Immunolocalization of the cell adhesion molecules ICAM-1 & VCAM-1 in pyogenic granuloma.Introduction
Pyogenic granuloma is a relatively common benign vascular lesion of the oral mucosa or skin that is considered to be non-neoplastic in nature . Pyogenic granuloma is now thought to represent exuberant tissue in response to local irritation or trauma . The lesion usually occurs in children and young adults as a solitary glistening red papule or nodule that is prone to bleeding and ulceration . Prominent capillary growth in hyperplastic granulation tissue is histopathologic characterstic of pyogenic granuloma . Some factors such as
inducible nitric ixide synthase (iNOS) ,Vascular Endothelial Growth Factor (VEGF) , basic Fibrblast Growth factor (bFGF)  or connective tissue Growth Factor (ct GF) [(8] are known to be involved in angiogenesis and rapid growth of pyogenic granuloma. Yam et al  proposed that pyogenic granuloma expressed significantly more VEGF and b FGF than healthy gingiva and periodontium. Also, angiostatin (angiogenesis inhibitor) was expressed significantly less in pyogenic granulomas than in healthy gingiva and periodontal inflammation. Cell adhesion molecules play important role in every aspect of human biology from the embryo where they are crucial for tissue and organ development, to the adult where they act as traffic signals to direct the sequential actions of immunesystem cells in wound healing, inflammation, cancer and even AIDS .
Intercellular adhesion molecules (ICAM-1) and Vascular cell adhesion molecules (VCAM-1) belong to Ig superfamily and both are involved in immune reaction . ICAM-1 is widely distributed on leukocytes, endothelial cells, fibroblasts and epithelial cells . The soluble form of ICAM-1 is regarded as a useful parameter in the diagnosis and monitoring of various inflammatory, neoplastic and immune disorders . VCAM-1 is expressed on the surface of mononuclear cells (monocytes, T-cells & eosinophils) [14,15,16]. Its expression on endothelial cells has been shown to increase the adhesiveness and migration of activated mononuclear cell .
In immunological and inflammatory reactions, ICAM-1 and VCAM-1 are surface glycoproteins that promote adhesion and subsequent recruitment of leukocytes . It has been reported that ICAM-1  and VCAM-1  expressed in periodontal diseases. ICAM-1 is induced by pro-inflammatory cytokines such as LL-1[beta], TNF-[alpha], IFN-[gamma] , IL-2, E. Coli lipopolysaccharides and VCAM-1 is induced by IL-1[beta]. Hence ICAM-1 & VCAM-1 play an important role in the pathogenesis and development of periodontal diseases , odontogenic keratocyst & ameloblastoma , however, no study up to our knowledge had investigated the expression and role of ICAM-1 and VCAM-1 in other common oral lesions like pyogenic granuloma.
The aim of this research is to study the expression of ICAM-1 & VCAM-1 in the main cell types of pyogenic granuloma and to investigate their effect on each other.
Materials and methods
Fifteen cases of oral pyogenic granuloma were fixed with formalin and routinely processed and embedded in paraffin wax. Samples were cut at 5^m sections and prepared for immunohistochemistry. The selected lesions were obtained from oral biopsy archive for both males and females between ages 15-25 years. After rehydration step, antigen retrieval was carried out by placement of slides in the autoclave for 3 minutes at 121[degrees]C. Internal peroxidase activities were suppressed with 1% H2O2 in methanol for 10 minutes. Tissue Sections were incubated overnight at 4[degrees]C with primary monoclonal antibody against ICAM-1 ((mouse monoclonal antihuman clone 6.5Bs Dakocytomation,USA) and against VCAM-1(mouse monoclonal anti-human clone 1.3C3 Dakocytomation,USA) for 30 minutes followed by application of secondary biotinylated antibodies (biotin labeled goat anti-rabbit & goat anti-mouse immunoglobulin in phosphate buffer saline "PBS") which was incubated for additional 30 minute. Diaminbenzidin (DAB) was then applied for 30 minutes for color development. Finally, the sections were washed in distilled water & counterstained with Meyer's Hematoxylin. Sections incubated in phosphate buffer saline instead of primary antibody were used as negative controls and normal tonsillar tissue were used as positive control. The staining intensity was evaluated according to the immunoreactivity-staining score, first introduced by Remmele et al  based on the semiquantitative scoring of cell staining as follows: grade (-), no staining; grade 1 (+), mild but definite staining; grade 2 (++), moderate staining; grade 3 (+++), strong staining.
