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Animal Model |
From the Department of Pathology and Animal Productions, Veterinary School, Bellaterra, Barcelona, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Systemic murine listeriosis is characterized by the rapid influx of leukocytes, especially neutrophils but also macrophages, into the site of initial bacterial replication, especially the liver and spleen. These cells have been shown to be essential for the early defense against the infection.7,8 Leukocyte recruitment to inflammatory sites consists of a complex series of interactions mediated by cell adhesion molecules expressed on the surface of inflammatory and endothelial cells. The initial step in the adhesion cascade is the tethering and rolling of leukocytes along the endothelium, which is mediated by the interaction of members of the selectin family and their carbohydrate ligands.9-11 E- and P-selectin, expressed on the endothelial cell surface, have been shown to support the initial rolling phase of neutrophils and monocytes in vitro and in vivo. Firm adhesion to endothelium and subsequent emigration through the vessel wall is dependent on leukocyte ß1 and ß2 integrin activation and their interaction with members of the immunoglobulin-like superfamily on the endothelium, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1).9,11
The use of the subcutaneous route of infection has been shown as a suitable model of systemic murine listeriosis, and can also reproduce central nervous system (CNS) lesions similar to those of listeria meningitis in humans and other species. The main feature of these lesions is the recruitment of inflammatory cells, especially macrophages and neutrophils, to the subarachnoid and ventricular space causing meningitis and choroiditis.6 Despite of being extensively used as an experimental model for the study of the pathogenesis of and host defense against intracellular bacteria, the expression and contribution of endothelial cell adhesion molecules to the pathogenesis of Listeria monocytogenes during experimental murine listeriosis have not been characterized yet. Therefore, the aim of this study was to detect by immunohistochemistry the expression of E-selectin, P-selectin, and ICAM-1, especially in the liver, lymphoid organs and CNS of mice during the course of experimental murine listeriosis using the subcutaneous route of infection, and to determine whether correlation exists between the expression of these adhesion molecules and the inflammatory infiltrate.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Forty-eight female 25-g SPF CD1 mice (Interfauna, Spain) were inoculated subcutaneously with 5 x 108 colony-forming units of viable L. monocytogenes in a 0.2 ml of solution in the lumbar zone. At various time intervals (days 1, 2, 3, 4, 5, 8, 11 after infection) (p.i.) groups of mice were killed by anesthetic (halothane) overdose. Immediately, blood collected from the cava vein was placed in heparin-coated microtubes (Becton Dickinson, Franklin Lakes, NJ) and 0.5 ml were directly plated on brain-heart infusion agar (BHIA). Samples of liver and spleen were aseptically removed, homogenized in Tris-buffered saline, and plated in 10-fold serial dilutions in BHIA for bacterial count. Plates were incubated for 24 hours at 37°C.
Samples of liver, spleen, lymph nodes, stomach, small and large intestine, pancreas, kidney, adrenal gland, urinary bladder, uterus, ovary, lung, heart, thymus, bone marrow, spinal cord, and brain were taken immediately after killing the animals, fixed for 48 hours in buffered formalin, embedded in paraffin, and processed for histopathology and immunohistochemistry.
Animal experiments were performed under the supervision of the Animal Care Committee of the Universitat Autònoma de Barcelona.
