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Technical Briefs |
1 Institut National de la Santé et de la Recherche Médicale (INSERM) U 525, 30 rue Lionnois, 54000 Nancy, France, and Centre de Médecine Préventive, 2 rue du Doyen Jacques Parisot, 54500 Vandoeuvre-lès-Nancy, France
aaddress correspondence to this author at: INSERM Unité 525, 30 rue Lionnois, 54000 Nancy, France; fax 33-03-83-32-13-22, e-mail Sophie.visvikis{at}cmp.u-nancy.fr
Intercellular adhesion molecule-1 (ICAM-1) and E-, P-, and L-selectin are cellular adhesion molecules involved in the recruitment of leukocytes on the activated vessel wall during inflammation (1) and play an important role in the early stages of atherosclerosis and its complications (2). Thus, the measurement of soluble adhesion molecules in serum may have diagnostic relevance in many inflammatory diseases (3). A profile of soluble adhesion molecule concentrations may allow better therapeutic decisions in inflammatory and autoimmune disorders, infection, cancer, and cardiovascular pathologies and may also aid in the prediction of cardiovascular events (4)(5). However, the use of these markers in clinical practice depends critically on knowledge of their reference values.
The purpose of the present study was to establish age- and sex-specific reference intervals for serum concentrations of soluble ICAM-1 and E-, P-, and L-selectin in healthy children (4–17 years) and adults (18–55 years).
Blood samples were taken from healthy individuals (157 boys and 146 girls 4–17 years of age and 245 men and 250 women 18–55 years of age) who were members of the Stanislas cohort (6). Participants were of French origin (Vosges and Meurthe et Moselle); free from serious and/or chronic illnesses, especially cardiovascular, hepatic, or renal diseases; and were not on treatment with lipid-lowering drugs. Volunteers with aspartate aminotransferase, alanine aminotransferase, or 
-glutamyltransferase activities >200 U/L, apolipoprotein E concentrations >200 mg/L, orosomucoid or haptoglobin concentrations >3 g/L, cholesterol or triglyceride concentrations >10 mmol/L, C-reactive protein concentrations >30 mg/L, or glucose concentrations >8 mmol/L were excluded. The study was approved by the local ethics committee of Nancy (France), and each participant gave written informed consent.
Venous blood samples were collected by venipuncture after an overnight fast (7) and centrifuged (1500g for 15 min at 4 °C) within 2 h after collection. Serum samples obtained were immediately frozen at -196 °C in liquid nitrogen until analysis (storage period, 21–39 weeks; mean, 31 weeks).
ICAM-1 and E-, P-, and L-selectin concentrations were measured with commercially available ELISAs (R&D System). The intra- and interassay CVs for the adhesion molecules were as follows: ICAM-1, 6.6% and 8.6%; E-selectin, 9.4% and 15%; P-selectin, 5.8% and 7.0%, and L-selectin, 8.9% and 12%.
Statistical analyses were performed with SAS software, Ver. 8.01 (SAS Institute Inc.). Because the distribution of serum concentrations of ICAM-1 and E-, P-, and L-selectin were skewed, log10 transformations were applied. ANOVA and pairwise comparison procedures with Bonferroni correction were performed to test differences in soluble ICAM-1 and E-, P-, and L-selectin concentrations between age and sex groups. Reference values were estimated using a nonparametric method (5th and 95th percentiles).
Relationships between the length of time of storage at -196 °C and the concentrations of the four adhesion molecules were examined, as recommended by Demerath et al. (8). We found no significant effect of storage length; we therefore did not adjust analyte values for this factor.
