Agnes H. Chan

Consequences of the photodynamic treatment of resting and activated peripheral T lymphocytes

The impact of the immunomodulatory photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) and visible light on the survival and surface receptor pattern of resting and activated murine T cells was evaluated. T cells treated for 48 h with immobilized anti-CD3 monoclonal antibody upregulated expression of the interleukin-2 receptor alpha-chain (CD25), transferrin receptor (CD71), the apoptosis-regulating Fas receptor (CD95), contained a greater level of the anti-apoptotic protein Bcl-2 and accumulated significantly more BPD-MA than their unactivated counterparts. Activated T cells displayed a modestly greater susceptibility to the photodynamic induction of DNA fragmentation than resting T cells. Resting T cells treated with sub-lethal levels of BPD-MA and light did not exhibit changes in surface levels of CD3, CD4, CD8, CD28, CD45 or T cell receptor (TCR) beta-chain structures. However, levels of major histocompatibility complex (MHC) class I antigens were decreased while the density of Thy-1.2 (CD90) increased on these cells. Photodynamically treated T cells failed to express optimal CD25 levels when exposed to the mitogenic anti-CD3 antibody. Activated T cells treated with sub-lethal levels of BPD-MA and light exhibited lower CD25 levels, a temporary block in cell cycle transition, but unaltered expression of MHC Class I, CD3, CD4, CD8, CD45, CD54, CD71, CD122 (IL-2R beta-chain) or TCR beta-chain antigens 24 h afterward. Resting and activated T lymphocytes differ in susceptibility to PDT-mediated apoptosis but both types are sensitive to anti-proliferative effects the treatment exerts at sub-lethal photosensitizer levels. The marked sensitivity of activated T cells to photodynamic inactivation likely contributes to the immunomodulatory action of BPD-MA.

Inhibition of contact hypersensitivity with different analogs of benzoporphyrin derivative

Four structural analogs of benzoporphyrin derivative (BPD), a potent anti-tumor photosensitizer, were evaluated for their capacity to influence the immunologically-mediated contact hypersensitivity (CHS) response against the hapten 2,4-dinitrofluorobenzene (DNFB). Immunocompetent hairless strain mice received BPD monoacid ring A (BPD-MA, verteporfin) and returned to normal housing conditions or treated with 690 nm red light (transcutaneous photodynamic therapy, PDT). Unexpectedly, we found that mice given BPD-MA exhibited significantly reduced CHS ear swelling responses to DNFB upon antigenic challenge, whether or not they had been treated with PDT. A significant reduction in the CHS response to DNFB was observed when BPD-MA or PDT was given 48 or 24 h prior to, on the same day, or 24 or 72 h after DNFB sensitization. However, the magnitude of the CHS response was unaffected if these treatments were given 96 h after DNFB sensitization, 24 h before challenge with DNFB. Significantly reduced CHS responses also occurred in Balb/c mice given BPD-MA with or without PDT. Mice given BPD-MA but retained in total darkness throughout the experimental period generated full-fledged ear swelling responses to DNFB indicating that CHS suppression with BPD-MA was light dependent. BPD monoacid ring B (BPD-MB) strongly reduced the CHS response of Balb/c mice kept under ambient light while BPD diacid ring A (BPD-DA) and BPD diacid ring B (BPD-DB) also lowered the CHS response but were less effective than the monoacid forms. Other photosensitizers including Photofrin, tin etiopurpurin, and zinc phthalocyanine did not alter the CHS response of Balb/c mice maintained under ambient light. The ability of different BPD analogs to inhibit the CHS response in mice held under ambient light conditions appears related to the potent photosensitizing activity of these compounds.

TRANSCUTANEOUS PHOTODYNAMIC THERAPY ALTERS THE DEVELOPMENT OF AN ADOPTIVELY TRANSFERRED FORM OF MURINE EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS

Abstract— Transcutaneous photodynamic therapy (PDT), utilizing benzoporphyrin derivative monoacid ring A (BPD, ver‐teporfin) and whole‐body light exposure, was assessed for its capacity to modify the course of adoptively transferred experimental autoimmune encephalomyelitis (EAE) in PL mice. Using a novel cell culture technique to facilitate the induction of this neurodegenerative condition, disease signs commenced 3–4 weeks after the transfer of myelin basic protein (MBP)‐reactive lymph node or spleen cells to naive syngeneic recipients. Mice administered MBP‐sensitized lymph node cells preincu‐bated with BPD followed by whole‐body 690 nm light irradiation (15 J/cm2) did not display symptoms of EAE. Although almost all animals given MBP‐sensitized spleen cells developed EAE, mice given BPD (1 mg/kg) and the light treatment 24, 48 or 120 h after spleen cell transfer exhibited significantly less severe disease symptoms than control animals. Mice given the photodynamic treatment 24 h after spleen cell transfer also exhibited a significantly later disease onset than the control animals. Treatment of mice with PDT 24 h prior to spleen cell transfer did not influence subsequent disease severity but modestly delayed its onset. In the absence of directed light, BPD did not influence the development of EAE. Spinal cord tissues were evaluated for the presence of T cell receptor (TCR) Vα4 mRNA transcripts that specifically encode for the TCR ot‐chain of MBP‐reactive T cells of PL mice. Using the polymerase chain reaction, Va4 TCR mRNA transcripts were present in spinal cord samples prepared from almost all control mice but in only about one‐half of spinal cord samples prepared from mice treated with PDT 24 h after spleen cell transfer. These observations indicated that PDT had limited the expansion of MBP‐specific Vot4+ T cells within the central nervous system. Transcutaneous PDT represents a new technique with which to approach the treatment of autoimmune disease.

Influence of photodynamic therapy on immunological aspects of disease - an update

Photodynamic therapy (PDT) utilises light-absorbing compounds combined with directed photo-irradiation to produce clinical effects. This review updates advances in the understanding of the biochemical pathways triggered by PDT within cells, its influence upon different immune parameters and progress in the use of PDT against human immune-mediated disease. Several works have further defined the notable capacity of PDT to foster anticancer immunity.

Clinical status of benzoporphyrin derivative

Benzoporphyrin derivative monoacid ring A (BPD) is currently in Phase II clinical trials for the treatment of cutaneous malignancies (basal cell carcinoma and cutaneous metastases) and psoriasis. Results to date suggest that this photosensitizer has potential in both of these areas. Recently, a clinical trial with BPD was initiated for the treatment of age related macular degeneration, a neovascular condition in the eye which leads to blindness. BPD is a lipophilic photosensitizer which is rapidly taken up by activated cells and the vascular endothelium of neovasculature. The PDT effects seen with BPD appear to be a combination of vascular occlusion and direct killing of target cells. Since many diseases involve either activated cells and/or neovasculature, PDT with photosensitizer with characteristics like those of BPD, has applications far wider than oncology. A new area of interest involving photosensitizers is that of immune modulation. A number of photosensitizers have been shown to effect immune modulation in animal models of immune dysfunction including autoimmunity (rheumatoid arthritis, lupus), cutaneous hypersensitivity and allografts. BPD and PHOTOFRINR have both been shown to be effective in ameliorating arthritic symptoms in a number of animal models. The mechanisms by which immune modulation is affected in these studies still remains to be resolved.

SENSITIVITY OF ACTIVATED MURINE PERITONEAL MACROPHAGES TO PHOTODYNAMIC KILLING WITH BENZOPORPHYRIN DERIVATIVE

Abstract— This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon‐γ (rIFN‐γ, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN‐γ‐activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD50= 14.4 ± 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm2) than cells (LD50= 18.2 ± 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN‐γ‐activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 μg/mL) took up more BPD than macrophages cultured in medium alone or with rIFN‐γ. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD50= 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN‐γ‐activated macrophages and the greater resistance of LPS‐activated macrophages, relative to medium‐cultured macrophages, to photodynamic killing with BPD is uncertain. However, the increased susceptibility of macrophages, activated with the immunomodulatory cytokine IFN‐γ, to treatment with BPD and light might indicate how photodynamic therapy could interfere with the development of experimental autoimmune disease, conditions in which activated macrophages are known to be involved.