Immunohistochemistry
Primary rat monoclonal antibodies against mouse ICAM-1 (clone KAT-1) (R&D Systems, Abingdon, UK), E-selectin (clone 10E9C) (Pharmingen, San Diego, CA), neutrophils (clone 7/9) (Immunokontact, Bioggio, Switzerland) and polyclonal rabbit against mouse P-selectin (Pharmingen) were used. Immunohistochemistry to detect ICAM-1 and E-selectin was performed as previously described.12 To detect P-selectin and neutrophils in formalin-fixed and paraffin-embedded tissues, sections were placed in 0.01 mol/L citrate buffer (pH 6) and heated for 10 minutes (P-selectin) or 5 minutes (neutrophils) in a microwave oven (Moulinex FM A735A, 850 W) for antigen retrieval. After blocking nonspecific binding, sections were incubated with the primary antibody diluted 1:250 (P-selectin) and 1:800 (neutrophils) in 0.05 mol/L Tris-buffered saline, pH 7.6, at 4°C overnight. A biotinylated goat anti-rabbit IgG and a biotinylated goat anti-rat IgG (Dako, Glostrup, Denmark) were used as a secondary antibodies diluted in Tris-buffered saline, pH 7.6, at 1:400 and 1:200, respectively. Reaction was developed with the avidin-biotin horseradish peroxidase complex using as chromogen 0.05% solution of 3,3'-diamenobenzidine (Sigma Chemical Co., St. Louis, MO) with 0.03% H2O2 in 0.1 mol/L imidazole buffer (pH 7.1). L. monocytogenes was immunohistochemically detected by a modified technique from Domingo et al13 using the avidin-biotin complex system (Dako). Sections incubated with isotype-matched antibodies served as negative controls in each technique.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Neither symptoms nor deaths were seen until day 4 p.i., with
the exception of one mouse that died on day 3 p.i. On day 4
p.i. many animals rested immobilized and showed hair bristling. From
day 5 p.i. to the end of the experiment, most animals showed
neurological signs such as ataxia and tremors, and three of them,
circling. During this period two animals died and many had to be
euthanized due to severe symptomatology. Results of bacterial counts in
the liver, spleen, and blood are shown in Figure 1
. Listeria was detected from
day 1 p.i. through the end of the experiment. The maximum
bacterial count in the liver (107
colony-forming units/g)
and the spleen (108
colony-forming units/g) occurred
between days 3 and 5 p.i., after which they gradually decreased
and became undetectable on day 11 p.i. Listeria was
detected in blood at low levels (10/ml) during the first two days but
reached levels of 103/ml on day 3 p.i. that lasted
until day 5 p.i., then declined until there were undetectable
levels on day 11 p.i. This period of bacteremia was coincident
with the period of maximal bacterial replication in the spleen and
liver.
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Control Animals
As previously reported12,14 no E-selectin expression was immunohistochemically detected in endothelial cells in any of the studied organs from control animals. Although P-selectin is preformed and stored in Weibel-Palade bodies of endothelial cells, platelets, and megakaryocytes of unstimulated animals,15 P-selectin was only detected in megakaryocytes and circulating platelets of control animals. ICAM-1 was constitutively expressed in all of the studied organs. In the liver, ICAM-1 was faintly expressed on sinusoidal endothelial cells, especially around the centrilobular vein and also in some branches of the portal vein and hepatic artery. In lymphoid organs, ICAM-1 expression was detected in some splenic red pulp sinusoidal endothelial cells and in the center of lymphoid follicles, as well as in the medullary and subcapsular sinuses of lymph nodes. In the CNS, ICAM-1 was expressed in subarachnoid venules and capillaries, especially those in the hippocampal sulcus, and with less intensity in periventricular vessels. Choroid epithelial cells also expressed ICAM-1 in their apical membrane. ICAM-1 was also detected in venules and capillaries of the rest of organs, as well as in the endocardium and pulmonary alveolar epithelial cells.
Infected Animals
Typical lesions of septicemic listeriosis affecting the liver, spleen, and lymph nodes1,3-5 began to appear on day 2 p.i. and were present until the end of the experiment. However, up-regulation and de novo expression of some of the adhesion molecules was already evident on day 1 p.i.
After inoculation, progressive ICAM-1 up-regulation in liver sinusoidal
endothelial cells was observed, and reached the highest level of
expression on days 2 to 3 p.i. and returned to basal levels on day
11 p.i. This up-regulation consisted not only of an increment in
the intensity of the reaction but also of the distribution, which was
markedly centrilobular on day 1 p.i. and became generalized during
days 2 to 3 p.i. At this time, small pyogranulomatous foci of
inflammation were randomly distributed throughout the hepatic
parenchyma with the presence of Listeria in the center of
the lesions. Sinusoidal endothelial cells immediately adjacent to these
areas showed a more intense ICAM-1 staining (Figure 2A)
. This overexpression around the
inflamed areas lasted to the end of the experiment, independently of
the inflammatory cell composition, and the expression level in the rest
of the sinusoidal cells. On days 3 to 4 p.i., lesions were
pyogranulomatous and necrotizing with great numbers of
Listeria and were most severe on day 5 p.i. On day
3 p.i., at the same time that sinusoidal expression was returning
to its basal levels, a marked progressive up-regulation was detected in
portal vessels associated with the presence of margination, diapedesis
and periportal infiltration of neutrophils and macrophages. Moreover,
ICAM-1 was de novo expressed in the latero-apical cell
surface of biliary epithelial cells (Figure 2B)
. This expression in the
portal area was maximum on day 5 p.i.; at this time inflammation
and bacterial burden was maximum in this organ. Between days 5 and
8 p.i., pyogranulomatous cholecystitis and the presence of free
and phagocytosed Listeria in bile ducts were common
findings. Expression of P-selectin in the liver could be detected in
centrilobular and portal veins and some portal capillaries on day
1 p.i. and became slightly reduced between days 2 and 3 p.i.
