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1 Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) in Pediatric Patients with Atopic Dermatitis Fatama M.K. Nasr, Eisha A. Ismail *, Ola A. M. Sharaki **, Layla K.Younis *** From the Departments of Pediatrics, Dermatology and Venereology *, Clinical Pathology, ** and Pathology ***, Faculty of Medicine, University of Alexandria, Egypt. Abstract There is increasing evidence that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) play an important role in allergic inflammation. They play an important role in establishing intercellular contacts necessary for immunologic reaction. Objective: The present study was designed to assess serum levels of soluble forms of ICAM-1 and VCAM-1 and skin biopsies were taken to assess their tissue level in atopic dermatitis patients. Methods: Serum levels of soluble ICAM-1 and soluble VCAM-1 were measured by immunoenzymatic assay in 25 patients with atopic dermatitis (AD) and 15 normal subjects as control group. The expression of ICAM-1 and VCAM-1 was studied by immunohistochemical procedure of skin biopsies from lesions. Results: Serum sicam-1 and svcam-1 were significantly (P<0.001) elevated in patients with AD compared to control. ICAM-1 staining was strongly positive in 60% of cases, moderately positive in 16%, and mildly positive in 24%. VCAM-1 staining showed relatively weaker positivity in all cases. Significant positive correlation was found between sicam-1, svcam-1 and IgE as well between sicam-1 and svcam-1. This suggests that, a common mechanism is responsible for increased production of serum IgE, sicam-1 and svcam-1 in AD. Conclusion : (1) Serum sicam-1 and serum svcam-1 are elevated in pediatric patients with AD. (2) Serum levels of sicam-1 and svcam-1 may be useful in monitoring disease activity in AD in childhood especially in treatment trials. Introduction Atopic dermatitis (AD) is an inflammatory skin disorder characterized by erythema, edema, intense pruritus, exudation, crusting and scaling. In the acute stages, intra-epidermal vesiculation (spongiosis) is present. There appears to be a genetically determined predilection. Infants with atopic dermatitis tend subsequently to develop allergic rhinitis and asthma. (1,2) Atopic dermatitis affects 2-8% of children and typically occurs in three stages with fairly distinctive features. The disease most often begins in infancy, usually during the first 2-3 months of life. The onset is sometimes delayed until the second or third year, 60% of patients are affected by 1 year of age and 90% by 5 years of age. (3) Despite many reports that immunologic disturbances contribute to many manifestations. (4,5) the pathogenesis of AD is still unclear. The presence of tissue eosinophilia is a feature of many allergic disorders including AD. The eosinophils may be a critical effector cells in the mechanisms of allergic inflammation. Thus, an increased understanding of basic mechanisms underlying eosinophil recruitment may have important therapeutic consequences. (6,7) There is increasing evidence that intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) play an important role in allergic inflammation. (8,9) They play an important role in establishing intercellular contacts necessary for immunologic reaction. Intercellular adhesion molecule I (ICAM-1) is a cytokine inducible adhesion molecule expressed on cells of multiple lineage including leukocytes, endothelial cells and keratinocytes. (10) It is involved in granulocyte extravasation, lymphocyte mediated cytotoxicity and in the development of specific immunologic responses requiring cell-cell interaction. The assessment of inflammatory markers in serum samples may be useful in monitoring the disease activity of AD, (11-13) especially in children, the assessment of disease activity may be complicated by controversial informations, thus objective parameters, Alexandria Journal of Pediatrics; Volume 12, Number 1; January

