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. 2006 Oct;146(1):76–84. doi: 10.1111/j.1365-2249.2006.03155.x

Relevance of neutrophils in the murine model of haemolytic uraemic syndrome: mechanisms involved in Shiga toxin type 2-induced neutrophilia

G C Fernandez *, M F Lopez *, S A Gomez *, M V Ramos *, L V Bentancor *, R J Fernandez-Brando *, V I Landoni *, G I Dran †, R Meiss ‡, M A Isturiz *, M S Palermo *
PMCID: PMC1809733  PMID: 16968401

Abstract

It has been demonstrated that infections due to Shiga toxins (Stx) producing Escherichia coli are the main cause of the haemolytic uraemic syndrome (HUS). However, the contribution of the inflammatory response in the pathogenesis of the disease has also been well established. Neutrophils (PMN) represent a central component of inflammation during infections, and patients with high peripheral PMN counts at presentation have a poor prognosis. The mouse model of HUS, by intravenous injection of pure Stx type 2 (Stx2), reproduces human neutrophilia and allows the study of early events in the course of Stx2-induced pathophysiological mechanisms. The aim of this study was to address the contribution of PMN on Stx2 toxicity in a murine model of HUS, by evaluating the survival and renal damage in mice in which the granulocytic population was depleted. We found that the absence of PMN reduced Stx2-induced lethal effects and renal damage. We also investigated the mechanisms underlying Stx2-induced neutrophilia, studying the influence of Stx2 on myelopoyesis, on the emergence of cells from the bone marrow and on the in vivo migration into tissues. Stx2 administration led to an accelerated release of bone marrow cells, which egress at an earlier stage of maturation, together with an increase in the proliferation of myeloid progenitors. Moreover, Stx2-treated mice exhibited a lower migratory capacity to a local inflammatory site. In conclusion, PMN are essential in the pathogenesis of HUS and neutrophilia is not merely an epiphenomenon, but contributes to Stx2-damaging mechanism by potentiating Stx2 toxicity.

Keywords: depletion, HUS, neutrophilia, PMN, Stx2

Introduction

Haemolytic uraemic syndrome (HUS) is an infrequent but serious complication of infection with Shiga toxin (Stx)-producing Escherichia coli (STEC) [1]. Haemolytic anaemia, thrombocytopenia and acute renal failure are the main features of HUS [2], and this disease occurs most frequently among children younger than 5 years of age, after a diarrhoeal prodrome characterized typically by bloody diarrhoea. Although Stx has been associated strongly with the disease, several authors have reported the importance of the inflammatory response in the development of HUS. In fact, the participation of polymorphonuclear neutrophils (PMN) in the disease has been suggested, as activation and degranulation of PMN have been demonstrated by the presence of high levels of elastase and interleukin (IL)-8 in the serum of patients, as well as by increased PMN adhesive capacity in vitro and subsequent endothelial damage [1,3–5]. Because endothelial dysfunction appears to be an important factor in the sequence of events leading up to the microangiopathic process of HUS, and as PMN possess mechanisms that can mediate tissue injury, it is reasonable to presume that activated PMN may contribute to endothelial damage in HUS.

Leucocytosis is observed frequently upon presentation with STEC-associated HUS and is a positive predictor of a poor outcome [4,6,7], although the underlying mechanisms leading to circulating leucocytosis remain unclear. Leucocytosis responds to several changing processes, including the production and migration of cells from the bone marrow into the blood, the demargination from blood vessel walls and the prevention of migration into tissues [8].

The lack of an animal model which reflects all features of human HUS is possibly related, at least in part, to interspecies differences in the expression of the Stx specific receptor, globotriaosylceramide (Gb3). In spite of the fact that the Gb3 receptor is absent in the glomeruli of mice, the mouse model of HUS by systemic injection of Stx2 reproduces the acute renal lesions, mainly by inducing tubular necrosis. Glomerular alterations are also observed, although they are due probably to systemic alterations such as the widespread thrombosis and the decreased renal flow secondary to haemodynamic imbalance [9,10]. Despite this limitation, the murine model of HUS is a useful tool that allows the investigation of early events in the course of HUS, which have probably already occurred in patients before diagnosis and are therefore impossible to study. Moreover, the mouse model reproduces other characteristic systemic alterations observed in HUS patients, such as platelet activation, thrombocytopenia and neutrophilia [10,11]. In this sense, neutrophilia correlates positively with renal damage in mice, and peripheral PMN from Stx2-treated mice show functional and phenotypic activated parameters [11,12]. However, decisive evidence is lacking demonstrating that the presence of PMN modulates Stx toxicity and that neutrophilia is not an epiphenomenon, but participates directly in the pathogenic mechanism of Stx2.