Effect of photodynamic therapy using benzoporphyrin derivative on the cutaneous immune response

In this study, the effect of transdermal photodynamic therapy (PDT) using benzoporphyrin derivative monoacid ring A (BPD) on the development of the immunologically mediated contact hypersensitivity (CHS) response against the hapten dinitrofluorobenzene (DNFB) and on the duration of skin allograft acceptance has been evaluated. In the CHS model it was found that the treatment of hairless strain mice with whole-body transdermal PDT using BPD (1 mg/kg) and LED light (15 J/cm2) resulted in a profound suppression of the CHS reaction if treatment was applied either 48 or 24 hours prior or up to 72 hours after sensitization of abdominal skin with DNFB. Less inhibition of the CHS response was observed if PDT was given one day before the ear challenge with DNFB which was applied 5 days following the initial DNFB sensitization. However, DNFB-exposed, PDT-treated mice retained the capacity to respond maximally to the unrelated contact sensitizer oxazolone. These results are consistent with other models of experimentally induced immune tolerance. allogeneic skin graft studies demonstrated that pretreatment of skin with BPD and light, at levels that did not cause significant tissue damage, significantly enhanced the length of engraftment. Using a separate protocol, photodynamic treatment of recipient mice at various times after transplant had no significant effect on allograft acceptance. Irradiation of skin in the presence of BPD may significantly inhibit the initiation of certain immunological responses within these tissues.

Immune modulation using transdermal photodynamic therapy

The photosensitizer benzoporphyrin derivative monoacid ring A (VerteporfinR or BPD) has maximum absorption characteristics (690 nm) and biodistribution characteristics which permit activation of the drug in capillaries of the skin without causing skin photosensitivity (transdermal PDT). This permits targeting of cells in the circulation for selective ablation. Since BPD has been shown to accumulate preferentially in activated lymphocytes and monocytes, studies have been undertaken to determine the effect of transdermal PDT on murine models for rheumatoid arthritis (the MRL/lpr adjuvant enhanced model) and multiple sclerosis (the experimental allergic encephalomyelitis (EAE) model in PL mice). Localized transdermal PDT with BPD was found to be completely successful in preventing the development of adjuvant enhanced arthritis in the MRL/lpr mouse as well as improving the underlying arthritic condition of these animals. In the EAE model, in which an adoptive transfer system was used, it was found that transdermal PDT of recipients was effective in preventing EAE if treatments were implemented up to 24 hours after cell transfer but was not effective if given later, indicating the requirement for circulating T cells for effective treatment.

Transcutaneous photodynamic therapy delays the onset of paralysis in a murine multiple sclerosis model

Photodynamic therapy (PDT) using benzoporphyrin derivative (BPD, Verteporfin) and whole body irradiation, can affect the course of adoptively transferred experimental allergic (autoimmune) encephalomyelitis (EAE) in PL mice. Murine EAE is a T cell-mediated autoimmune disease which serves as a model for human multiple sclerosis. Using a novel disease induction protocol, we found that mice characteristically developed EAE within 3 weeks of receipt of myelin basic protein (MBP)-sensitized, in vitro-cultured spleen or lymph node cells. However, if animals were treated with PDT (1 mg BPD/kg bodyweight and exposed to whole body 15 Joules cm2 of LED light) 24 hours after receiving these cells, disease onset time was significantly delayed. PDT-treated mice developed disease symptoms 45 +/- 3 days following cell administration whereas untreated controls were affected within 23 +/- 2 days. In contrast, application of PDT 48 or 120 hours following injection of the pathogenic cells had no significant effect upon the development of EAE. Experiments are in progress to account for the protective effect of PDT in this animal model. These studies should provide evidence on the feasibility of PDT as a treatment for human autoimmune disease.

Photodynamic immune modulation (PIM)

Photodynamic Therapy (PDT) is accepted for treatment of superficial and lumen-occluding tumors in regions accessible to activating light and is now known to be effective in closure of choroidal neovasculature in Age Related Macular Degeneration. PDT utilizes light absorbing drugs (photosensitizers) that generate the localized formation of reactive oxygen species after light exposure. In a number of systems, PDT has immunomodulatory effects; Photodynamic Immune Modulation (PIM). Using low- intensity photodynamic regimens applied over a large body surface area, progression of mouse autoimmune disease could be inhibited. Further, this treatment strongly inhibited the immunologically- medicated contact hypersensitivity response to topically applied chemical haptens. Immune modulation appears to result from selective targeting of activated T lymphocytes and reduction in immunostimulation by antigen presenting cells. Psoriasis, an immune-mediated skin condition, exhibits heightened epidermal cell proliferation, epidermal layer thickening and plaque formation at different body sites. In a recent clinical trial, approximately one-third of patients with psoriasis and arthritis symptoms (psoriatic arthritis) displayed a significant clinical improvement in several psoriasis-related parameters after four weekly whole-body PIM treatments with verteporfin. The safety profile was favorable. The capacity of PIM to influence other human immune disorders including rheumatoid arthritis is under extensive evaluation.