2 easily assessable would be helpful in performing clinical trials. The aim of this work was to study serum ICAM-1 and svcam-1 in AD in pediatric age group, as Subjects and Methods This study comprised 25 children and infants with atopic dermatitis (AD). Patients were attendants of Outpatient Clinic of El-Shatby University Children Hospital and the Outpatient Clinic of Skin and Venereal Diseases, Alexandria Main University Hospital. They ranged in age from 0.5 to 12 years. Ethical consent of their parents was obtained. Patients have been selected in such a way to exclude those with parasitic diseases and systemic illness and those having applied local steroids for the preceding two weeks or having used any drugs which could possibly affect their immunological status. All patients were subjected to thorough history taking and careful clinical examination. The diagnosis of AD relied upon (14) : [1]- Pruritus and scratching; [2]-Chronically relapsing course; [3]-Typical morphologic varieties of skin lesions; [4]-Typical personal or family history of asthma, allergic rhinitis or AD; and [5]-Duration of more than six weeks (to exclude transient irritant reactions). For assessment of severity of AD, the disease was classified as mild (less than 5% involvement); moderate (between 5 and 30% involvement), and severe (over 30% involvement). (15) According to this classification, 7 patients (28%) presented with mild, 12 patients (48%) with moderate, and 6 patients (24%) with severe forms of AD.Fifteen healthy infants and children were included as controls. Laboratory assessment : Specimen collection and preparation : Blood samples were aspirated form all patients and controls in the morning. EDTA blood was used for eosinophilic count. Serum samples were stored at -20 o C in Ependorf tubes until used for detection of immunoglobulin E (IgE). EDTA plasma was removed rapidly and carefully after centrifugation. Samples were labelled and stored in dispense aliquots in sterile Ependorf tubes at -20 o C until examined for soluble cell adhesion molecules well to study tissue ICAM-1 and VCAM-1 in skin biopsies taken from these patients. Eosinophil count was done by a countingchamber method. (16) IgE was detected in human serum using Enzyme Immunoassay Technique (QUORUM DIAGNOSTICS). (17) Measurement of soluble forms of intercellular adhesion molecule-1 (sicam-1) and vascular cell adhesion molecule-1 (svcam-1) by using Immunoenzymatic assay - Immunotech; A Coulter Company.. (18,19) Principle : The immunoenzymatic assay of sicam-1 or svcam-1 is a sandwich type assay. The first step leads to the capture of sicam-1 or svcam-1 by monoclonal anti-sicam-1 or anti-s VCAM-1 antibody bound to the wells of micortiter plate and the binding of the second monoclonal anti-sicam-1 or antisvcam-1 antibody which is biotinylated, to the solid phase antibody-antigen complex. In the second step, the streptavidin-peroxidase conjugate binds to the biotinylated antibody. After incubation, the wells are washed and the binding of the streptavidin-peroxidase via biotin is followed by the addition of a chromogenic substrate of peroxidase. The intensity of the colour produced is proportional to the sicam-1 or svcam-1 concentration in the sample or standard. Histopathological examination : Skin biopsies were taken from children and infants with atopic dermatitis during relapse phase of the disease. Biopsies were chosen from the most representative skin lesion (except the face). Specimens were rapidly frozen and stored at -70 o C till the time of immunostaining. Immunohistochemical procedure for ICAM-1 and VCAM-1 : Staining was performed using a labelled streptavidin-biotin technique (Universal kit, Diagnostic Products Corporation), using a mouse monoclonal antibody ICAM-1 (CD54) (Novocastra, UK) at 1:800 dilution and a mouse monoclonal antibody VCAM-1 (CD106) (Novocastra, UK) at 1:100 dilution. AEC (3 amino-9-ethyl carbozole) was used as a chromogen and Mayers haematoxylin as a counterstain. Negative controls were used by substituting the primary Alex.J.Pediatr. 12 (1), Jan