Statistical analysis was performed using a computer software package, SPSS version 17 (SPSS Inc., Chicago,IL) p# 0.05 was considered statistically significant.
Results and discussion
The majority of epithelial cells of pyogenic granuloma have revealed mild to moderate ICAM-1 immunostaining (41-47%), while inflammatory cells (mononuclear cells) expressed moderate to strong ICAM-1 staining (38.4%). On the other hand, endothelial cells expressed moderate ICAM-1 activity (53.8%) (figure 1). Epithelial cells showed wide range of staining pattern for VCAM-1, while inflammatory cells expressed mild to moderate VCAM-1 activity (38.46%). Interestingly, the majority of the endothelial cells exhibit mild VCAM1 expression (69.2%) (figure 2). The percentage of stained cells for ICAM-1 & VCAM-1 in epithelial cells, inflammatory cells and endothelial cells are shown in table (1). Total ICAM-1 expression showed a significant difference among cell types, whereas VCAM-1 expression did not show any significant difference among these cells (table 2). Chi square test was applied on each cell type to measure the staining differences between ICAM-1 and VCAM-1. There was no significant statistical difference between the expression of the two molecules in epithelial cells and inflammatory cells, however highly significant difference was observed between the expression of the two molecules in the endothelial cells (table 3).
The development of an inflammatory response involves coordinated and sequential adhesive interaction between leukocytes & endothelial cells that are manifested as leukocyte rolling, adherence & migration . Both ICAM-1 & VCAM-1 are members of immunoglobulin gene super family that appear to mediate the firm adherence and emigration of leukocytes across endothelial cell monolayer . These findings have led investigators to associate the role of ICAM-1 & VCAM-1 in the pathogenesis of acute & chronic inflammatory diseases. Studies performed on monolayers of cultured endothelial cells have revealed that ICAM-1 and, to a lesser extent, VCAM-1 are both constitutively expressed on these cells [28,29]. The results of the present investigation revealed that ICAM-1 was strongly expressed on inflammatory cells. However, moderate staining were demonstrated in the endothelial cells while week reaction were exhibited in epithelial cells of pyogenic granuloma. The weak ICAM-1 staining in epithelial cells may be due to small population of antigen presenting cells that respond to oral antigens which induce inflammatory response. Regarding VCAM-1, it was shown that marked increase (6 times) in endothelial VCAM-1 expression is constitutively associated with active inflammation 2[9,30]. Research studies showed that VCAM-1 is not a constitutive adhesion molecule of human umbilical vein endothelial cells but is inducible by TNF-[alpha] & IL-1 [32,33]. TNF is produced by macrophages as a consequence of lipopolysaccharide (LPS) release from the gram negative bacteria, since LPS is a major component of their cell walls . TNF production up-regulate VCAM-1 expression which plays an important role in regulation the movement of leukocytes from the blood to the foci of inflammation. The present study revealed a weak expression in endothelial cells of the majority of pyogenic granuloma cases suggesting that these lesions are toward regression becauseacute inflammation is subsided and inflammatory mediators such as IL-1 and TNF were vanished. Inhibition of VCAM-1 expression and up regulation of ICAM-1 expression by TGF-[beta] may be related to selective T-helper 1 (Th1) infiltration in pyogenic granuloma. In conclusion, our findings provide a direct evidence about the involvement of cell adhesion molecules ICAM-1 and VCAM-1 in the pathogenesis of pyogenic granuloma. Furthermore ICAM-1 and VCAM-1 expression can be used as reliable markers for inflammatory neo-vasculairization to measure micro-vessel density due to inflammatory response. Therefore, these molecules can be used as a marker to differentiate inflammatory pyogenic granuloma from other developmental vascular lesions.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
[1.] Vilmann, A., P. Vilman, H. Vilman, 1986. Pyogenic granuloma: Evaluation of oral conditions, Br J Oral Maxillofac Surg, 24: 37682.