In this study, we evaluated the toxic effects of Stx2 in mice in which the granulocytic population was depleted using a specific anti-serum, and found that the absence of PMN categorically reduced Stx2-induced lethal effects and decreased renal damage. Moreover, we also investigated the mechanisms underlying Stx2-induced neutrophilia, and found that Stx2 administration induced an increase in the proliferation of myeloid progenitors, an acceleration of leucocyte appearance into the peripheral circulation and a reduced migration into tissue. In this regard, the impaired migratory capacity of PMN could not be attributed to a PMN deficiency, as when Stx2-treated PMN were transferred to non-treated mice the migration into tissues was increased.

Materials and methods

Mice

BALB/c and non-transgenic C57BL/6 mice were bred in the animal facility of the Department of Experimental Medicine, Academia Nacional de Medicina, Buenos Aires. Male mice aged 9–16 weeks and weighing 20–25 g were used throughout the experiments. They were maintained under a 12-h light–dark cycle at 22 ± 2°C and fed with standard diet and water ad libitum. Green fluorescent protein (GFP)-expressing C57BL/6-Tg(ACTbEGFP)1Osb/J hemizygous transgenic mice were obtained originally from Jackson Laboratories (Bar Harbor, Maine, USA) and were kindly provided by Dr Oscar Campetella from Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, Buenos Aires, Argentina. The experiments performed herein were conducted according to the principles set forth in the Guide for the Care and Use of Laboratory Animals (National Institute of Health, 1985).

Stx2 preparation

Dr Sugiyama Junichi kindly provided Stx2 from Denka Seiken CO Ltd (Nigata, Japan). Purity was analysed by the supplier, which showed only one peak in high performance liquid chromatography (HPLC). Stx2 preparation was checked for endotoxin contamination by the Limulus amoebocyte lysate assay, given that 1 IU/ml is equal to 0·1 ng/ml of United States Pharmacopea standard E. coli endotoxin [13]. The Stx2 preparation contained less than 40 pg lipopolysaccharide (LPS)/µg of Shiga toxin protein.

In vivo treatments

Stx2 lethality was evaluated by treating mice with serial dilutions of Stx2 in pyrogen-free saline 0·2% fetal calf serum (FCS), via intravenous (i.v.) injection in the retro-orbital plexus. The dose selected was approximately 1 ng/mouse, which induced a ∼50% mortality between 96 and 120 h after injection. The same batch and dosage of Stx2 was used for all experiments.

Peripheral blood myeloid cell determination

Blood samples were obtained by puncture of the retro-orbital plexus at different times after Stx2 injection and cells were counted in a Neubauer chamber. The percentage of circulating myeloid cells (band and segmented PMN and monocytes) was determined after differential cell counts on May–Grünwald–Giemsa-stained blood smears.

Bone marrow cell collection

The animals were killed with an overdose of ether, and the right femur bone marrow was eluted with 5 ml of sterile saline, washed and the cell count was performed with a Neubauer chamber.

Release of leucocytes into the circulation

The emergence of leucocytes in the blood was measured following an i.v. injection of 50 µCi/kg of [3H]-thymidine (20 Ci/mmol; Perkin Elmer, Boston, MA, USA) as described previously [14]. Saline or Stx2 were co-injected with the isotope. Blood samples were obtained daily for 3 days, erythrocytes were lysed by hypotonic shock, and cells were counted. The radioactivity was determined by liquid scintillation counting; the results were expressed as the total radioactivity per 106 cells [counts per minute (cpm) per 106 cells] and plotted as a function of time after injection of the isotope.

In vitro progenitor cell assay

Bone marrow cells were plated in 0·3% soft agar (Britania Laboratory, Buenos Aires, Argentina) in Iscove’s modified Dulbecco’s medium (IMDM; HyClone, Logan, UT, USA) containing penicillin–streptomycin, 20% FCS, 1% glutamine and 10 ng/ml of recombinant mouse granulocyte–macrophage colony-stimulating factor (GM-CSF; Sigma, St Louis, MO, USA) in six-well plates at 0·5 × 106 cells per well. The plates were incubated at 37°C in 5% CO2 for 7 days. Granulocyte–macrophage colony-forming units (GM-CFU) were scored using an inverted microscope with phase contrast.

