Circulating forms of intercellular adhesion molecule (ICAM)-1 in mice lacking membranous ICAM-1 by Natasja K. van den Engel, Edmund Heidenthal, Antje Vinke, Hubert Kolb, and Stephan Martin Blood Volume 95(4):1350-1355 February 15, 2000 ©2000 by American Society of Hematology

cICAM-1 concentrations in serum of wild-type and ICAM-1 mutant mice cICAM-1 concentrations in serum of wild-type and ICAM-1 mutant mice.Sera from 5 C57BL/6 wild-type and 5 ICAM-1–exon 4 mutant mice were assayed for cICAM-1 by ELISA-1 in duplicate. cICAM-1 concentrations in serum of wild-type and ICAM-1 mutant mice.Sera from 5 C57BL/6 wild-type and 5 ICAM-1–exon 4 mutant mice were assayed for cICAM-1 by ELISA-1 in duplicate. Mean values are indicated by lines. Natasja K. van den Engel et al. Blood 2000;95:1350-1355 ©2000 by American Society of Hematology

Comparison of membrane integrated and circulating ICAM-1 in wild-type and ICAM-1–exon 4 mutant mice.(A) Visualization of the ICAM-1 expression on leukocytes isolated from wild-type and ICAM-1–exon 4 mutant mice. Comparison of membrane integrated and circulating ICAM-1 in wild-type and ICAM-1–exon 4 mutant mice.(A) Visualization of the ICAM-1 expression on leukocytes isolated from wild-type and ICAM-1–exon 4 mutant mice. Isolated spleen cells were stimulated with 100 U/mL IFN-γ and 10 ng/mL LPS for 18 hours to increase ICAM-1 expression. Cells were incubated with FITC-conjugated anti-CD45 (1:7) and PE-conjugated anti-ICAM-1 (1:7). In each measurement PE-fluorescence intensity of 10 000 FITC-positive cells was determined. (B) cICAM-1 production by in vitro cultured spleen cells. Freshly isolated spleen cells (5 × 105 cells/well) were cultured for 3 days in RPMI supplemented with 10% FCS and 0.08% β-mercaptoethanol. Supernatant (150 μL) was taken after 1 hour and at the end of cultivation. cICAM-1 concentrations were determined by ELISA-2 in duplicate. Natasja K. van den Engel et al. Blood 2000;95:1350-1355 ©2000 by American Society of Hematology

RT-PCR analysis of alternatively spliced ICAM-1 RNA isoforms RT-PCR analysis of alternatively spliced ICAM-1 RNA isoforms.RT-PCR was performed on spleen RNA from wild-type and ICAM-1 mutant mice using 5′UTR and 3′UTR primers. RT-PCR analysis of alternatively spliced ICAM-1 RNA isoforms.RT-PCR was performed on spleen RNA from wild-type and ICAM-1 mutant mice using 5′UTR and 3′UTR primers. (A) RT-PCR products resolved on a 1.5% agarose gel. For identification, fragments were transferred to a nitrocellulose membrane, which was hybridized with an exon 2-specific probe. Positive fragments were cloned and sequenced. Indicated on the diagram are short sequences of the exons joining the alternatively spliced sites. (B) Schematic representation of the ICAM-1 isoforms of ICAM-1–exon 4 mutant mice. The isoforms contain 2 or 3 extracellular domains, a transmembrane domain, and a cytoplasmic region. Natasja K. van den Engel et al. Blood 2000;95:1350-1355 ©2000 by American Society of Hematology

Comparison of cICAM-1 in wild-type and mutant C57BL/6 mice by fast protein liquid size exclusion chromatography.Sterile filtered serum (500 μL) from wild-type (+/+), ICAM-1–exon 4 mutant mice (-/-) and a mixture of +/+ and -/-serum were chromatographed on a... Comparison of cICAM-1 in wild-type and mutant C57BL/6 mice by fast protein liquid size exclusion chromatography.Sterile filtered serum (500 μL) from wild-type (+/+), ICAM-1–exon 4 mutant mice (-/-) and a mixture of +/+ and -/-serum were chromatographed on a Superdex-200 column in separate runs. Collected fractions of 0.5 mL were analyzed for the presence of cICAM-1 by ELISA-1. The molecular weight of proteins in the different fractions was estimated from the elution profile of marker proteins of molecular weight in the range of 12 to 200 kd. Natasja K. van den Engel et al. Blood 2000;95:1350-1355 ©2000 by American Society of Hematology

Circulating ICAM-1 concentrations in serum from LPS-treated mice Circulating ICAM-1 concentrations in serum from LPS-treated mice.Mice were given a single dose of 40 mg/kg of LPS intraperitoneally. Circulating ICAM-1 concentrations in serum from LPS-treated mice.Mice were given a single dose of 40 mg/kg of LPS intraperitoneally. Blood samples were taken from the retro-orbital plexus with heparinized capillaries before (0 hour) and at indicated times after LPS injection. At different time points, different groups of animals were used. Serum ICAM-1 concentrations were analyzed by ELISA-2 in duplicate. Results are expressed as the mean (± SEM) ICAM-1 immunoreactivity. Natasja K. van den Engel et al. Blood 2000;95:1350-1355 ©2000 by American Society of Hematology

Overview of the different splice variants encoded by the mRNA detected in wild-type, ICAM-1–exon 4, and ICAM-1–exon 5 mutant mice.Extracellular domains are indicated: +, detected; −, not detected. Overview of the different splice variants encoded by the mRNA detected in wild-type, ICAM-1–exon 4, and ICAM-1–exon 5 mutant mice.Extracellular domains are indicated: +, detected; −, not detected. Natasja K. van den Engel et al. Blood 2000;95:1350-1355 ©2000 by American Society of Hematology