However, on days 5 and 8 p.i. most of the animals had again marked
expression of P-selectin in the centrilobular and portal veins and also
in capillaries located near or within inflamed areas (Figure 2C)
.
Between days 4 and 8 p.i. P-selectin was markedly expressed in the
cytoplasm of circulating megakaryocytes in hepatic sinusoids. From day
8 p.i. to the end of the experiment, lesions became smaller and
populated by a great proportion of macrophages and lymphocytes. On day
11 p.i. inflammation was almost absent, hepatocytes were
vacuolated, and there was extramedullary hematopoietic activity with
numerous megakaryocytes circulating within sinusoids and showing less
P-selectin expression. Endothelial P-selectin expression had
disappeared and ICAM-1 was at its basal levels with the exception of
sinusoidal cells adjacent to small granulomas. E-selectin was only
detected in some capillary and venule endothelial cells located in
inflamed portal areas of the liver of two animals on days 3 and 4
p.i.
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Neither P-selectin nor E-selectin was immunohistochemically detected in splenic endothelial cells from infected animals. Only megakaryocytes and platelets in the red pulp showed cytoplasmic P-selectin expression. On day 1 p.i., there was an up-regulation of the ICAM-1 constitutive expression in the spleen, and some animals also showed positive reaction in some nodular and trabecular arteries. Intralesional macrophages and those within the red pulp also showed ICAM-1 expression in the cell membrane. Endothelial expression in the red pulp was maximal on day 5 p.i., and returned to basal levels on day 11 p.i.
Lymph node lesions consisted of paracortical pyogranulomatous infiltrates associated with bacterial antigen. Although some animals had lesions in the inguinal and lumbar lymph nodes on day 1 p.i., most were present from day 2 p.i. through day 8 p.i. P-selectin and ICAM-1 but not E-selectin were de novo expressed on the endothelium of some veins in the medullary cords and high endothelial venules of some animals with lesions. Circulating macrophages in the medullary sinuses also showed intense ICAM-1 expression in their membrane.
CNS lesions appeared on day 3 p.i. and were present through
experiment. The course of lesions is summarized in Table 1
. As in the liver and spleen, induction
and up-regulation of the studied adhesion molecules were detected soon
after infection. ICAM-1 was already up-regulated on day 1
p.i., especially in subarachnoid venules and capillaries (Figure 2D)
,
and was also expressed in large veins and arterioles. Subarachnoid
vessels also showed de novo P-selectin expression (Figure 2E)
, which was more intense in the vessels of the hippocampal sulcus.
Meningeal connective tissue, especially in this sulcus, also expressed
ICAM-1 (Figure 2D)
. Some parenchymal vessels, and sinusoidal
endothelial cells in the choroid plexus also had faint expression of
P-selectin.
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. Lesions first appeared
on day 3 p.i. and consisted of the presence of few inflammatory
cells, predominantly neutrophils in the subarachnoid space. This
inflammatory infiltrate was always more prominent in the hippocampal
sulcus and hippocampus-associated internal meninges (Figure 2H)
.