Mouse peripheral blood neutrophil isolation

Mice were bled from the retro-orbital plexus with heparin. Blood was diluted 1 : 2 with saline, and peripheral blood PMN cells were obtained by centrifugation on a Ficoll-Hypaque density gradient (δ = 1·090; Ficoll Pharmacia, Uppsala; Hypaque, Wintthrop Products, Buenos Aires, Argentina) and dextran sedimentation, as described previously [15]. Contaminating erythrocytes were removed by hypotonic lysis. After washing, the cells (> 96% PMN on May–Grünwald–Giemsa-stained preparations) were resuspended in saline.

Mouse peritoneal exudate cells

Thioglycollate-elicited PMN were collected at different times after Stx2 administration by peritoneal lavage with pyrogen-free saline 4 h after mice were injected intraperitoneally (i.p.) with 3 ml of sterile fluid thioglycollate 3%, as described previously [16]. Cells were then washed and counted.

Phagocytosis

Zymozan (Zy) particles (2 mg/ml) were conjugated with fluorescein isothiocyanate (FITC) (2 mg/ml) in carbonate-bicarbonate pH 9·5 1 M (Zy-FITC) for 4 h at room temperature. Peritoneal exudated PMN (2 × 105 cells) were incubated with 100 µl Zy-FITC (0·1 mg/ml) for 30 min at 37°C 5% CO2. Immediately afterwards, the cells were treated with trypsin 1% in phosphate-buffered saline (PBS) for 5 min at 37°C to remove particles deposited on the surface of the cells and finally, the cells were fixed with 0·5% paraformaldehyde and measured by flow cytometry.

PMN transfer experiments

PMN of saline and Stx2-treated GFP C57BL/6 mice were isolated 72 h after Stx2 administration and 1 × 106 PMN were injected i.v. to non-transgenic C57BL/6 mice. After 30 min, thioglycollate was injected i.p. and peritoneal exudate cells were obtained as described above. Cells were counted and green fluorescence (FL-1) was determined by flow cytometry.

PMN depletion studies

Neutrophils from control BALB/c mice were obtained as stated above. Adult rabbits were injected i.v. with a suspension of 1 × 107 PMN per dose at days 1, 10 and 20. At day 30, animals were bled by puncture of the ear vein and serum was maintained at −20°C. Rabbits were boosted monthly. In preliminary experiments, we determined that an i.p. injection of a 1 : 2 dilution of 100 µl of the anti-serum induced a ∼75% depletion of mouse peripheral PMN 4 h after injection (PMN n = 1·44 ± 0·23 × 109/l for control and 0·36 ± 0·06 × 109/l for treated mice) with no statistically significant differences in mononuclear cells (MN; MN n = 2·32 ± 0·27 × 109/l for control and 1·94 ± 0·25 × 109/l for treated mice). Different protocols of depletion were performed: (1) the depletion was maintained throughout the experiment by a first injection of the anti-serum 4 h before Stx2 administration and successive injections every 12 h; (2) the depletion was maintained for only the first 24 h after Stx2 injection; or (3) the depletion was induced 48 h after Stx2 administration and maintained until the end of the experiment. To evaluate the effect of Stx2 in the presence of PMN, mice received a non-immune rabbit sera every 12 h instead of the anti-PMN anti-serum.

Urea studies

Blood was obtained by puncture of the retro-orbital plexus. Biochemical determinations of urea in serum were performed in an autoanalyser CCX Spectrum (Abbott Diagnostics System, Buenos Aires, Argentina) following standardized instructions.

Histological studies

Animals from the saline-treated and the different PMN-depleted experimental groups were killed 72 h after Stx2 injection and subjected to necropsy. Both kidneys from each mouse (three per group) were bisected longitudinally and fixed in 10% neutral formalin and processed routinely. Sections of paraffin-embedded tissue were stained with haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) and examined by light microscopy. Glomerular injury was evaluated by the presence of hypercellularity, crescent formation and thrombosis. Tubular injury was evaluated by the presence of alterations in tubular epithelium, basement membrane integrity and necrosis. Vascular interstitial congestion and the presence of PMN within the kidneys were also assessed.

