-activated cell sorter (FACS) wash [PBS containing 0.2% bovine serum albumin (BSA) and 0.1% sodium azide]. Aliquots (10 µl 4x107 cells/ml concentration) were seeded onto a 96-well plate, and a 1:1 mixture of blocking immunoglobulin G (IgG; human 
globulin, 6 mg/ml, in FACS wash) and anti-CD11b antibody (clone 5C6 [24
], a generous gift of Dr. Neil Gozzard, Celltech R&D, Slough, UK) or corresponding isotype control (final concentrations 5 µg/ml) was placed on the cells. After 30 min incubation on ice, the plate was washed, and a second incubation was carried out, consisting of a 1:1:1 mixture of Gr1-fluorescein isothiocyanate (FITC; BD Biosciences, Oxfordshire, UK), F4/80-phycoerythrin (Serotec, Oxfordshire, UK), and Streptavidin Tricolor (Caltag Laboratories, Northamptonshire, UK; final concentrations of 1.67 µg/ml, 1.00 µg/ml, and 0.33 µg/ml, respectively) for further 30 min. After washing, cells were fixed with ice-cold cell fixative (CellFixTM, BD Biosciences) and analyzed using the CellQuestTM software on a Power Macintosh G3 computer. For each sample, a minimum of 10,000 events was acquired. Gates were constructed around two populations, the FL-1-positive events (Gr1+) and FL-2-positive events (F4/80+). The percentage of total events in each population, together with the level of CD11b expression, was measured.
Intravital microscopy of the mouse cremaster muscle
The mouse cremaster muscle microcirculation was prepared for intravital microscopy as described previously [25
]. Briefly, following anesthesia with a mixture of xylazine (7.5 mg/kg) and ketamine (150 mg/kg), the left jugular vein was cannulated with polyethylene tubing (PE10) for drug administration. The cremaster was dissected free of skin and fascia, opened longitudinally, and then pinned out flat against the viewing platform of a plexiglass stage. The preparation was mounted on a Zeiss Axioskop "FS" microscope (original magnification: 40x; Carl Zeiss, Welwyn Garden City, UK) and transilluminated with a 12-V, 100-W halogen light source to observe the microcirculation. Superfusion of the cremaster muscle with bicarbonate-buffered solution (BBS; g/liter: 7.71 NaCl, 0.25 KCl, 0.14 MgSO4, 1.51 NaHCO3, and 0.22 CaCl2, pH 7.4, at 37°C, gassed with 5% CO2/95% N2) at a rate of 2 ml/min began immediately. A Hitachi charged-coupled device color camera (KPC571; Tokyo, Japan) acquired images displayed on a Sony Trinitron color video monitor (PVM 1440QM; Tokyo, Japan) and recorded on a Sony super-VHS videocassette recorder (SVO-9500 MDP) for subsequent off-line analysis. A video time-date generator (FOR.A video timer, VTG-33, Tokyo, Japan) projected the time, date, and stopwatch function onto the monitor.
Platelet-activating factor (PAF)-induced inflammation
After a 30-min stabilization period of BBS superfusion, a postcapillary venule (diameter between 20 and 40 µm; length >100 µm) was selected and superfused with 1 nM PAF (C16 form: C26H54NO7P; Sigma-Aldrich) to inflame the tissue. Recordings were then made every 15 min up to a total of 90 min. Off-line analyses were performed to calculate WBC velocity (V) in µm/s, the time taken for a leukocyte to roll along 100 µm of a vessel wall; extent of leukocyte adhesion, i.e., number of adherent leukocytes (stationary for at least 30 s) in the 100-µm vessel length over a 5-min recording; and extent of leukocyte emigration, measured as the number of leukocytes that had emigrated up to 50 µm either side of the vessel. Wall shear rate (WSR) was calculated by the Newtonian definition: WSR = 8000 x (Vmean/diameter) and expressed in s–1. Centerline red blood cell (RBC) velocity was also measured on-line using an optical Doppler velocimeter.
The mouse cremaster was also inflamed with zymosan particles as described recently [26 ]. Briefly, fluorescent zymosan (Alexa Fluor 488-labeled, Molecular Probes, Eugene, OR) was injected into the scrotum at a dose of 30 µg in 400 µl saline at time 0, and the microcirculation was analyzed at the 6-h time-point. One to three randomly selected, postcapillary venules were observed for each mouse. Leukocyte rolling and adhesion were analyzed as above, whereas leukocyte emigration was determined per field of view (one to three fields per vessel) [26 ].