[2.] Neville, B.W., D.D. Damm, C.M. Allen and J.E. Bouquot, 2005. Oral & Maxillofacial Pathology. 2nd Ed.Saunders Company.
[3.] Patrice, S.J., K. Wiss, J.B. Mulliken, 1991. Pyogenic granuloma (Lobular Capillary hemangioma): a clinico pathologic study of 178 cases, Pediatric Dent, 8: 267-76.
[4.] Yuan, K., Y.T. Jin, M.T. Lin, 2000. Expression of Tie-2, angiopoietin -1, angiopoietin-2, ephrinB2 & ephrin B4 in pyogenic granuloma of human gingival implicates their roles in inflammatory angiogenesis, J Periodontal Res., 35(3): 165-71.
[5.] Shimzu, K., S. Naito, Y. Urato, I. Sekine, T. Kondo, I. Katayama, 1998. Inducible Nitirc Oxide Synthase is expressed in granuloma pyogenicum, Br J Dermatol, 138: 769-73.
[6.] Bragado, R., E. Bello, L. Requenal, G. Renedo, E. Texeiro, M.V. Alvarez, et al. 1999. Increased expression of vascular endothelial growth factor in pyogenic granuloma, Acta Derm Venerol, 79: 422-25.
[7.] Hagiwara, K., N.M. Kashhely, H. Uezato, S. Nonakas, 1999. Mast cell 'densities' in vascular proliferation. A preliminary study of pyogenic granuloma, portwine stain, cavernous hemangioma, cherry angioma, Kaposi's sarcoma and malignant hemangioendothilioma, J Dermatol, 26: 577-86.
[8.] Igarash, A., N. Hayashi, K. Nashiro, K .Takehara, 1998. Differential expression of connective tissue growth facto gene in cutaneous fibrohistiocytic and vascular tumors, J Cut Pathol, 25: 143-48.
[9.] Yuan, K., Y.T. Jin, M.T. Lin, 2000. The detection and comparison of angiogenesis-associated factors in pyogenic granuloma by Immunohistochemistry, J Periodontal., 71: 701-9.
[10.] Springer, T., 1997. Adhesion receptors of the immune system, Nature, 346: 425-34.
[11.] Mojcik, C.F., E.M. Shevach, 1997. Adhesion molecules: a rheumatologic prospective, Arthritis Rheum, 40: 991-1004.
[12.] Buck, C.A., 1992. Immunoglobulin superfamily: Structure, function and relationship to other receptor molecules, Semin Cell Biol, 3: 179-88.
[13.] Seth, R., F. Raymond, M. Makgada, 1991. Circulating ICAM-1 isoforms: diagnostic prospects for inflammatory & immune disorders, Lancet, 338: 83-84.
[14.] Elices, M., N. Osborn, Y. Takada, C. Crouse, S. Luhowsky, M. Hemler and R. lobb, 1990. VCAM-1 on activated endothelium interacts with the leukocytes integrin VLA-4 at a site distinct from VLA-4/ fibronectin binding site, Cell., 60: 577-84.
[15.] Almendro, N., T. Bellon and C. Rins, 1996. Cloning human platlet endothelial cell adhesion molecule, structural & functional characterization, J immunol., 12: S411-21.
[16.] Stolpe, V., V. Saag, 1996. Intracellular adhesion molecule-1, J Mol Med., 74: 13-33.
