David A. Musser

CUTANEOUS PHOTOSENSITIZING AND IMMUNOSUPPRESSIVE EFFECTS OF A SERIES OF TUMOR LOCALIZING PORPHYRINS

Abstract— A series of tumor localizing porphyrins was evaluated with respect to their ability to elicit cutaneous photosensitivity and systemic immunosuppression, two of the most common side effects associated with photodynamic therapy. Using the murine ear swelling response as an indicator, it was found that all the non‐metalloporphyrins caused cutaneous photosensitization. Immunosuppressive effects were noted using hematoporphyrin derivative (HPD) and meso‐tetra(4‐sulfonatophenyl)porphine if sensitization occurred immediately after photoirradiation, but none were evident using Photofrin II (PII) or meso‐tetra(4‐carboxyphenyl)porphine (TCPP). Subsequent studies indicated that PII and TCPP manifested a delayed type immunosuppression similar to that found following UVB photoirradiation. Manganese (III) meso‐tetra(4‐sulfonatophenyl)porphine, a prototype magnetic resonance imaging contrast agent, was also evaluated because of its reported demetallation in vivo. It was found to cause neither cutaneous photosensitivity nor immunosuppression.

IL-10 does not play a role in cutaneous Photofrin photodynamic therapy-induced suppression of the contact hypersensitivity response.

Abstract Photodynamic therapy (PDT) treatment of both malignant and benign skin diseases has proven to be effective, and its use is increasing worldwide. However, preclinical studies using murine models have shown that PDT of the skin inhibits cell-mediated immune reactions, as measured by the suppression of the contact hypersensitivity (CHS) reaction. We have previously demonstrated that PDT enhances IL-10 expression in treated skin, and that the kinetics of induction of IL-10 is similar to the kinetics of suppression of systemic CHS reactions by cutaneous PDT. In the following report we have expanded upon these studies to demonstrate that cutaneous PDT, using Photofrin®, induces elevated levels of systemic IL-10 that persist for at least 28 days following treatment. The increase in systemic IL-10 correlates to a prolonged suppression of CHS of at least 28 days following cutaneous PDT. IL-10 has been implicated as the causative agent in the suppression of cell-mediated immune reactions by UVB and transdermal PDT. However, in the studies reported here we demonstrate that the suppression of CHS by cutaneous PDT occurs via an IL-10 independent mechanism, as administration of anti–IL-10 antibodies had no effect on the ability of PDT to induce CHS suppression. These results were further confirmed using IL-10 knockout (KO) mice. Cutaneous PDT of IL-10 KO mice resulted in CHS suppression that was not significantly different from suppression induced in wild-type mice. Thus, it appears as though IL-10 does not play a role in CHS suppression by cutaneous PDT. Suppression of cell-mediated immune reactions by UVB and transdermal PDT is reversible by IL-12, which is critical for the development of these reactions. We show that administration of exogenous IL-12 is also able to reverse CHS suppression induced by cutaneous PDT, suggesting that whereas suppression of cell-mediated immune reactions by UVB, transdermal PDT and cutaneous PDT occurs via different mechanisms, a common regulatory point exists.

Inhibition of DNA dependent RNA synthesis by porphyrin photosensitizers

Abstract Select porphyrin photosensitizers were studied to determine their effects on DNA-dependent RNA synthesis in the presence and absence of visible light. All of the porphyrins were found to inhibit wheat germ polymerase II to some degree in the dark. In the presence of light, the inhibitory effects of the porphyrins was found to result from both inactivation of the enzyme and impairment of the ability of DNA to serve as a template.