Zymosan phagocytosis
Use of fluorescent zymosan also allowed the quantification of phagocytosis in vivo [26
]. For PMN phagocytosis, the cremaster preparation was handled as described above, and at the end of experimentation, fluorescent particles were visualized using a silicon-intensified, low-level light camera (Hamamatsu Photonics, Enfield, UK). Phagocytosis was quantified as the number of transmigrated leukocytes containing fluorescent particles per field of view (more than nine fields of view per mouse were analyzed). The phagocytic response was normalized for the total number of infiltrating leukocytes, and data are presented as index of phagocytosis: (phagocytosing PMN/total PMN) x 100.
In vitro protocols
PMN CD11b expression
Blood was collected by cardiac puncture under halothane (3%) anesthesia from four to six mice from each genotype. Aliquots (1 ml) were then incubated with or without PAF (0.3–3 µM), phorbol myristoyl acetate (PMA; 1–10 µM), or formyl-Met-Leu-Phe (fMLP; 1–30 µM) for 15 min at 37°C. Cells were then washed with 5 ml PBS supplemented with NaN3 (3% w:v). Smaller aliquots of blood (200 µl) were then labeled with 50 µl rat anti-mouse CD11b (5 µg/ml; clone 5C6) for 60 min at 4°C, using a protocol described previously [17
]. After two washes, cells were then stained with 50 µl FITC-conjugated rabbit anti-rat IgG antibody (1:50; Serotec) for 5 min at room temperature. Following two further washes in PBS, RBC lysis was performed with Immuno-LyseTM (Coulter, Luton, UK). Flow cytometry was performed using a FACScan analyzer (Becton Dickinson, Cowley, UK) with an air-cooled 100 mW argon laser, tuned to 488 nm, connected to an Apple Macintosh G3 computer running Cell Quest II software. Forward- and side-scatter characteristics were used to distinguish amongst the three distinct cell populations (lymphocytes, monocytes, and granulocytes) and CD11b levels measured in the FL-1 channel (wavelength of 548 nm), expressing data as units of median fluorescence intensity (MFI) [17
].
Chemotaxis
Blood-isolated mouse neutrophils were produced by negative immunomagnetic separation as described in ref. [27
], using a chilled separation column connected to a VarioMACSTM magnet (Miltenyi Biotec, Bisley, UK). Briefly, mouse blood was incubated with a cocktail of rat anti-mouse monoclonal antibodies: anti-CD2, anti-CD5, and anti-CD45R (5 µg/ml; BD Biosciences), anti-F4/80 (1 µg/ml, Serotec), and anti-CD54 (3 µg/ml, generous gift from Dr. Chapman, Celltech R&D) for 30 min at 4°C and then washed and incubated with anti-rat IgG MicroBeads (20 µl per 107 cells) for a further 15 min prior to running the leukocyte/MicroBead mixture through the column and collection of neutrophil-rich effluent. The neutrophil-rich population (>95% PMN) was then resuspended (4x106 cells/ml in RPMI 1640, supplemented with 0.1% BSA), and 25 µl was loaded on the top well of a NeuroProbe ChemoTx-101-3TM 96-well plate equipped with a 3-µm pore-size filter (NeuroProbe, Gaithersburg, MD). As chemoattractants, 0.1–1 nM fMLP or PAF was added in the bottom well in a volume of 27 µl and incubated for 2 h at 37°C in 5% CO2/95% O2 atmosphere. The ChemoTx-3TM plate was then centrifuged (312 g, 5 min) to pellet cells migrated on the undersides of the filter. Migrated neutrophils were counted after a 1:1 dilution in Turk’s solution, using a Neubauer haematocymometer.
Statistical analysis
All values are expressed as mean ± SE of mean, with number (n) of animals per group where stated. Statistical analysis for the intravital microscopy studies was assessed by Student’s t-test (two groups) or by one-way ANOVA followed by Bonferroni post-hoc test (more than two groups). Differences among groups for the in vitro experiments were determined by the Mann-Whitney U test. In all cases, a probability value of P < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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No differences in the extent of zymosan-induced WBC rolling velocity or adhesion to cremaster postcapillary venules were observed between the two genotypes (Fig. 2a and 2b ), However, AnxA1 null mice displayed a greater degree of cell emigration compared with wild-type control mice (Fig. 2c) . Table 1 shows that no differences in hemodynamic parameters were observed.
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Mouse cremaster microcirculation activated by PAF
Next, to determine whether the inhibitory effect of endogenous AnxA1 was limited to zymosan or could also be seen with another stimulus, the inflammatory response of the cremaster microvascular beds by PAF superfusion was compared with buffer control.