[17.] Thornhill, M., S. Wellicome, D. Mahiouz, J. Lanchbury, Kyan-A, U. Ung and D. Haskard, 1991. Tumor necrosis factor combines with IL-4 or IFN-y to selectively enhance endothelial cell adhesiveness for T-cell , J Immunol., 146: 5926.
[18.] Hosokawa, Y., I. Hosokawa, K. Ozaki, H. Nakae, T. Matsue, 2006. Cytokines differentially regulate ICAM-1 and VCAM-1 expression on human gingival fibroblasts, Clin Exp Immunol., 144: 494-502.
[19.] Gemmell, E., L.J. Walsh, N.W. G.J.Savage, G.J. Seymour, 1994. Adhesion molecule expression in chronic inflammatory periodontal disease tissue , J periodontal Res., 29: 46-53.
[20.] Del Castillo, L.F., R. Schlegel Gomez, M. Pelka, O.P. Hornstein, A.C. Johannessen, Von den P. Driesch, 1996. Immunohistochemical lcalization of very late activation integrins in healthy and diseased human gingiva, J Periodontal Res., 31: 36-42.
[21.] Ozawa, A., H. Tada, R. Tamai, et al. 2003. Expression of IL-2 receptor beta and gamma chains by human gingival fibroblasts and upregulation of adhesion to neutrophils in response to IL-2, J leukoc Biol., 74: 352-9.
[22.] Hayashi, J., I. Saito, I. Ishikawa, N. Miyasaka, 1994. Effects of cytokines and periodontopathic bacteria on the leukocyte function-associated antigen1/ intercellular adhesion molecule 1 pathway in gingival fibroblasts in adult periodontitis , Infect Immun, 62: 5205-12.
[23.] Joe, B.H., J.L. Broke, M. Keskintepe, P.J. Hanes, J.M. Mailhot, B.B. Sigh, 2001. Interleukin-1 beta regulation of adhesion meolecules on human gingival and periodontal ligament fibroblasts, J Periodontal, 72(7): 865-70.
[24.] Wang, J., M. Zhong, L.Z. Zhang, Y. Wang and Z.Y. Wang, 2003. Expression of ICAM-1 & VCAM-1 in human ameloblastoma & odontogenic keratocyst, Shanghai Kou Qiang Yi Xue., 12(4): 273-276 (abstract).
[25.] Remmele, W., U. Hildebrand, H.A. Hienz, P.J. Klein, M. Vierbuchen, L.J. Behnken, B. Heiche and E. Scheidt, 1986. Comparative histological, histochemical, immunohistochemical & biochemical studies on estrogen receptors, lectin receptors, and Barr bodies in human breast cancer, Virchows Arch A Pathol Anat Histopathol, 409: 127-147.
[26.] Panes, J. and D. Granger, 1998. Leukocyteendothelial cell interactions: molecular mechanisms & implications in gastrointestinal disease, Gastro-Enterology, 114: 1086-90.
[27.] Komatsu, S., R. Berg, J. Russel, Y. Nimura and N. Granger, 2000. Enteric microflora contribute to constitutive ICAM-1 expression on vascular endothelial cells, Am J Physiol Gastrointestinal Liver Physiol, 279: 186-191.
[28.] Myers, C., S. Wertheimer, J. Schembri-King, T. Park and R. Wallace, 1992. Induction of ICAM-1 by TNF-[alpha], IL-1[beta] & LPS in human endothelial cells after down-regulation of PKC, Am J Physiol, 262: C57-C64.
[29.] Henseleit, U., C. Steinbrink, M. Sunderkotter, M. Goebeler, J. Roth, C. Sorg, 1994. Expression of murine VCAM-1 in vitro & in different models of inflammation in vivo: correlation with emigration of monocytes, Exp Dermatol, 3: 249-57.
[30.] Wong, D. and K. Donovini-Zis, 1995. Expression of vascular cell adhesion molecule-1 (VCAM-1) by human brain microvessel endothelial cells in primary culture, Microvas Res., 49: 325-330.
[31.] Frenette, P. and D. Wagner, 1996. Adhesion molecules: Part II: Blood vessels and blood cells, N Eng J Med., 335: 43-45.
