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THU0023 Chronic Inflammation: Costimulatory Molecule CD86 Expression on Human Cultured Endothelial Cells
  1. M. Cutolo1,
  2. R. Brizzolara1,
  3. P. Montagna1,
  4. S. Soldano1,
  5. P. Contini2,
  6. B. Villaggio3
  1. 1Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine
  2. 2Division of Clinical Immunology, Department of Internal Medicine
  3. 3Clinical Academic Unit of Nephrology, Department of Internal Medicine, University of Genova, Genova, Italy

Abstract

Background Vascular endothelium is involved in several immune mediated diseases and in chronic inflammation. An important matter of debate is whether endothelial cells (ECs), both in resting or activated state, express the repertoire of costimulatory molecules such as CD86 (B7.2), for adequate T cell interaction/activation [1,2].

Objectives In order to evaluate the CD86 presence on ECs of different origin, we studied in vitro two distinct human ECs lines, both resting or activated with different immune/inflammatory stimuli (γIFN, IL-17, IL-1β).

Methods Human Umbilical Vein Endothelial Cells (HUVEC, Lonza, Switzerland) and Human Microvascular Endothelial Cardiac Cells (HMVEC-C, Lonza, Switzerland), at fourth culture passage, were stimulated for 48 hours with γIFN (500 U/ml, Sigma, Milan, Italy) in order to activate them [3]. In addition, HMVEC-C were stimulated for 48 hours with two further stimuli: human IL-1β (500 U/ml, Adipogen, Incheon, South Korea) or IL-17 (100 ng/ml, Biovision, CA, USA). Thus, in both HUVEC and HMVEC-C, the expression of EC phenotypic markers was detected by flow cytometric analysis, using PE anti-human CD31 and CD105 mouse antibodies (Milteny Biotec Inc, CA, USA). In addition, the expression of CD86 in resting or activated ECs was detected by flow cytometric analysis, using a FITC anti-human CD86 mouse antibody (BD, Biosciences, NY, USA). In every three experiments specific isotype control was included.

Results At flow cytometric analysis, the phenotypic characterization of the ECs by CD31 and CD105 specific staining was confirmed. CD31 and CD105 markers were found in respectively 99% and 96% of the unstimulated HUVEC, as well as in 93% and 96% of the unstimulated HMVEC-C.

ECs in resting condition, expressed mild level of CD86 (57% on HUVEC and 60% on HMVEC-C) at 48 hours. The same ECs after 48 hours of γIFN stimulation, showed an evident increase of the fluorescence for CD86 expression (85% on HUVEC and 68% on HMVEC-C).

Further evaluations limited to HMVEC-C, showed that IL-1β stimulation did not change the percentage of CD86 positive cells (61%) after 48 hours, compared with untreated cells (60%). Conversely, IL-17 treatment induced a light increase of CD86 positive cells (71%) after 48 hours, compared to untreated cells (60%).

Conclusions Our study shows that ECs (both HUVEC and HMVEC) express constitutively the costimulatory molecule CD86, and the expression is increased in activated state. In particular, microvascular ECs activated with γIFN and IL-17, but not with IL-1β, show an increased CD86 expression compared to their resting condition. These results suggest the involvement and contribution of ECs during the immune/inflammatory response for example by facilitating T cell adhesion and migration.

References

  1. Kreisel D et al. J Immunol 2002;169(11):6154-61.

  2. Lozanoska-Ochser et al. J Immunol 2008;181;6109-6116.

  3. Batten P et al. Immunol 1996;87:127-133.

Disclosure of Interest None Declared

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