HLA-II - induced Cytokines from Gingival Fibroblasts Promote HUVEC Proliferation

Objectives: The role of human leukocyte histocompatibility antigen (HLA) class II molecules (HLA-II) on non-antigen presenting cells has aroused controversy. We previously reported that HLA-II on human gingival fibroblasts (HGFs) do not present antigens, but transduce signals into the cells, resulting in the expression of several cytokines and chemokines. However, exact role of these cytokines as well as other cytokines secreted from HGFs in the pathogenesis of chronic periodontal inflammation is not fully understood. The aim of this study was to see the effects of HLA-II-induced cytokines on the proliferation of human umbilical vein endothelial cell (HUVEC).

Methods: HGFs were obtained from periodontally healthy donors during the extraction of impacted third molar. Recombinant human interferon-ƒ× was used to up-regulate the expression of HLA-II on cell surface. Furthermore, HGFs were stimulated with either anti-HLA-DR antibodies or isotype-matched IgG2a. Antibody-based cytokine-microarray analysis was performed to detect secreted cytokines associated with angiogenesis. Next, the secreted cytokines productivity was quantified using ELISA. Then, cell proliferation assay was performed to see if these cytokines actually promoted the proliferation of HUVEC using thiazolyl blue tetrazolium bromide (MTT) assay. In addition, neutralizing antibodies against these cytokines and their receptors were used in cell proliferation assay to see the association of these cytokines with HUVEC proliferation.

Results: 1) Interleukin (IL)-8 and growth-related oncogene (GRO) were identified as angiogenesis-associated-chemokines from HLA-II-stimulated-HGFs. 2) Cell culture supernatant from HLA-II-stimulated-HGFs promoted the growth of HUVEC. 3) Addition of anti-IL-8 neutralizing antibody and anti-CXCR1 antibody inhibited the growth of HUVEC dose-dependently, while that of anti-GRO antibody did not.

Conclusion: Our results suggest that HLA-II-induced IL-8 in HGFs is associated with angiogenesis, possibly via CXCR1, in chronic periodontal inflammation.