As AnxA1 null mice have never been used to investigate potential changes in the microcirculation, initially, we monitored the effects of superfusion with control buffer. Superfusion of the preparations with buffer provoked, over time, minimal alterations in cell adhesion (Fig. 3a ) and emigration (Fig. 3b) . Similarly, no changes in cell rolling (velocity values being in the range of 50–80 µm/s over the time course analyzed, with no difference between genotype) were detected up to 90 min. It is important that AnxA1 null mice displayed an identical response with respect to all these cellular parameters, and this apparent lack of "physiological role" for AnxA1 in these settings was also evident from the analysis of the hemodynamic parameters (Table 2 ).
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40% on net values) at 60 and 75 min superfusion. Analysis of the hemodynamic parameters did not show significant differences between the genotypes (not shown), and values were essentially in line with those reported in Table 2
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Effect of AnxA1 gene deletion on PMN functional responses
These in vivo analyses were then complemented with a series of in vitro experiments to determine if the AnxA1 null PMN exhibited a different degree of activation in response to stimulation. Figure 4
summarizes the data obtained when CD11b expression was monitored by flow cytometry. Addition of PAF to peripheral blood aliquots for 15 min led to a concentration-dependent increase in CD11b expression on the PMN, which was evident from 0.3 µM onward (Fig. 4a)
. It is interesting that CD11b up-regulation was higher in AnxA1 null cells, and significance was observed at 1 µM concentration, and maximal activation was measured at the highest concentration tested of 3 µM PAF. This pattern of PMN activation was also observed when two other stimuli were used. AnxA1 null PMN responded maximally to 3 µM fMLP, whereas a 10-µM concentration was required to produce a maximal response in wild-type PMN (Fig. 4b)
. The higher susceptibility of AnxA1 null PMN to fMLP was also evident at the lowest concentration tested of 1 µM. Finally, in absolute terms, PMA (10 µM) produced the most marked cellular response (Fig. 4c)
, with significant increases in AnxA1 null cells at 3 and 10 µM concentrations.
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Chemotaxis of AnxA1 null PMN
Finally, to link in vitro with in vivo analyses, PMN chemotaxis in a modified Boyden chamber assay was evaluated. Purified blood PMN responded with directed locomotion to PAF or fMLP, when applied at a concentration as low as 0.1 or 1 nM (Fig. 5
). Higher concentrations of either chemoattractant failed to produce a stronger response (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The inhibitory role of AnxA1 on the process of leukocyte trafficking has predominantly been studied using a pharmacological approach. For instance, systemic treatment with AnxA1 or its bioactive peptides dampens PMN recruitment in several vascular beds and in response to several distinct stimuli [15 16 17 , 28 ], with the lung being an exception [29 ]. Less clear from such studies, though, is the role that the endogenous mediator might be playing in regulating the cellular inflammatory response. Passive immunization protocols have revealed a major role for AnxA1 in promoting the resolution phase of inflammation, such that prolonged PMN recruitment and edema formation could be measured in animals receiving neutralizing antisera [30 ]. The same holds true for acute joint inflammation [16 ].
The recent availability of the AnxA1 null mouse provides the optimal tool to address this aspect in a more detailed manner. Initial analyses in these mice indicated specific time-related alterations in sensitivity to inflammatory stimuli. For instance, in the mouse paw edema model, absence of AnxA1 did not modify the early phase of the response (2–4 h), whereas an augmented response was measured at later time-points (e.g., >20% at 24 h) [18 ]. In a second study using the antigen-induced arthritis model, a higher degree of joint inflammation was determined in the AnxA1 null mouse [19 ]. In the present study, we started our analysis from the zymosan peritonitis model, revealing a higher degree of PMN recruitment in AnxA1-deficient mice. This was determined by light microscopy and flow cytometry, monitoring Gr1+ cells. Associated with higher Gr1+ cells, a lower content of F4/80+ cells in the AnxA1 mice was measured at the 4-h time-point. In this model of peritonitis, rapid disappearance of macrophages occurs as early as 1–2 h [31 ], with a partial reappearance at 4 h post-zymosan injection. This "macrophage disappearance response" is possibly linked to higher cell adhesion to serosal mucosæ as well as to rapid mobilization to mesenteric lymph nodes [32 , 33 ] and is indicative of a prolonged activation status. These in vivo results complement previous data describing an early defect in phagocytosis and altered profiles of activation of AnxA1 null macrophages [21 ].