Statistical analysis

All data correspond to the mean ± s.e. of individual mice. The log-rank χ2 test was used to compare survival curves. Statistical differences were determined using the unpaired Student’s t-test, and for multiple group comparisons one-way analysis of variance (anova) was used. P < 0·05 was considered significant.

Results

Stx2 toxicity in the absence of PMN

The relevance of PMN in the toxicity of Stx2 was evaluated in animals in which the neutrophilic population was removed by means of a polyclonal rabbit anti-serum. Mice were treated using different protocols of depletion in order to establish whether PMN were important in the early or late phases of Stx2 effects. Therefore, depletion of PMN was maintained throughout the experiment (Stx2 + throughout depletion group), maintained for only the first 24 h after Stx2 inoculation (Stx2 + early depletion group) or induced from 48 h after Stx2 injection until the end of the experiment (Stx2 + late depletion group). All PMN-depleted groups were compared with a group of mice injected with Stx2 that also received injections of rabbit serum from non-immunized rabbits (Stx2 + non-immune serum). Figure 1a shows that Stx2-induced mortality was significantly reduced when PMN were absent throughout the experiment. This experimental group also showed a decrease in the level of plasmatic urea at 72 h post-Stx2 injection (Fig. 1b), an indicator generally used in the murine model of HUS to follow the course of Stx2-induced renal damage [17]. Injection of non-immune rabbit serum alone was innocuous and urea levels of these mice were similar to normal mice.

Fig. 1.

Fig. 1

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Modulation of Stx2 toxicity in polymorphonuclear neutrophils (PMN)-depleted mice. Depletion of mouse peripheral PMN was achieved by repeated injection of a polyclonal anti-PMN antibody obtained as described in Materials and methods. Shiga toxin type 2 (Stx2) was injected at time 0 together with a non-immune rabbit anti-serum or with an anti-PMN anti-serum. Different protocols of PMN depletion were performed: the depletion was maintained throughout the experiment (Stx2 + throughout depletion), the depletion was only maintained for the first 24 h after Stx2 injection (Stx2 + early depletion), or the depletion was induced 48 h after Stx2 administration and maintained until the end of the experiment (Stx2 + late depletion). Results were expressed as the mean ± s.e. of four independent experiments with six mice per group per experiment (n = 24 mice per group). (a) Survival percentages of the different groups. *P < 0·05 compared to Stx2 + non-immune serum, Stx2 + early depletion and Stx2 + late depletion. (b) Plasmatic urea values were determined at 72 h after Stx2 inoculation. #P < 0·05 compared to non-immune serum and Stx2 + throughout depletion.

To confirm whether the decreased Stx2 toxicity in PMN-depleted mice, assessed as survival rate and urea levels, was conveyed by a reduction in renal damage, we evaluated histological lesions of the different experimental groups (Fig. 2). Histological sections of mice given either saline or a non-immune rabbit serum only were completely normal (data not shown). Figure 2a,b shows that the Stx2 + non-immune serum-treated group showed the typical Stx2-associated focal cortical necrosis involving tubular epithelial swelling and disrupted basement membrane, together with intraluminal deposits of amorphous material. Some glomeruli contained capillary congestion and acidophilic material consistent with thrombosis. No polymorphonuclear infiltrating cells were observed within the renal tissue or glomerular capillaries of Stx2-treated mice. Similar alterations were observed in the Stx2 + early and late depletion groups (data not shown). The Stx2 + throughout depletion group showed only minimal tubular alterations and the architecture of the kidney was conserved (Fig. 2c,d), confirming that Stx2-induced renal damage is attenuated in the absence of PMN.

Fig. 2.

Fig. 2

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Histology of kidneys from polymorphonuclear neutrophils (PMN)-depleted mice treated with Shiga toxin type 2 (Stx2) (haematoxylin and eosin, PAS staining). (a, b) Cortical renal tissue showing a glomerulus (a) and proximal tubules (b) of mice treated with Stx2 + non-immune rabbit anti-serum. This group developed focal tubular necrosis with severe dilation of tubule lumens and extensive loss of epithelial cells. The glomerular capillaries show PAS-positive material, consistent with incipient thrombus (arrows). (c, d) Renal sections of the Stx2 + throughout depletion group showing a glomerulus (c) and proximal tubules (d). Mice within this group showed only minor tubular alterations. The magnification of all figures is × 400.