Mechanism of the localization of manganese (III) mesotetra(4-sulfonatophenyl)porphine i in mice bearing L1210 tumors

In accordance with earlier work the manganese (III) derivative of meso-tetra(4-sulfonatophenyl)porphine (TPPS4) is found to accumulate in the tumors of L1210-bearing mice. The tumor/liver ratio of porphyrin extends from 1.5 to 3.6 over a range of dose and time periods. The subcellular distribution of porphyrin in L1210 tumor and liver, and the tissue distribution (cellular, stroma, soluble) in L1210 tumor indicates that the porphyrin tends to be located predominantly in soluble and stromal fractions. These data are interpreted in terms of the physiology and composition of neoplastic tissue to formalize a mechanism for the localization of Mn(III)TPPS4 in L1210 tumor and a general working hypothesis for the localization of porphyrins in neoplastic tissue. The in vivo stability of Mn(III)TPPS4 is also addressed and is found to be demetallated to a degree of approximately 1% in liver and kidney.

Characteristics of the Immunosuppression Induced by Cutaneous Photodynamic Therapy: Persistence, Antigen Specificity and Cell Type Involved¶

Abstract Relatively little is known about the immunosuppression induced in mice which have received cutaneous photodynamic therapy (PDT). Consequently, experiments were undertaken using mice which received dorsal PDT using Photofrin® as the photosensitizer in an attempt to characterize the overall nature of the immunosuppression. Photoirradiation of mice at various times after injection indicated there was no correlation between photosensitivity and immunosuppression. The suppression was found to be adoptively transferable and antigen specific suggesting the generation of suppressor cells. Selective cell depletions prior to adoptive transfer indicated a CD4+ T cell to be responsible for the immunosuppression. Interestingly, using allogeneic spleen cells, no effect on the delayed type hypersensitivity (DTH) response was found. The results indicate that the suppression induced by cutaneous PDT, with the exception of the lack of DTH suppression, is similar to that induced by UVB irradiation but unlike that reported using laser PDT of the peritoneal cavity. This suggests that not only the type of photoirradiation but also the site of photoirradiation might determine the character of the induced immunosuppression.

The Anatomic Site of Photodynamic Therapy is a Determinant for Immunosuppression in a Murine Model

Abstract— The role of the irradiation site in the induction of suppression of the contact hypersensitivity (CHS) response following photodynamic therapy (PDT) was examined in a murine model. Laser irradiation on the flanks of nontumor‐bearing Photofrin®‐injected mice caused suppression of the CHS response. If the irradiation was conducted on a subcutaneously implanted foil disc on the flank no immunosuppression occurred, indicating that no suppressive factor (s) of sufficient quantity to cause suppression was released from the skin, but rather irradiation of internal organs was the cause. Irradiation of tumors implanted on the flanks of mice reduced the suppression, suggesting an immunopotentiating effect of PDT. Irradiation on the thigh in the presence or absence of a tumor gave no immunosuppression. These results suggest that the anatomic site of irradiation is one determinant for the elicitation of suppression of the CHS response.

Photodynamic therapy affects the expression of IL-6 and IL-10 in vivo

Photodynamic therapy (PDT), which can effectively destroy malignant tissue, also induces a complex immune response which potentiates anti-tumor immunity, but also inhibits skin contact hypersensitivity (CHS) and prolongs skin graft survival. The underlying mechanisms responsible for these effects are poorly understood, but are likely to involve meditation by cytokines. We demonstrate in a BALB/c mouse model that PDT delivered to normal and tumor tissue in vivo causes marked changes in the expression of cytokines interleukin (IL)-6 and IL-10. IL-6 mRNA and protein are rapidly and strongly enhanced in the PDT treated EMT6 tumor. Previous studies have shown that intratumoral injection of IL- 6 or transduction of the IL-6 gene into tumor cells can enhance tumor immunogenicity and inhibit tumor growth in experimental murine tumor systems. Thus, PDT may enhance local anti-tumor immunity by up-regulating IL-6. PDT also results in an increase in IL-10 mRNA and protein in the skin. The same PDT regime which enhances IL-10 production in the skin has been shown to strongly inhibit the CHS response. The kinetics of IL-10 expression coincide with the known kinetics of PDT induced CHS suppression and we propose that the enhanced IL-10 expression plays a role in the observed suppression of cell mediated responses seen following PDT.