To determine whether the augmented PMN trafficking in response to zymosan was associated to a tonic effect of endogenous AnxA1 on the early events of the leukocyte-endothelium interaction process, we turned to intravital microscopy, monitoring the zymosan response in the cremaster microcirculation [26 ]. Intrascrotal injection of zymosan provoked a marked degree of activation within the microcirculation that was selectively altered by AnxA1 gene deficiency: no changes in the extent of cell rolling and adhesion could be measured, whereas there was a much higher number of emigrated leukocytes. It is conceivable that this augmented response in AnxA1-deficient mice could be related to multiple effects including abnormal activation of resident cells (e.g., macrophage, discussed above) and of the mechanisms that lead to PMN trafficking; in any case, the selectivity of effect of endogenous AnxA1 on the leukocyte emigration step of the trafficking cascade was unequivocal. Noteworthy, pharmacological studies with AnxA1 and its peptido-mimetics pointed to alteration of postadhesion phenomena as the site of action for the antimigratory properties of these compounds [15 , 17 ].
This aspect was further investigated using a direct PMN stimulant. Superfusion of PAF onto the cremaster produced the expected cascade of leukocyte rolling, adhesion, and emigration events [34 , 35 ], congruent with the accepted model of cell recruitment [36 , 37 ]. Again, endogenous AnxA1 exerted a tonic inhibitory control selectively on WBC emigration across the endothelial wall, demonstrating that its counter-regulatory effect was not restricted to a specific stimulus. In line with these new findings with the AnxA1 null mouse, PAF-induced activation of the hamster cheek-pouch microcirculation revealed a role for endogenous AnxA1 in mediating the antimigratory effect of dexamethasone, with alteration of the fate of the adherent leukocytes, provoking detachment and preventing cell emigration [38 ].
Another interesting point to highlight is the lack of alterations in the AnxA1 null mouse when the cremaster microcirculation was superfused with buffer (up to 90 min). Thus, the AnxA1 counter-regulatory effect is unlikely to have a function in resting conditions, but rather, it becomes operative in pathological settings, such as after zymosan- or PAF-induced activation of the leukocyte trafficking in the microcirculation.
Prompted by the results obtained with PAF and intravital microscopy, the final part of the study focused on the analysis of PMN functions using two in vitro assays of cell activation. The results obtained indicate that AnxA1 deficiency is associated with exaggerated PMN activation. However, this occurs in a discrete and stimulus-independent manner. Using a whole blood assay to minimize cell activation as a result of purification procedures, it was found that AnxA1 null PMN responded more swiftly to fMLP, PAF, or PMA, as measured by up-regulation of CD11b on the plasma membrane. It is of interest, in this context, to recollect that a series of biochemical studies have highlighted a role for this protein and its N-terminal domain in mediating fusion of PMN granules with membrane preparations [7 , 39 ]. It is therefore possible that the PMN cytoplasmic pool of endogenous AnxA1 [10 ] might localize at the fusion areas between specific granules and plasma membrane. It is relevant that activation of specific granules with consequent increased cell-surface CD11b expression has been postulated to "serve" the PMN machinery during subendothelial cell chemotaxis [40 , 41 ]. The specific intracellular process(es) tonically inhibited by AnxA1 are currently unclear, although initial determinations of F-actin formation suggest that the site of action for this anti-inflammatory protein is down-stream of this pivotal process (S. Yona, unpublished data).
Along the same lines are the data obtained in the experiments of PMN chemotaxis, in which two distinct activators were applied. Again, a subtle defect was observed in the absence of AnxA1, with an augmented degree of cell chemotaxis at low-intermediate concentrations of stimulants. This set of data matches well with the in vivo observations made in the mouse cremaster microcirculation and discussed above and confirms that endogenous AnxA1 plays an important inhibitory role in PMN locomotion.
In conclusion, analysis of PMN activation assays in vitro showed alterations in selected processes (i.e., CD11b up-regulation and PMN chemotaxis), likely to impact on those operating in the inflamed microcirculation. The results obtained with this first study support the hypothesis that endogenous AnxA1 plays an important regulatory role for this endogenous mediator on PMN activation in vitro and recruitment in vivo.
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ACKNOWLEDGEMENTS |
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Received April 20, 2005; revised May 23, 2005; accepted June 6, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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4 integrin in the inflammatory response of neutrophils J. Exp. Med. 194,219-226This article has been cited by other articles:
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) were superfused (starting at time 0) with BBS (buffer control; a, b) or 1 nM PAF (c, d). The extent of leukocyte adhesion and emigration was assessed every 15 min over a 90-min time course. Data are mean ± SEM of six mice per group. *, P < 0.05, versus respective wild-type value.
