Characterization of Endogenous Protoporphyrin IX Induced by δ-Aminolevulinic Acid in Resting and Activated Peripheral Blood Lymphocytes by Four-Color Flow Cytometry

Abstract

Lymphocytes treated with delta-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CD8. In activated cells treated with 0-6 mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX accumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71+ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4+ and 85% of the CD8+ T cells accumulated PpIX. The PpIX levels of CD8+ cells were about 5% greater than CD4+ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.

Cellular and Antitumor Activity of a New Diethylene Glycol Benzoporphyrin Derivative (Lemuteporfin)†

“…The uptake selectivity of lemuteporfin for activated versus nonactivated cells was assessed (12, 16–18). Selectivity for activated cells over quiescent, normal cells may be indicative of an adequate therapeutic window for PDT when treating diseases such as cancer.…”

Selective Action of the Photosensitizer QLT0074 on Activated Human T Lymphocytes¶

“…Mouse T cells activated with the anti-CD3 antibody exhibited greater verteporfin uptake and underwent apoptosis at lower concentrations of this photosensitizer than did nonactivated T cells (7). Mitogen-stimulated human T cells synthesized higher amounts of the endogenous photosensitizer protoporphyrin IX (PPIX) than did their resting equivalents after incubation with the PPIX precursor aminolevulinic acid (8,9). This feature was associated with a selective killing of the activated T cell population when combined with light exposure (8,9).…”

Topical photodynamic therapy with 5‐aminolaevulinic acid does not induce hair regrowth in patients with extensive alopecia areata

“…5‐Aminolaevulinic acid (ALA) is a small, hydrophilic precursor of protoporphyrin IX (PpIX) that can be used topically as a PDT photosensitizer. The observation that lymphocytes accumulate around hair follicles in AA, and the recent demonstration that ALA is preferentially metabolized to PpIX in activated lymphocytes, provide a rationale for exploring the use of ALA‐based PDT for treating AA 4 . In addition, hair regrowth in AA has been reported for PDT with a topical formulation of the photosensitizer haematoporphyrin 5…”