Study of the mechanisms underlying Stx2-induced leucocytosis

We have observed previously that Stx2-treated mice showed a slow but sustained neutrophilia, which was maximal 72 h after Stx2 administration [11]. In order to study the mechanisms related to this phenomenon, the effects of Stx2 on the emergence of labelled leucocytes into the blood was studied after intravenous co-injection of tritiated thymidine, which is incorporated in dividing cells in the bone marrow. Figure 3 shows that Stx2 induced an increase in the appearance of cells into the circulation from the bone marrow that was evident at 72 h post-Stx2 injection.

Fig. 3.

Fig. 3

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Emergence of leucocytes from the bone marrow after Shiga toxin type 2 (Stx2) treatment. Mice were injected with [3H]-thymidine together with saline (control) or Stx2, and leucocytes from blood samples obtained daily for 3 days were counted. The radioactivity was determined by liquid scintillation counting. The results were expressed as the total radioactivity per 106 cells [counts per minute (cpm) per 106 cells], and plotted as a function of time after injection of the isotope. Each point represents the mean ± s.e. of two independent experiments using three mice in each time-point per experiment. *P < 0·05 compared to control.

As previous cytometric analysis revealed that Stx2 causes an increase in the percentage of peripheral CD11b positive cells, a marker of the myeloid population, with no alterations in other leucocyte populations [11], we performed an in vitro assay to determined whether Stx2 could be inducing the release of cells of the myeloid lineage from the bone marrow by accelerating the proliferation of their progenitors. The number of colony-forming units of the granulocyte–macrophage lineage was increased in mice treated with Stx2 at 24 h post-treatment and this effect persisted at 72 h (Fig. 4).

Fig. 4.

Fig. 4

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Proliferation of myeloid progenitors after Shiga toxin type 2 (Stx2) injection. Bone marrow cells were collected and the number of granulocyte–macrophage-colony-forming units (GM-CFU) was determined as described in Materials and methods. The bars represent the mean ± s.e. of control (n = 9), and Stx2-treated mice at 24 (n = 8) and 72 h (n = 10) after Stx2 administration. *P < 0·05 compared to control.

The contribution of the different myeloid subpopulations to the leucocytosis induced by Stx2 was studied in blood smears of mice treated with Stx2 (Table 1). Stx2 induced a significant increase in the relative and absolute number of band and segmented PMN, whereas the monocytic population was not affected. The kinetics of band and segmented PMN increases were different. Whereas Stx2 induced an early band PMN appearance in the peripheral blood, which was maintained at 72 h after toxin injection, segmented PMN increased gradually.

Table 1.

Characterization of blood myeloid subpopulations after Stx2 administration.

Control Stx2 24 Stx2 72
Band PMN
 Absolute number† 154·4 ± 13·4 289·5 ± 41·0* 295·1 ± 51·1*
 Percentage‡ 4·13 ± 0·35 7·74 ± 1·11* 7·20 ± 1·25*
Segmented PMN
 Absolute number 891·4 ± 65·1 1221 ± 90·9* 2203·0 ± 177·0*
 Percentage 23·83 ± 1·73 32·63 ± 2·43* 53·73 ± 4·31*
Monocytes
 Absolute number 139·1 ± 13·2 145·8 ± 14·3 102·1 ± 59·2
 Percentage 3·71 ± 0·35 3·90 ± 0·38 2·49 ± 0·43
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*

P < 0·05 compared to control,

†

absolute number in cells/mm3

‡

percentage considering the entire leucocyte population as 100%. PMN: polymorphonuclear neutrophils; Stx2: Shiga toxin type 2.

Stx2 effects on the migratory capacity of PMN

As leucocytosis can result from increased production and release from the marrow into the blood, but also from prevention of migration into tissues, we evaluated the in vivo migratory capacity of PMN towards inflammatory stimulus. Mice treated with Stx2 at different time-points (24 and 72 h post-Stx2 administration) were injected with thioglycollate in the peritoneum and the PMN exudate was collected 4 h later. Low numbers of PMN were present in the peritoneum before thioglycollate injection and no significant increase in PMN accumulation in the peritoneum was detected following injection of diluent (PBS) alone. After the thioglycollate administration, Stx2-treated mice exhibited a lower accumulation of peritoneal exudated PMN compared to control animals both at 24 and 72 h, although statistically significant differences were reached only at 72 h post-Stx2 administration (Fig. 5a). However, PMN recovered from peritoneal lavages of Stx2-treated mice were not functionally impaired, as they showed a higher phagocytic activity compared to PMN from control mice (Fig. 5b).