Presence of L. monocytogenes was associated with these
lesions. At this time, a marked overexpression of P-selectin and ICAM-1
(Figure 2, G and F)
was seen in meningeal venules and capillaries
adjacent to the lesions, as well as, in the case of ICAM-1, in the
meningeal connective tissue. On day 4 p.i., macrophages expressing
ICAM-1 on the cell membrane were prominent in the meningeal lesions and
some cells were in the ventricular lumen causing ventriculitis. On day
5 p.i. expression of ICAM-1 in meninges was maximal and all of the
animals showed an intense pyogranulomatous meningitis with numerous
macrophages, and most of them also suppurative ventriculitis with
Listeria-laden neutrophils. In most of these animals,
ventriculitis was associated with choroiditis and ependymitis, with
necrosis of choroidal and ependymal cells. Animals with
ventriculitis-choroiditis also showed increased expression of ICAM-1 on
the choroid epithelial cells (Figure 2I)
and, most, de novo
expression on ependymal cells (Figure 2J)
. Listeria-laden
neutrophils were detected adhered to ICAM-1-expressing ependymal cells
(Figure 2J)
. In animals with ventriculitis, expression of ICAM-1 but
not P-selectin, in periventricular vessels, especially venules, was
clearly up-regulated in association with adhesion and diapedesis of
neutrophils and macrophages. Some animals, from days 8 to 11 p.i.,
also showed perivascular accumulation of macrophages and neutrophils in
the rombencephalum, with little Listeria antigen. These
vessels showed intense expression of ICAM-1 while the rest of
parenchymal vessels were faintly positive. P-selectin expression was
not detected in these inflammatory lesions but some meningeal and
parenchymal vessels, apparently unrelated to the infiltrate, had a
slightly positive expression. On day 11 p.i. vascular and
epithelial expression of ICAM-1 had returned to basal levels and only
faint meningeal expression of ICAM-1 was still present. As in other organs, E-selectin expression in the CNS was detected in few animals (4/47) and few vessels. It was expressed in some meningeal venules and capillaries in the subarachnoid space of the hippocampal sulcus. On day 5 p.i., expression in periventricular capillaries and in sinusoidal endothelial cells of the choroid plexus was detected in two animals.
In other organs, consistent lesions were multifocal pyogranulomatous adrenalitis (40%) and myocarditis (37%). These lesions were most severe and affected most animals on day 5 p.i. and between days 5 and 8 p.i., respectively. Between days 5 and 8 p.i. most animals had pyogranulomatous myocarditis involving atria and ventricles and some also showed pyogranulomatous valvular endocarditis. In these organs there was not expression of selectins, and the basal expression of ICAM-1 on the adrenal capillary network and on the endocardium and myocardial capillaries and venules was clearly up-regulated after infection. Venules and capillaries adjacent to inflammatory lesions in the heart had intense ICAM-1 expression.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Thus, the high up-regulation
observed during our experiment suggests that sinusoidal ICAM-1
expression may play a crucial role in the early migration of
neutrophils and macrophages to the liver. Furthermore, ICAM-1 has been
shown to be essential for the neutrophil-dependent injury of
hepatocytes seen during endotoxemia in galactosamine-sensitized
mice.17
After the extensive ICAM-1 sinusoidal expression
that lasted to day 3 p.i., only sinusoidal up-regulation
surrounding inflammatory lesions and portal vessels was detected
through the experiment. This more restricted expression suggests that
the organism may direct and recruit inflammatory cells to those places
where Listeria has been able to survive and multiply. This
expression was maximum on day 5 p.i., when bacterial replication
and inflammation in the liver were most pronounced. Intense periportal
inflammation and the presence of Listeria free and within
inflammatory cells in the lumen of bile ducts could be seen at the same
time that expression of ICAM-1 was induced in biliary epithelial
cells, maybe reflecting a mechanism to reduce both inflammation and
bacterial burden from the liver by bile excretion. It has been shown
that during the period of maximal hepatic bacterial replication in
systemic murine listeriosis, Listeria is eliminated through
the biliary system.18 As in the liver, no expression of any selectin was detected through the experiment in splenic red pulp sinusoidal cells, and only up-regulation of ICAM-1 was demonstrable. The sinusoidal nature of the vascular system in the spleen where blood cells are allowed to have intimate contact with endothelial cells and macrophages in the marginal sinuses of the red pulp could explain, in the same manner that in the liver, the absence of selectin expression and the no necessity of the selectin-mediated rolling in this organ. In lymph nodes even though the immunohistochemical reaction was faint, expression of both P-selectin and ICAM-1 was correlated with leukocyte recruitment to lymph nodes through high endothelial venules.
