Pentoxifylline inhibits human T-cell adhesion to dermal endothelial cells.

Pentoxifylline (PTX), a methyl xanthine derivative with phosphodiesterase inhibitory activity, has been shown to have antiinflammatory effects. Previous studies have demonstrated that PTX can suppress TNF alpha production and function, and can inhibit the adhesion of neutrophils and monocytes to endothelial cells. In the present study, we sought to determine whether PTX also interferes with the adhesion of human peripheral blood T lymphocytes to cells of the human dermal endothelial cell line HMEC-1. Using a cell adhesion immunoassay, the effect of different doses of PTX (10(-5)-10(-2) M) on the binding of unactivated or PMA-activated T cells to unstimulated or TNF alpha-stimulated endothelial cells was investigated. In addition, blocking experiments with monoclonal antibodies against pairs of adhesion molecules known to be involved in endothelial cell/T-cell adhesion were performed. Unactivated T cells showed minimal adhesion to unstimulated endothelial cells. PMA-activated T cells showed an eightfold increased binding to TNF alpha-stimulated endothelial cells, which was found to be mediated largely by LFA-1/ICAM-1. PTX inhibited the binding of PMA-activated T cells to TNF alpha-stimulated endothelial cells in a dose-dependent manner. This inhibition was only found when PTX was present during the adhesion assay. A similar inhibition was found when PTX was replaced by isobutylmethylxanthine, another methyl xanthine derivative, or by a combination of two cAMP analogues. The results suggest that interference with T-cell/endothelial cell adhesion, which forms an essential step in the migration of T cells from the peripheral blood into sites of inflammation, may be another explanation for the beneficial effect of PTX in several inflammatory dermatoses.

Pentoxifylline inhibits T-cell adherence to keratinocytes.

In many inflammatory dermatoses leukocyte function-associated antigen-1/intercellular adhesion molecule-1 mediated T-cell/keratinocyte adhesion is considered to play an important role. Pentoxifylline (PTX), a methylxanthine derivative widely used for the symptomatic treatment of various vascular disorders, was recently found to have anti-inflammatory effects. PTX can suppress tumor necrosis factor-alpha production and function, and inhibits leukocyte-endothelial cell adherence. The aim of the present study was to investigate whether PTX also interferes with T-cell/keratinocyte binding. Peripheral blood T cells were activated with phorbol myristate acetate and co-incubated with interferon-gamma- or tumor necrosis factor-alpha-stimulated keratinocytes (SVK 14 cells) in the presence or absence of PTX. Using an enzyme-linked immuno cell adhesion assay PTX was found to inhibit T-cell/keratinocyte adhesion in a dose-dependent manner. A similar inhibition was found when PTX was replaced by isobutylmethylxanthine, another methylxanthine derivative, or by a combination of two cyclic adenosine monophosphate analogues. No major effect on T-cell/keratinocyte adherence was observed when PTX was present during the pre-incubation of keratinocyte monolayers with tumor necrosis factor-alpha or interferon-gamma prior to the adhesion assay. In keratinocyte monolayers the interferon-gamma or tumor necrosis factor-alpha induced intercellular adhesion molecule-1 expression could not be inhibited by PTX. However, when PTX was added to short-term organ cultures of normal human skin biopsies, the lipopolysaccharide- and tumor necrosis factor-alpha-induced keratinocyte intercellular adhesion molecule-1 expression was blocked completely. The interferon-gamma-induced ICAM-1 expression was not blocked by PTX. The results presented herein suggest that impaired T-cell/keratinocyte binding may be one of the mechanisms by which PTX exerts a beneficial effect in certain inflammatory dermatoses.

CD44 antibody stimulates adhesion of peripheral blood T cells to keratinocytes through the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 pathway.

Close contact between T lymphocytes and keratinocytes is an important feature of many inflammatory skin diseases. In vitro studies showed that stimulation of keratinocytes with interferon-gamma or tumor necrosis factor-alpha and of T cells with phorbol esters results in a leukocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1-mediated adhesion. The present study was performed to investigate the role of the CD44 molecule in keratinocyte/T-cell binding. The CD44 class of lymphocyte adhesion receptors is involved in lymphocyte binding to high endothelial venules and to extracellular matrix compounds and is therefore important in lymphocyte recirculation and homing. Moreover, CD44 can act as a co-stimulating signal in T-cell activation and promotes homotypic adhesion of in vitro cultured CD3-stimulated T cells. Using a cell adhesion assay a sixfold increase in T-cell/keratinocyte adhesion was found after pre-incubating the T cells with anti-CD44. This increased adhesion was found to require an intact cytoskeleton, to be energy and magnesium dependent, and could be completely inhibited by anti-LFA-1 and anti-ICAM-1. Pretreatment of T cells with hyaluronic acid, a ligand for CD44 and an extracellular matrix compound in the dermis and epidermis, did not affect T-cell/keratinocyte adhesion.

Spectophotometric quantification of cell-cell adherence by an enzyme-linked immuno-cell adhesion assay.

In the present study a spectophotometric method was developed to determine the number of T cells binding to human epidermal keratinocytes (KC). In this cell adhesion assay the adherent T cells were detected with a cell-specific monoclonal antibody conjugated to horseradish peroxidase and the coloured substrate quantified in an ELISA reader at 492 nm. A correlation was demonstrated between the number of T cells and the extinction values measured. The enzyme-linked immuno-cell adhesion assay (ELICAA) was used to quantify KC/T lymphocyte adherence in a series of experiments designed to evaluate its reliability and reproducibility. Compared with the 51Cr-labelled adherence assay, the ELICAA was a safe, rapid and accurate method avoiding the use of radioactive material.