Fig. 5.

Fig. 5

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Migration properties of polymorphonuclear neutrophils (PMN) to an inflammatory site after Shiga toxin type 2 (Stx2) administration. (a) Control (n = 20) and 24 h or 72 h Stx2-treated mice (n = 12) were injected with thioglycollate 3% and peritoneal exudate PMN were collected after 4 h and counted as described in Materials and methods. The results were expressed as the mean ± s.e. of the number of PMN. (b) The phagocytic capacity of peritoneal exudated PMN was measured 72 h after Stx2 administration using fluorescein isothiocyanate (FITC)-labelled Zymozan (Zy-FITC) and results were expressed as the mean ± s.e. of the mean fluorescence intensity (MFI). (c) PMN of saline-treated and 72-h Stx2-treated transgenic green fluorescence protein (GFP) mice were isolated and 1 × 106 cells were injected intravenously to control, non-transgenic, mice (n = 11). After 30 min, thioglycollate was injected intraperitoneally and peritoneal exudated cells were obtained. Cells were counted and green fluorescence (FL-1) was determined by flow cytometry. Results were expressed as the mean ± s.e. of the number of GFP migrating PMN. *P < 0·05 compared to control.

Stx2 could be affecting the intrinsic migratory properties of PMN themselves, or could be impairing the trafficking signals that mediate the attraction of cells towards the inflammatory site. In order to address this issue, we transferred purified saline and 72-h Stx2-treated C57BL/6 transgenic fluorescent PMN to normal C57BL/6 non-transgenic (recipient) animals, and followed the migration of these cells into the peritoneum 4 h after thioglycollate injection. In preliminary experiments, and as described by others, we observed that inoculation of Stx2 caused similar toxicity in BALB/c and C57BL/6 mice, evidenced by mortality rates, increased uraemia and LPS potentiation effects [18]. Moreover, C57BL/6 mice also showed a lower number of peritoneal exudated PMN 72 h after Stx2 administration, as observed in Fig. 5a for BALB/c mice (data not shown). As shown in Fig. 5c, a higher number of fluorescent PMN from Stx2-treated mice migrated to the peritoneal cavity of non-transgenic recipient animals compared to fluorescent PMN from saline-treated animals.

Discussion

One remarkable observation of our study was that the absence of PMN considerably reduced Stx2 toxic effects, assessed as survival rates, urea levels and renal histological examination. Although different authors have investigated the role of PMN in the development of HUS, to our knowledge this is the first time that the importance of PMN in the course of Stx2-induced toxicity has been addressed conclusively. Moreover, protection was accomplished only when depletion was maintained throughout the experiment, and the presence of PMN only in the early or late phases of Stx2 action was sufficient to exacerbate Stx2 toxicity. Activation of PMN is essential for host defence against microbial infections but can also be associated with the pathological side-effects of tissue destruction [19,20]. PMN-mediated mechanisms involved in tissue injury that may contribute to endothelial damage in HUS include the production of toxic reactive oxygen species (ROS), and the proteolytic capacity of enzymes stored in granules [19]. In this sense, as activation of PMN and increased ROS generation have been described after Stx2 administration [11,12], we suggest that these mechanisms coupled to PMN activation are exacerbating Stx2 toxicity in our model.

During an acute inflammatory response, complex events are triggered to bring about the recruitment of PMN to the site of infection. Regulation of the exit of leucocytes from the circulation to peripheral inflammatory sites is well known as the four-step paradigm of rolling, adhesion, diapedesis and chemotaxis. In vivo, the migration of PMN to an inflammatory focus in Stx2-treated mice was found to be decreased. These findings have two implications: one concerning neutrophilia that will be discussed later, and the other related to Stx2 pathophysiology. In this sense, in the presence of Stx2 failure of transmigration of PMN bound to the endothelial surface may be pathological, as adherent and activated PMN may cause vascular occlusion and damage. Although we did not observe adherent or infiltrated PMN within the kidney of Stx2-treated mice to validate this hypothesis, the absence of PMN does not discard the potential damaging role of reactive oxygen species or proteases released upon PMN activation. In agreement with this observation, histological kidney sections of patients with HUS do not reveal a prominent infiltration of PMN. Van Setten et al. found only a minor infiltration of PMN within the renal tissue of patients [21], and although Inward et al. reported an increased number of PMN within the glomeruli of patients they analysed only fatal cases of HUS, and the number of PMN observed in five of the 13 cases analysed was similar to the number observed within the glomeruli of control children [22]. Despite this, the importance of PMN in the course of HUS is unquestionable, as a great deal of evidence supports the relevance of PMN in the pathophysiology of HUS in children [4,6,23–25]. Moreover, as HUS is a systemic disorder, it cannot be excluded that PMN exacerbate systemic Stx2-induced endothelial cell damage, causing a more pronounced generalized thrombotic state that, in turn, can also contribute to renal injury.

