Circulating intercellular adhesion molecules in blood and bronchoalveolar lavage in Behçet's disease

The aim of this study was to evaluate circulating intercellular adhesion molecule-1 (cICAM-1) in serum and in bronchoalveolar lavage (BAL), as a marker for the inflammatory process in patients with active Behçet's disease (BD). Circulating ICAM-1 was tested by an enzyme linked immuno-sorbent assay in serum and in BAL of patients with BD. These values were compared to those of patients with tuberculosis and to healthy controls. Increased levels of circulating ICAM-1 were found in serum from patients with active BD compared to healthy controls (p < 0.01). Similar levels of serum cICAM-1 were found in BD and tuberculosis. Additionally, both BD and tuberculosis patients exhibited high levels of cICAM-1 in BAL fluid, suggesting that this increase may be a result of the immune system activation in inflammatory sites. Circulating ICAM-1 seemed to have a good discriminative power in identifying active BD, being elevated in all active stages (p < 0.01) compared to remission BD stage. No differences were found in active BD patients depending upon the clinical manifestations. These results suggest that cICAM-1 may be involved in leucocyte adhesion and migration into the vessel wall of the lung. Circulating forms are derived from molecules expressed on the surface of activated cells, as a result of an inflammatory process.

Introduction leukin-8 (IL-8) and interferon-gamma (IFN-/), act as leukocyte chemotactic factors, enhance Behget's disease (BD) is now recognized as a adhesion molecule expression and mediate systemic disease with the vascular complications endothelial cell damage, through increased proof aneurysm formation and venous thromboduction of oxygen free radicals. 7' Our previous sis. ' 2 The underlying pathological process is a studies showed that integrins were increased in multifocal vasculitis involving veins, capillaries sera, in cerebrospinal fluid and in bronchoand arteries. 3'4 Involvement of the pulmonary alveolar lavage cells from BD patients. 9'1 Among vascular tree in BD has been extensively studied several adhesion molecules that mediate by Erkan and Cavdar 4 who evaluated its freadhesive interactions, intercellular adhesion quency at 5-10%. The pathological process molecule-1 (ICAM-1), and its ligand are known affecting arteries in BD was considered a result to play a pivotal role in both the migration of of immune complex deposition in small vessels activated lymphocytes across endothelium and leading to complement fixation and poly-basement membranes and their adherence to morphonuclear cell activation, resulting in target tissues where putative antigens are degeneration and occlusion of visa vasorum, located. In addition to membrane-bound ICAM-More complex cellular pathways are actually 1, a circulating, functionally active form of this considered as the infiltrating cells invading molecule has been described, and is denoted inflammatory sites are mainly CD4 + lympho-clCAM-1. cytes, macrophages and B cells, present in the In the present study we searched for elevated vascular adventia and media. 5'6 These inflamma-levels of clCAM-,1 in sera and in bronchoalveolar tow cells together with endothelial cells produce lavage (BAL) from patients with active BD. Two cytokines, 6 some of which, such as interleukin-1 groups were studied, tuberculous patients and (IL-1), turnout necrosis factor (TNF-a), inter-healthy controls. Materials and Methods Immunoassays: Circulating ICAM-1 levels were measured by a commercially available ELISA Patients: Forty patients with BD were charactersystem (Bender & Co., Vienna). This kit uses a ized according to the criteria of the International horseradish peroxidase conjugated monoclonal Study Group of Behget's Disease. Twenty-eight anti-human clCAM-1 antibody. The company's were in active stage, and had recurrent oral and suggested procedures were followed without genital ulcerations and uveitis associated with modification, and the plates were read at a waveone or more other clinical manifestations as length of 450nm in an ELISA reader. Circulating shown in Table 1. These patients were not ICAM-1 concentrations were determined by corntreated. Eleven patients with active BD were susparing the mean absorption of duplicate samples pected of having pulmonary manifestations and with that of a standard curve. Forty healthy underwent a bronchofibroscopy with bronchodonors (aged 45 + 9 years) served as controls to alveolar lavage. Macroscopic lung involvement establish normal values of clCAM-1. was confirmed in seven cases (chronic cough associated to interstitial shadows on the chest Peripheral blood samples: Peripheral venous roentgenogram or pulmonary aneurysms). As blood was taken from all patients and from all control subjects, ten patients (male, mean age control subjects. Serum was separated from 45 years, range 25-49 years) with no evidence clotted whole blood after centrifugation at of interstitial lung disease were studied. They 400 x g for 10 min at room temperature and were undergoing routine bronchoscopy for sus-stored at -70C before measuring circulating pected bronchial carcinoma. In all of them, the adhesion molecules. lung lavage was roentgenographically normal, and BAL cytology showed a normal differential Bronchoalveolar lavage: The chosen lobe was count. Another seven patients (male, mean age lavaged with 50ml aliquots of sterile 0.9% 50 years, range 37-56 years)with confirmed sodium chloride, as reported recendy. 2 The pulmonary tuberculosis were also studied, lavage fluid was gently aspirated after each Informed consent was obtained from all subjects aliquot and collected into a sterile, siliconized for the study, glass botde and maintained at 4C. The lavage bMeningoencephalocele, seizures, cranial nerve palsy, quadriparesis.
Cpulmonary interstitial shadows (aneurysm on the right main branch or on the upper lobe pulmonary artery).
Adhesion molecules in Behet's disease fluid was filtered through coarse gauze and centrifuged at 350 x g at 4C. The supernatants were concentrated by evaporation and reconstituted in I ml normal saline and studied by ELISA for clCAM-1.
Statistical analysis: All results are expressed as mean _+ standard deviation (S.D.). Only p < 0.01 degree of significance was accepted. The Mann-Whitney U-test was used to compare the levels of circulating adhesion molecules between the different groups.
Patients with BD were studied according to their clinical stage. Patients with active BD had high levels of circulating clCAM-1 (694 _ _ 37 ng/ ml), when compared to BD patients in remission stage (387-t-69ng/ml) ( Table 2). Patients with active BD were compared for circulating adhesion molecules, according to their clinical manifestations. No differences were found between patients with pulmonary or with extrapulmonary manifestations ( Table 2). BAL fluid level of cICAM-I: BAL fluid from ten BD patients, seven tuberculous patients and ten healthy controls was studied for circulating adhesion molecules (Fig. 2). BAL fluid from healthy controls showed limit levels of clCAM-1 (105 -t-28ng/ml). BD patients with pulmonary manifestations exhibited high levels of clCAM-1 (635 +__ 47 ng/ml). Levels in BAL fluid from patients with tuberculosis (734 _+ 96 ng/ml) were increased to levels similar to those in BD patients.