CNS involvement is a common feature during listeriosis of humans and other animal species.6,19 However, neither the precise mechanism of leukocyte migration across the blood-brain-barrier nor the signals attracting leukocytes to the CNS are completely understood. In this study, and a previous report,6 the occurrence of CNS lesions was a late phenomenon after infection and was coincident with the onset of a period of bacteremia. As previously reported, persistent bacteremia is required for the invasion of the murine CNS by L. monocytogenes during experimental murine listeriosis using the intravenous route of infection.2 In our experiment, and in the same way as in the liver and spleen, induction and up-regulation of P-selectin and ICAM-1 in the CNS occurred on day 1 p.i. However, neither inflammation nor Listeria antigen were evident until day 3 p.i., when animals showed slight pyogranulomatous leptomeningitis associated with Listeria antigen. Interestingly, leptomeningeal inflammation was more evident in the hippocampal sulcus where endothelial P-selectin and ICAM-1 as well as meningeal ICAM-1 were strikingly up-regulated at the time lesions appeared. This indicates that both molecules may play a key role in the recruitment of leukocytes and development of meningitis during experimental murine listeriosis. Supporting these findings, after intracerebral lipopolysaccharide (LPS) inoculation, neutrophil cuffing around ICAM-1-positive vessels in the hippocampal sulcus,20 and P-selectin expression in almost all veins and venules in the leptomeninges were observed.21 Contribution of endothelial selectins to the development of meningitis has been shown in a cytokine-induced meningitis model in mice deficient for P- and E-selectin.22 ICAM-1 has also been reported to be critically involved in the early phase of bacterial meningitis in rats after intracisternal inoculation of pneumococcal cell wall.27 Expression of ICAM-1 in the subarachnoid connective tissue has been previously shown after LPS inoculation of mice and could contribute to leukocyte adhesion and migration along the subarachnoid space after vascular extravasation.24 In our experiment ventriculitis/choroiditis followed leptomeningitis and was not associated with P-selectin and ICAM-1 expression on the choroid sinusoidal endothelial cells but with the expression of ICAM-1 on choroidal and ependymal cells. Furthermore, Listeria was first detected in association with meningeal inflammation whereas choroid plexus did not show any lesion at that time. This suggests that in this model, inflammatory cells first enter the subarachnoid space through meningeal vessels and not through the choroid plexus as it had been previously suggested.6 Similar results regarding the time-course of CNS inflammation has been previously reported after intracerebral infection with L. monocytogenes.25 Although our results suggest the possible route taken by leukocytes to reach the subarachnoid space, the way through which Listeria arrives to the CNS remains unclear. It has been suggested that some neurotropic bacteria gain access to the cerebrospinal fluid (CSF) through the choroid plexus carried by bacteria-laden macrophages.6,23 In our conditions, it seems unlikely that L. monocytogenes enters the CNS through the choroid plexus, suggesting that probably reaches the CSF through meningeal vessels. In fact, early lesions affecting the ventricular system respected the integrity of the choroidal epithelial cells without apparent inflammatory exocytosis through them, and consisted of an accumulation of inflammatory cells together with numerous Listeria in the ventricles. The time course and the histopathological characteristics of the observed lesions indicate that the development of choroiditis could be the consequence of passive accumulation of Listeria free and within phagocytes from the subarachnoid space. Furthermore, inflammatory cells could reach the ventricular space from periventricular vessels, which had marked ICAM-1 expression associated with margination and diapedesis when ventricular inflammation was present. Constitutive expression of ICAM-1 on choroid plexus epithelial cells has been reported to function as a costimulatory molecule in antigen presentation and to maintain the proper scavenger function of epiplexus cells.24,26 Intense inflammation in the CSF could be responsible for the marked up-regulation of ICAM-1 epithelial expression on the choroid plexus as well as the induction on some ependymal cells promoting the adhesion of leukocytes helping them to move along the ventricles to scavenge their surface.
As the CNS, the heart was a commonly affected organ during the late phase of the infection. Endothelial expression of ICAM-1 seems to be involved in the development of myocardial inflammatory infiltrates, which could play an important role on mortality during systemic murine listeriosis.
In summary, strong up-regulation of P-selectin and ICAM-1 occurs during the course of experimental murine listeriosis. This expression is correlated with leukocyte recruitment during murine listeriosis, especially to the liver and CNS. Expression of P-selectin and up-regulation of ICAM-1 in meningeal vessels, especially in those located in the hippocampal sulcus, associated with the initial meningeal inflammation suggest that leukocytes probably reach the CNS through these vessels during experimental murine listeriosis. Additional studies are needed to find out the route taken by L. monocytogenes to enter the CNS.
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Footnotes |
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Supported by the Comisión Interdepartamental de Ciencia y Tecnología (CICYT), AGF93-C02–02.
Accepted for publication June 15, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-granule membrane protein, is also synthesized by vascular endothelial cells and is localized in Weibel-Palade bodies. J Clin Invest 1989, 84:92-99
in vivo. Cell Adhes Commun 1994, 2:7-14[Medline]
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), and blood
(
). Data are expressed
as the mean ± SD (five to eight mice per
time point).