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Intercellular adhesion molecule1
(ICAM1 or CD54) and Eselectin (CD62E) play an important role in leukocyte
adherence to endothelium and extravasation at sites of inflammation (Bevilacqua
et al, 1987; Springer, 1990). Immunohistochemical techniques on frozen sections
have been used to detect these molecules in animal and human tissues (Fries
et al, 1993; Henseleit et al, 1996; Kuzu et al, 1993; Neumann et al, 1996).
Using two commercially available antibodies against mouse ICAM1 and Eselectin
and standard antigen unmasking protocols, we have now detected these molecules
in formalinfixed, paraffinembedded tissues from normal and lipopolysaccharidestimulated
mice. No immunohistochemically detectable constitutive expression of Eselectin
was found in any normal tissue. Ninety minutes after lipopolysaccharide
inoculation, Eselectin expression was detected on endothelial cells in most
evaluated organs, except for liver sinusoidal endothelial cells and vessels
of neural parenchyma. ICAM1 was constitutively expressed, and it was upregulated
in all organs 90 minutes after lipopolysaccharide inoculation. Although
the distribution and patterns of expression of both molecules examined in
this study parallel previous reports in which immunohistochemistry on frozen
sections was used, the present methods have the advantages of improved resolution
and easier handling of tissue samples. The possibility of detecting these
adhesion molecules in routinelyprocessed tissues permits retrospective study
as well as improved morphologic resolution.
After routine processing of tissues for histopathologic examination, a decrease
in antigenicity of these molecules is produced, and the use of antigenretrieval
techniques is required. Tissue sections (5 to 6 µm) on adhesive coated
slides were deparaffinized, rehydrated, and placed for 30 minutes in a 3%
H202;solution in distilled water for endogenous peroxidase inhibition. After
washing in Trisbuffered saline (TBS, pH 7.6), sections were treated for
antigen retrieval. To unmask Eselectin, slides were placed in a 0.1% pronase
E (Sigma Chemical Company, St. Louis, Missouri) solution in distilledwater
at room temperature for 8 minutes. For ICAM1, sectionswere placed in 0.01
M citrate buffer (pH 7.4) and heated for 5minutes (5 rounds of 1 minute
each) in a microwave oven (850W, Moulinex FM A735A; Moulinex Espana, Madrid,
Spain). After washing sections in TBS, endogenous biotin was blocked with
a commercially available blocking kit (Vector Laboratories, Inc., Burlingame,
California). Nonspecific binding was blocked with a 10% goat normal serum
solution in TBS for 1 hour at room temperature. Sections were incubated
with primary antibodies at the working dilutions at 4°C overnight. The
secondary antibody was incubated for 1 hour at room temperature for E-selectin
and at 37°C for ICAM1 detection.
Reaction was developed with the avidinbiotin horseradish peroxidase complex
(Dako, Glostrup, Denmark) using as chromogen a 0.05% solution of 33' diamenobenzidine
(Sigma) with 0.03% H2O2 in 0.1 M imidazole buffer (pH 7.1) for 4 minutes.
Sections were counterstained with Mayer's hematoxylin. Sections incubated
with isotypematched antibodies served as negative controls. Primary rat
monoclonal antibodies against mouse ICAM1 (Clone KAT1) and Eselectin (Clone
10E9.6), which were tested for use on frozen sections, were purchased from
R&D Systems (Abingdon, United Kingdom) and Pharmingen (San Diego, California),
respectively. Working dilutions were 1:100 for ICAM1 and 1:50 for Eselectin,
both of which were diluted in 0.05 M TBS (pH 7.6). The secondary biotinylated
rabbit anti-rat IgG antibody was obtained from Dako and diluted 1:200 in
TBS (pH 7.6).
Animals used in this study were 10 female Swiss mice given an intraperitoneal
injection of bacterial lipopolysaccharide (1 55:B5, Escherichia coli; Sigma
Chemical Company, St. Louis, Missouri) at a single dose of 500 µg
in 0.5 ml of saline. Five animals inoculated with saline were used as controls.
Samples from brain, liver, spleen, kidney, mesenteric lymph node, salivary
gland, lung, heart, skin, small and large intestine, mesentery, and genital
system were placed in neutral buffered formalin for 48 hours, embedded in
paraffin, and processed for immunohistochemistry.
Typical results are shown in Figure 1. E-selectin was predominant on endothelial
cells of venules, arterioles, capillaries, and large diameter vessels. (Fig.
1, a and b), as well asin renal glomeruli from lipopolysaccharidestimulated
mice (Fig.1 c). A faintly positive reaction was distributed along the endocardial
endothelium. In the Iymphoid system, E-selectin expression was reduced to
a few scattered venules in the medullarycords, and it was weak in high endothelial
venules. A few nodular arterioles in the splenic white pulp and very few
red pulpsinusoidal endothelial cells showed a faintly positive reaction.
ICAM1 was expressed on endothelial cells of venules, capillaries, and some
larger veins and arteries, as well as on choroid plexus epithelial cells
(Fig. 1, d to h), basal keratinocytes, and pulmonary alveolar epithelial
cells (Kang et al, 1996; Silber et al, 1 994; Steffen et al, 1 996). It
was also expressed in the center of secondary follicles in the Iymph nodes,
Peyer patches, and spleen, possibly corresponding to high levels of expression
by follicular dendritic cells (Koopman et al, 1991).
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Received April 28, 1997. Affiliations: Department of Pathology and Animal
Productions, Veterinary School, Bellaterra, Barcelona, Spain. This work
was supported by the Commision Interdepartmental de Cienciny Tecuologia
(AGF93C0202). Address reprint requests to: Dr. S L. Ortigosa, Histologia
i Anatomia Patologica, Department de Patologia i Produccions Animals, Facultat
de Vetennaria, UniversitatAutonoma de Barcelona, 08193 Bellaterra, Barcelona,
Spain. Fax: 34 3 5813142. |