3 antibody with PBS. Activated endothelial cells were used as positive internal control. Specific staining pattern was identified by a red membranous reaction. ICAM-1 and VCAM-1 positivity were graded on a scale from zero to three +++, where zero represents negative staining, + represents mild intensity, ++ represents moderate intensity and +++ represents strong staining intensity. Results Among the 25 infants and children with AD, 48% were males and 52% were females. Ages ranged from years (mean 4.97 ± 5.77 years). Twelve patients (48%) had infantile phase of AD, they showed features of mild acute to subacute eczema mainly on the cheeks and lateral aspects of the legs, thirteen patients (52%) showed the childhood type, they were presented with features of subacute eczema mainly in the cubital and poplitial fossa. Positive consanguinity was detected in 40% and positive family history in 64%. Associated bronchial asthma was present in 20% and associated rhinitis in 16%. Disease severity was mild in 28%, moderate in 48% and severe in 24%. There was a statistically significant difference of both eosinophilic count and IgE level between patients and controls (table I). Serum ICAM-1 ( ng/ml) was significantly elevated (P<0.0001) in 25 patients with AD compared to 15 healthy controls ( ng/ml). Also serum VCAM-1 (mean ng/ml) was significantly elevated (P<0.002) in patients compared to healthy controls ( ng/ml) (table I). Correlation coefficient (r) was performed between eosinophilic count, serum IgE, sicam-1 and svcam-1 in both AD patients and controls (figures 1-4 Discussion Atopic dermatitis is a chronic or chronically relapsing, inflammatory skin disorder. Despite many reports that immunologic disturbances contribute to many manifestations, (4,5) the pathogenesis of atopic dermatitis is still unclear. High levels of total serum IgE antibodies, as frequently seen in patients with atopic dermatitis, appear to be a result of lymphocyte activation and of an imbalance of the interleukin-4/interferon-gamma Statistical analysis : The collected data were fed to a Computer Program (SPSS/Win) version 6. Analysis and tabulation of data were conducted using the following tests of significance: [1]-Student's "t" test; [2]-ANOVA (F test); [3]-Chi square (X 2 ); [4]-Correlation coefficient (r); and [5]-Spearman correlation coefficient (r sp ). A 5% level of significance was chosen. and tables II, III, IV). There was statistically significant positive correlation between sicam-1 and serum IgE, svcam-1 and serum IgE and between sicam-1 and svcam-1. Also, a significant positive correlation was noted between duration of the disease and svcam-1. IgE, sicam-1 and svcam-1 correlates with disease severity. Histology: H&E stained sections showed mild papillary dermal edema, perivascular lymphocytic infiltrates, with varying number of admixed eosinophils particularly in the upper dermis. Acanthosis and elongation of rete ridges were noted. Also epidermal atrophy was noted in some cases. Immunostaining: ICAM-1 staining was strongly positive (+++) in 15 cases out of 25 (60%) (figures 5, 6) moderately positive (++) in 4 cases (16%) (figures 7, 8), mildly positive (+) in 6 cases (24%). Membranous positivity was noted in dermal lymphocytes and macrophages, endothelial cells and fibroblasts. VCAM-1 staining showed relatively weaker positivity in all studied cases. Moderate staining was noted in 8 cases (32%), mild staining in 11 cases (44%), negative staining in 6 cases 24%. Positivity was seen in endothelial cells and basal epidermal cells (figure 9) ratio (20,21). ICAM-1 is involved in granulocyte extravasation and lymphocyte-mediated cytotoxicity, in the development of specific immune responses requiring cell-to-cell interaction, (22-24) and it has been assumed to be a marker of the early inflammatory reaction. (25) VCAM-1 binds to antigen on lymphocytes and is considered to be important for their migration into tissues and for initiation of chronic inflammation. (26) 171 Alex. J. Pediatr. 12 (1), Jan 1998

4 In infants and children, the assessment of disease activity may be complicated by controversial information; thus easily assessable, objective parameters,, would be helpful in performing clinical trials. Several investigators have demonstrated that the assessment of inflammatory markers in serum samples may be useful in monitoring the disease activity of AD. (11-13) Alex.J.Pediatr. 12 (1), Jan

5 Table I: Comparison between Age, Eosinophilic count, serum IgE, sica M and svcam of patients and controls Patients (n = 25) Controls (n = 15) t test Age (in years) Range Mean± S.D 4.97 ± ± 3.50 Eosinophil count (x10 9 /L) Range * Mean± S.D 0.94± ± 0.27 Serum IgE (IU/ml) Range ** Mean±S.D ± ± sicam-1 (ng/ml) Range ** Mean±S.D ± ± svcam-1 (ng/ml) Range ** Mean±S.D ± ± Significant * p<0.05, ** p<0.01 Table II : Relation between serum IgE, sicam-1 and svcam-1 with other associated atopic diseases AD only (n=16) AD with BA (n=5) AD with rhinitis (n=4) F test Serum IgE (IU/ml) sicam-1 (ng/,l) svcam-1 (ng/ml) P > 0.05 (no significant difference) Table III: Relation between serum IgE, sicam-1 and svcan-1 with disease severity Mild (n=7) Moderate (n=12) Severe (n=6) F test Serum IgE (IU/ml) sicam-1 (ng/,l) svcam-1 (ng/ml) Significant, P < Table IV: Spearman correlation coeffecient (rsp) between disease severity, ICAM-1 and VCAM-1 expressions in relation to other parameters Disease severity ICAM-1 expression VCAM-1 expression Age in years Duration of disease E.count (x10 9 /L) Serum IgE (IU/ml) sicam-1 (ng/ml) svcam-1 (ng/ml) Disease ** 0.708** 0.783** severity ICAM-1 expression 0.734** VCAM-1 expression * Alex. J. Pediatr. 12 (1), Jan 1998