Discussion
Circulating ICAM-1 levels in serum and in BAL fluid from patients with active BD and having 8()().. pulmonary manifestations were increased at the same level as in tuberculous patients, compared to healthy controls. The pathological importance of circulating adhesion molecules has not yet been fully delineated. The release of ICAM-1 from the surface may be a mechanism to control cell adhesion. Circulating ICAM-1 may be a byproduct of alteration in cell adhesion molecule expression and cell adhesion, characteristic of inflammation. BD in the active stage was characterized by an increase of clCAM-1 compared to BD in the remission phase. The elevated serum concentration of 1CAM-1 in active BD suggests that it may be a useful marker of disease activity.
Thus, circulating ICAM-1 was raised in all active BD patients, independently of the lesion sites. This increase was specific to the activated stage, reflecting immune system activation. The most important point was the increased clCAM-1 in BAL fluid from BD patients. A similar increase was previously reported in pulmonary tuberculosis 13 and idiopathic pulmonary fibrosis, 3 that are characterized by extensive inflammation. In BD, lymphocytes are responsible for the recruitment of inflammatory cells to the lung, 14 by releasing a wide array of cytokines. Patients with active BD are characterized by an increase of INF-3,, 15  production. These cytokines can up-regulate adhesion molecule expression on some endothelial cells. 7 The presence of circulating adhesion molecules in BAL from BD patients provides a possible mechanism by which these inflammatory leukocytes are attracted to the lesion site, leading to development of the disease. The main features of lung involvement in BD consist of vasculitis forming multifocal aneurysm and thrombosis of the pulmonary artery.  Histological examination of the aneurysm wall reveals an inflammatory exudate containing lymphocytes and polymorphonuclear leukocytes invading the adventia of the artery. 17 ICAM-1, the LFA-1 receptor of which is expres- 21 sed on all leukocytes, probably mediates the adhesion of neutrophils, monocytes and lymphocytes to vascular endothelium. LFA-1 was found to be increased in BAL lymphocytes from active BD patients, 9 facilitating their transendothelial migration. Atherosclerotic aortic aneurysms and plaques 2-23 are also associated with elevated clCAM-1. The detection of cICAM-1 may serve as a useful diagnostic tool in diseases where leukocyte endothelial cell interactions play an important role.