[32.] Crook, M.F., A.C. Newby and K.M. Southgate, 2000. Expression of intracellular adhesion molecules in human saphenous veins: effects of inflammatory cytokines and neointima formation in culture , Atherosclerosis, 150: 33-41.
[33.] Haraldsen, G., D. Kvale, B. Lien, I. Farstad and P. Brandtzaeg, 1996. Cytokine-regulated expression of E-selectin, intercellular adhesion molecule -1 (ICAM-1) & vascular cell adhesion molecules -1 (VCAM-1) in human intestinal microvascular endothelial cells, J Immunol, 156: 2558-65.
(1) Natheer H.Al-Rawi BDS, (2) Sausan Al Kawas, (3) Wasan Hamdi Younis
(1), (2) Department of Oral & Craniofacial Health Science, College of Dentistry, University of Sharjah ,UAE. (3) BDS, Ph.D, Assistant Professor, Department of Oral Diagnosis, College of Dentistry, University of Baghdad, Iraq.
Sausan Al Kawas D.D.S, Ph.D, FICD., Department of Oral & Craniofacial Health Sciences College of Dentistry, University of Sharjah, P.O. Box: 27272 Sharjah, United Arab Emirates.
Mobile: 00971-507929735: Fax: 00971-65585641
Table 1: Percentage of ICAM-1& VCAM-1 expression in different cells of pyogenic granuloma ICAM-1 Mild (grade 1) Moderate(grade 2) Epithelial cells 8 (47%) 7 (41%) Inflammatory cells 3 (23.07%) 5 (38.46%) Endothelial cells 4 (30.76%) 7 (53.8%) VCAM-1 Mild (grade 1) Moderate(grade 2) Epithelial cells 6 (46.15%) 2 (15.3%) Inflammatory cells 5 (38.46%) 5 (38.46%) Endothelial cells 9 (69.2%) 1 (7.6%) ICAM-1 Strong (grade 3) Epithelial cells 2 (12%) Inflammatory cells 5 (38.46%) Endothelial cells 2 (15.5%) VCAM-1 Strong (grade 3) Epithelial cells 5 (38.46%) Inflammatory cells 3 (23.7%) Endothelial cells 3 (23.7%) Table 2: Total ICAM-1 & VCAM-1 expression among different cells of pyogenic granuloma ICAM-1 Mild (grade 1) Moderate(grade 2) Epithelial cells 8 7 Inflammatory cells 3 5 Endothelial cells 4 7 X2 9.11 DF 4 VCAM-1 Mild (grade 1) Moderate(grade 2) Epithelial cells 6 2 Inflammatory cells 5 5 Endothelial cells 9 1 X2 5.245 DF 4 ICAM-1 Strong (grade 3) Epithelial cells 2 Inflammatory cells 5 Endothelial cells 2 X2 P<0.05 DF VCAM-1 Strong (grade 3) Epithelial cells 5 ( Inflammatory cells 3 Endothelial cells 3 X2 DF p>0.05 Table 3: Immunohistochemical staining differences among different cells of pyogenic granuloma Epithelial cells Mild (grade 1) Moderate(grade 2) ICAM-1 8 7 VCAM-1 6 2 [chi square] 4.17 DF 2 Inflammatory cells Mild (grade 1) Moderate(grade 2) ICAM-1 3 5 VCAM-1 5 5 [chi square] 1 P>0.05 DF 2 Endothelial cells Mild (grade 1) Moderate(grade 2) ICAM-1 4 7 VCAM-1 9 1 [chi square] 6.62 DF 2 Epithelial cells Strong (grade 3) ICAM-1 2 VCAM-1 5 [chi square] DF P>0.05 Inflammatory cells Strong (grade 3) ICAM-1 5 VCAM-1 3 [chi square] DF Endothelial cells Strong (grade 3) ICAM-1 2 VCAM-1 3 [chi square] DF P<0.02