The decreased migration of PMN towards an inflammatory stimuli is in apparent disagreement with the results reported by Gomez et al., who found that PMN from Stx2-treated mice showed enhanced migration properties evaluated in vitro in response to a chemotactic gradient of formylpeptides (fMLP) [12]. However, when purified transgenic fluorescent Stx2-treated PMN were transferred to control non-transgenic mice, and the in vivo migration of these cells was induced by i.p. injection of thioglycollate, an even higher number of exudated cells was found compared to fluorescent saline-treated PMN. Altogether, these results allow us to disregard that the decreased in vivo migratory capacity of Stx2-treated PMN is related to an impaired intrinsic chemotactic response. Moreover, the phagocytic capacity of exudated PMN from Stx2-treated mice was found to be increased, indicating an activated rather than an impaired functional state of these PMN. In line with this hypothesis, it has been reported previously that PMN from mice treated with Stx2 showed an increased CD11b expression and adhesion to blood vessels [11]. In some inflammatory conditions, such as endotoxaemia, migratorially depressed yet activated PMN accumulate in the lumen of microvessels [26]. It has been demonstrated that, in vivo, PMN migration into sites of local inflammation is impaired in animals treated systemically with LPS, fMLP or IL-8 [27–29]. Moreover, naive PMN perfused over activated endothelial cells were able to adhere to and penetrate the monolayer, but failed to transmigrate when exposed to exogenous activators, such as fMLP, IL-8 and C5a [30]. Therefore, early systemic exposure to an agent may desensitize the response to another agent or even to the same agent required for transmigration [31–33].

Infection and inflammatory processes are associated invariably with leucocytosis, and many microbial products or components, such as endotoxin (LPS) and fMLP, have been reported to induce leucocytosis [8]. Our results showed that Stx2-induced neutrophilia arises from a sum of different events, including acceleration in the release of bone marrow cells, increased in the proliferation of progenitor myeloid cells and prevention of migration into tissues. As indicated by differential counts of peripheral myeloid subpopulations, Stx2 had an effect on only the neutrophilic population, which egressed not only at a faster rate but also at an earlier stage of maturation. In the bone marrow, immature myeloid cells adhere to the stromal network by means of interactions between oligosaccharides and lectins, and between adhesion molecules. Mobilization is the result of the action of peripheral chemokines and remodelling of the matrix and basement membranes by proteases. Although several cytokines and colony-stimulating factors, i.e. IL-1β, IL-8 and granulocyte–colony-stimulating factor (G-CSF), have been implicated in leucocytosis [8,34–35, ], recent studies have shown that activation of PMN leads to release of proteases which are central in cytokine-induced leucocytosis and stem cell mobilization [36–38]. Whether this mechanism is involved in Stx2-induced neutrophilia needs further investigation.

In conclusion, the results presented in this work shed light on the mechanisms underlying Stx2-induced neutrophilia, and show decisive evidence that demonstrate that neutrophilia is not merely an epiphenomenon, but contributes to the pathophysiology of Stx2 by exacerbating Stx2-induced mortality and renal damage. These results have important clinical implications, as if host factors such as PMN and their products contribute to HUS pathogenesis, it is reasonable to consider the targeted blockade of these factors as a novel therapeutic strategy for STEC treatment.

Acknowledgments

The authors thank Dr Oscar Campetella and María Virginia Tribullati from Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín, Buenos Aires, Argentina, for providing the transgenic GFP mice; and Nora Galassi, Marta Felippo, Héctor Costa and Antonio Morales for their excellent technical assistance. This work was supported by grants from Alberto J. Roemmers, and Agencia Nacional de Promoción Científica y Tecnológica, Argentina.

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