The mean (SD), median, and 5th and 95th percentile concentrations of the four adhesion molecule for three groups of children (4–9, 10–14, and 15–17 years) and three groups of adults (18–34, 35–44, and 45–55 years) are presented in Table 1
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From 4 to 17 years, log-transformed ICAM-1 and E-, P-, and L-selectin concentrations significantly decreased in both boys and girls. The P values for the differences in the concentrations of ICAM-1 and E-, P-, and L-selectin across the three age groups were 
0.001, 0.001, 0.05, and 0.001, respectively (ANOVA tests). No significant sex-related differences were detected in children. From 18 to 55 years, L-selectin concentrations decreased with age in both sexes (P 0.001), whereas E-selectin concentrations increased only in women (P 0.05). ICAM-1 and P-selectin concentrations did not vary significantly across the three age groups, independent of gender. ICAM-1, E-selectin, and P-selectin concentrations were significantly higher in men than in women (P 0.05, 0.001 and 0.001, respectively, t-test), whereas no significant sex effect was observed for L-selectin concentrations.
Circulating soluble isoforms of adhesion molecules in blood result either from the release of membrane-bound molecules by proteolytic cleavage of the extracellular portion (ICAM-1 and E-, P-, and L-selectin) or from alternative splicing of the mRNA lacking the transmembrane domain (P-selectin) (3). The concentrations of these soluble isoforms presumably reflect their relative abundance as membrane-bound molecules. The key role played by these molecules in the recruitment of leukocytes by the endothelium suggests that measurement of their circulating concentrations may be useful for monitoring the process of inflammation of the vessel wall. The measurement of adhesion molecule concentrations is thus of clinical interest. The ready availability of commercial ELISAs for determination of soluble adhesion molecules has greatly facilitated research in the clinical significance of their concentrations. Higher concentrations have been reported in various inflammation-related diseases, mainly immune disorders, inflammation, cancer, and cardiovascular disease (3). Such high concentrations of circulating adhesion molecules may be of interest for diagnostic and prognostic purposes, as demonstrated for ICAM-1 and P-selectin, which provide useful information in healthy individuals and in patients with cardiovascular diseases (9). However, reference values for healthy children and adults have not been defined.
In the current study, we measured serum ICAM-1 and E-, P-, and L-selectin concentrations and determined their age and sex-specific reference intervals over a wide age range (4–55 years) in a healthy population of both sexes.
Earlier studies dealing with serum adhesion molecule (ICAM-1 and E-selectin) concentrations in healthy pediatric populations pointed to an age dependency (10)(11)(12). In this study, we observed that mean serum ICAM-1 and E-, P-, and L-selectin concentrations steadily decrease during childhood. ICAM-1 and P-selectin concentrations did not vary with age in adulthood, whereas L-selectin decreased in both sexes and E-selectin increased in women only. Although the decrease in adhesion molecule concentrations with age in children has already been described, its physiologic significance during normal development is unknown. Authors of previous studies in adults observed that ICAM-1, E-selectin, and P-selectin concentrations did not vary with age between 18 and 65 years (3)(5)(8)(10)(13)(14), but the decrease in L-selectin during adulthood has not been described.
ICAM-1, E-selectin, and P-selectin concentrations display a significant sex dependency only in adults, with men having higher concentrations than women. These sex-related differences, as reported previously in healthy adults (8)(14)(15)(16), are probably partly attributable to steroid hormones, especially estrogen. Indeed, healthy postmenopausal women on hormone replacement therapy had lower concentrations of these adhesion molecules than controls not on replacement therapy (7)(17)(18), and in vitro studies have shown negative regulation of adhesion molecule expression by estrogen (19).
In conclusion, we have shown that serum ICAM-1 and E-, P-, and L-selectin concentrations are age-dependent in childhood and sex-dependent in adulthood. Application of age- and sex-adjusted reference intervals appears to be necessary. These findings emphasize the need to use age- (for children) and sex-matched controls (for adults) in all analyses of the possible clinical impact of circulating concentrations of adhesion molecules.
Acknowledgments
We thanks Prof. G. Siest, Dr. C. Sass, and the Centre de Médecine Préventive of Vandoeuvre-lès-Nancy, France, for management of the Stanislas cohort. This work was supported by an IDS grant (INSERM no. 4D00SE/1999). Anne Ponthieux has received a grant from Société Française d’Hypertension Artérielle.
References
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