Biology Contributions
Irradiation induces increase of adhesion molecules and accumulation of β2-integrin-expressing cells in humans

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Abstract

Purpose: The purpose of our investigation was to describe the dose- and time-dependent histomorphologic alterations of the irradiated tissue, the composition of the infiltrate, and the expression patterns of various adhesion molecules.

Methods and Materials: We analyzed immunohistochemically alterations in oral mucosa in 13 head and neck cancer patients before radiotherapy and with 30 Gy and 60 Gy. All had oral mucosa irradiation, with a final dose of 60 Gy using conventional fractionation. Snap-frozen specimens were stained using the indirect immunperoxidase technique. Histomorphology was studied in paraffin-embedded sections. In addition, we determined the clinical degree of oral mucositis.

Results: Histomorphologic evaluation showed no vascular damage. Irradiation caused a steep increase of β2-integrin-bearing cells (p < 0.01), whereas the percentage of β1-integrin-positive cells remained at low levels. Additionally we found an increase in the expression of endothelial intercellular adhesion molecule-1 (ICAM-1) (p < 0.01) and E-selectin (p < 0.05), while endothelial vascular cell adhesion molecule-1 (VCAM-1) expression remained at very low levels.

Conclusion: Our findings indicate that in radiation-induced oral mucositis there is no marked vascular damage until the end of radiotherapy. For recruitment of leukocytes, β2 is more involved than β1. Pharmaceuticals that block leukocyte adhesion to E-selectin or ICAM-1 may prevent radiation-mediated inflammation in oral mucosa.

Introduction

One of the most severe early side effects of radiotherapy in head and neck cancer patients is oral mucositis, defined as the reactive inflammatory process of the oropharyngeal mucosa (1). Although there are numerous studies of the clinical symptoms (2) the pathohistological alterations in irradiated oral mucosa are poorly described. Edema and leukocyte infiltration are the predominant histological changes in irradiated tissues. In addition, vascular damage has also been observed in early radiation injury associated with total obliteration of the lumen of small blood vessels (3). Following local single exposure (75 Gy) to ionizing radiation in a rabbit model, margination and occasional sticking of leukocytes in blood vessels were observed. Furthermore migration of polymorphonuclear and mononuclear leukocytes across the endothelium was also seen (4). In more recently published studies, increased leukocyte adhesion was observed following ionizing radiation of 6 Gy in a rat model 5, 6. In agreement with those in vivo observations, neutrophils were found to bind to irradiated endothelial cells in vitro in a time-dependent manner after single exposure of 5 Gy 7, 8. Considering those investigations it is still unclear how fractionated radiotherapy, which is characterized by multiple low-dose irradiation (2 Gy), affects the morphology and the function of the vessels and leukocyte infiltration.

To elucidate these alterations, studies are required which observe tissue changes at different time points during the course of radiotherapy. There are distinct pathways by which these leukocytes can emigrate from the vessels. Studies by intravital microscopy have identified a sequence of adhesive interactions involved in leukocyte emigration from the bloodstream to extravascular sites of inflammation (9). This coordinated sequence of events is mediated by at least three families of adhesion receptors: selectins (E-selectin), integrins leukocyte function-associated antigen 1 (LFA-1), very late activation antigen 4 (VLA-4), Mac-1 (member of the integrin family), and the Ig gene superfamily intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) 10, 11. E-selectin (CD62E), a member of the selectin family, is selectively expressed on activated endothelial cells (12) and mediates leukocyte rolling 13, 14, but is not responsible for cell arrest under conditions of flow 15, 16. Firm cell arrest and finally transmigration are mediated by proteins of the Ig gene superfamily, expressed on endothelium, and their counterstructures on leukocytes (integrins) 10, 11, 17.

ICAM-1 (CD54) is routinely expressed on endothelial cells at low levels (18), but its expression is upregulated by inflammatory stimulation 18, 19. VCAM-1 (CD106) is expressed only on activated endothelium 20, 21. Each CAM has specific ligands, which show distinct distribution patterns on leukocytes. LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), two β2-integrins, are ligands for ICAM-1 and are most important for neutrophil transmigration 22, 23, 24 although there is one in vitro study which recently described a novel CD18-independent migration mechanism for polymorphonuclear leukocytes (25). Monocytes use the β2-pathway too (26). VLA-4, a β1-integrin, is the major ligand structure for VCAM-1 (10, 11, 17) and is present on monocytes and lymphocytes, but not on polymorphonuclear leukocytes 10, 17, 27, 28.

Recently, an in vitro study showed different dose- and time-dependent expression patterns of various adhesion molecules on endothelial cells during ionizing radiation (29). In this study 1 Gy presented a threshold single-exposure dose for the expression of E-selectin whereas the threshold single-exposure dose for ICAM-1 was 5 Gy. E-selectin expression began to increase at 2 h after exposure and returned to baseline at 20 h. It is unclear if endothelial ICAM-1 and E-selectin expression increase during fractionated radiotherapy. An in vitro investigation can only in part reflect the in vivo conditions 30, 31.

Dose- and time-dependent alterations in irradiated oral mucosa have not been evaluated in humans. The aim of our prospective study was to describe the dose- and time-dependent histomorphologic alterations of the tissue, the composition of the infiltrate, and the expression patterns of various adhesion molecules in vivo. Besides the clinical assessment of the irradiated oral mucosa, we studied the endothelial expression of ICAM-1, VCAM-1, and E-selectin by immunohistochemistry. In addition, we determined the total number and the percentage of integrin-bearing cells in the connective tissue.

Section snippets

Patients

Thirteen patients (age 45–71) with squamous cell carcinoma of the head and neck participated in this study. All of them underwent postoperative radiotherapy receiving external irradiation, 2 Gy/day, five times a week to a total dose of 60 Gy. None of those patients received additional systemic medical treatment before or during radiotherapy. A preradiotherapy dental examination and treatment were undertaken to eliminate foci of infection or mechanical irritation. Samples of the nontumorous

Increase of the clinical degree

Comparing the number of patients who developed different mucositis scores, we found an increase of patients with degree 2 and 3 mucositis after 40 Gy. There was no inflammation detectable in any cases at 10 Gy. All patients showed a mucositis score of 2 or 3 at 60 Gy (Fig. 1).

Irradiation induces subepithelial infiltration

No tumor was found in any specimen. The number of infiltrating cells increased during radiotherapy and was highest in cases with ulceration (mucositis score 3). The inflammatory infiltration was dominated by mononuclear

Discussion

Responses of mammalian tissue to ionizing radiation were described as vascular damage (3), inflammation, and transendothelial migration of leukocytes (4). This transmigration results from interactions between receptor-ligand pairs on both the leukocyte and the endothelial cell. Because it is known that E-selectin, ICAM-1, and VCAM-1 play a leading part in inflamed tissue (10, 11), we determined the endothelial expression of these adhesion molecules in irradiated tissue. Considering that not

Acknowledgements

We are very grateful to Dr. A. Bankfalvi for making the microscope available to us and to Ms. K. Fischer, Ms. S. Merfeld, and Ms. K. Focke for patient and accurate technical assistance.

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