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7 175 Alex. J. Pediatr. 12 (1), Jan 1998

8 Figure 5 : A case of atopic dermatitis showing strong ICAM-1 immunopositivity in dermal papillae (Strep ABC x 100). Figure 6 : High power view of previous case showing membranous staining of dermal Lymphocytes and macrophages (Strep ABC x 400). Alex.J.Pediatr. 12 (1), Jan

9 Figure 7 : Note dermal oedema, ICAM-1 moderate staining in activated endothelial cells, fibroblasts and perivascular lymphocytes (Strep ABC x 100). Figure 8: High power view of previous slides showed ICAM-1 positivity of perivascular lymphocytes and endothelial cells (x 400). 177 Alex. J. Pediatr. 12 (1), Jan 1998

10 Figure 9 : VCAM-1 moderate positivity in dermal endothelial cells and basal epidermal cells (Strep ABC x 400). The results of this study demonstrated increased serum levels of sicam-1 and svcam-1 in patients with atopic dermatitis compared with healthy controls. Also we demonstrated high levels of total serum IgE and eosinophilic count compared with healthy control. Positive correlations were noted between sicam-1 and serum IgE, svcam-1 and serum IgE and between sicam-1 and svcam-1. On the other hand, no similar correlations were found in control subjects. This suggests that a common mechanism is responsible for increased production of serum IgE, sicam-1 and svcam-1 in atopic dermatitis. The expression of a series of adhesion molecules in snap-frozen skin biopsies of patients with AD, showed a significant increase of VCAM-1 and ICAM-1 expression in skin lesions. ICAM-1 showed stronger immunostaining intensity in all patients compared to VCAM-1. This could be explained by the fact that most of our cases were in subacute phase. In chronic lesions, significant reduction of VCAM-1 expression was noted. (27) Thus the levels of ICAM-1 and VCAM are correlated to the duration of the disease. The increase in tissue ICAM-1 and VCAM-1 may be caused by the release of cytokines from endogenous skin. This hypothesis was supported by Jung et al (1996), (28) who found that skin organ culture without exogenous cytokines up-regulates all adhesion molecules in healthy appearing skin of AD,but not in normal skin. Masinovsky et al (1990), (29) reported that mast cells residing in uninvolved atopic skin have been found to contain significant amount of IL-4. Mast cells may be a cellular source for cytokines which induce the high levels of ICAM-1 and VCAM-1 observed in the skin biopsies taken from patients with AD. It can be concluded that resident cells in atopic skin constitutively release cytokines which up-regulate certain adhesion molecules leading to an increased susceptibility to inflammatory reactions. This work clarified that the determination of sicam-1 and svcam-1 could be, like eosinophil count and others, a useful marker for monitoring AD activity. Also changes in sicam-1 levels may be a reflection of the leukocyte endothelial cell interaction, which is crucial for leukocyte infiltration into inflammatory skin lesions. In conclusion, serum levels of sicam-1 and svcam-1 may both be reliable and useful markers of disease activity, especially in children with AD. In addition, the assessment of inflammatory markers may be used to monitor drug efficacy in clinical trials. Alex.J.Pediatr. 12 (1), Jan

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12 29. Masinovsky B, Urdal D, Gallatin WM. IL-4 acts synergistically with IL-1 to promote lymphocyte adhesion to microvascular endothelium by induction of vascular cell adhesion molecule-1. J Immunol 1990; 145(9): Alex.J.Pediatr. 12 (1), Jan