Prolongation of murine skin allograft survival by the systemic effects of 8-methoxypsoralen and long-wave ultraviolet radiation (PUVA)

Systemic administration of the photoactive drug 8-methoxypsoralen to a group of mice bearing cutaneous allografts, followed by exposure to long-wave ultraviolet radiation (UVA, 320-400 nm) (PUVA) daily for 14 days at a site distant from the allograft, significantly increased the survival time of the allografts. This effect was seen both in donor-recipient combinations that differ at the major histocompatibility complex and in those differing only at minor histocompatibility loci. Treatment with 8-methoxypsoralen or long-wave UV radiation alone was ineffective in prolonging allograft survival, as were doses of mid-wave UV radiation (UVB, 280-320 nm) that produced greater inflammation than the PUVA protocol. Allografted, PUVA-treated animals also demonstrated decreased alloantigen reactivity against donor-strain spleen cells during the period of treatment by cytotoxicity assays. Allografts of skin in the murine system are highly immunogenic and are generally rejected faster than organ allografts; thus PUVA treatment appears to exert a potent effect on prolonging allograft survival. The systemic nature of the effect and the fact that adverse side effects from PUVA are largely limited to the skin suggest that PUVA might have a role in clinical organ transplantation management.     

Activation of keratinocytes with psoralen plus UVA radiation induces the release of soluble factors that suppress delayed and contact hypersensitivity.

Exposure of mice to psoralen plus ultraviolet A (320-400 nm) radiation or midrange ultraviolet B (280-320 nm) radiation causes a systemic suppression of the immune response. Although the mechanisms involved in the induction of suppression are not entirely clear, recent studies have demonstrated that ultraviolet B--irradiated keratinocytes release soluble factors that depress delayed-type hypersensitivity to alloantigens and activate the suppressor cell pathway. The purpose of this study was to determine whether PUVA-treated keratinocytes could also cause the release of such immunosuppressive factors. Treatment of keratinocytes with psoralen and UVA radiation induced the release of a factor that depressed the delayed-type hypersensitivity reaction to alloantigen. The suppressive factor was released regardless of whether the psoralen formed monofunctional or bifunctional adducts with DNA and regardless of its phototoxicity. In addition, keratinocytes treated with psoralen and lower doses of UVA radiation released a factor that inhibited contact but not delayed-type hypersensitivity, suggesting that more than one immunosuppressive factor is released following treatment of keratinocytes with appropriate doses of psoralen and UVA radiation. Our findings provide evidence that immunosuppressive factors released from keratinocytes may play a role in the induction of systemic immune suppression following PUVA treatment. Moreover, they demonstrate that PUVA treatment, unlike UVB treatment, can cause the release of more than one immunosuppressive factor from keratinocytes.     

Immunosuppression by factors released from UV-irradiated epidermal cells: selective effects on the generation of contact and delayed hypersensitivity after exposure to UVA or UVB radiation

Exposure of murine epidermal cells to UV radiation in vitro causes the release of immunoregulatory factors that mimic some of the immunosuppressive effects of in vivo UV irradiation. The purpose of this study was to investigate the spectrum of immune responses affected following i.v. injection of supernatants obtained from cultures of epidermal cells exposed in vitro to UV radiation. Treatment of primary epidermal cell cultures or transformed keratinocytes (Pam 212 cells) with UVB (280-320 nm) radiation caused the release of factors that suppressed the induction of delayed hypersensitivity to alloantigen and trinitrophenyl-modified self-antigens in syngeneic and allogeneic mice. Contrary to expectations, however, the injection of supernatants from UVB-irradiated epidermal cells had no effect on the induction of contact hypersensitivity to trinitrochlorobenzene. On the other hand, treatment of the keratinocytes with UVA radiation (320-400 nm, filtered to remove wavelengths in the UVB region) resulted in the release of a factor that suppressed contact but not delayed hypersensitivity. Neither the UVA-induced nor the UVB-induced suppressive factor inhibited the generation of an antibody response to sheep erythrocytes, indicating that, like the suppression that occurs after in vivo exposure to UV radiation, the suppression induced by factors from UV-irradiated keratinocytes is selective in nature. These data support the hypothesis that soluble keratinocyte-derived suppressive factors are involved in the induction of systemic immune suppression by UV radiation. In addition, they suggest that multiple suppressive factors, having different immunosuppressive properties, are produced by different wavelengths of UV radiation.     

Enhancement of cutaneous delayed hypersensitivity reactions by a single exposure to UV-A or PUVA

Summary The influence of irradiation with UV-A and PUVA (8-methoxypsoralen and UV-A) on delayed hypersensitivity reactions to microbial antigens was studied in healthy human individuals. Skin reactions to Candidaalbicans antigen and PPD were enhanced by UV-A als well as by PUVA compared with nonirradiated tests. A statistically significant difference was reached with UV-A for both antigens. For PUVA, erythemogenic doses to Candida tests produced a significant increase of response.     

Enhancement of the elicitation phase of the murine contact hypersensitivity response by prior exposure to local ultraviolet radiation

Local exposure of the sites of elicitation (ears) of contact hypersensitivity (CHS) in mice to relatively modest doses of ultraviolet radiation (FS40 sunlamps) daily for 4 days prior to challenge in previously immunized animals results in significant enhancement of the CHS response as measured by 24-h swelling. This effect was seen in both C3H/HeJ and A/J mice and with the use of both trinitrochlorobenzene and dinitrofluorobenzene as contact sensitizers. Doses of PUVA (parenteral administration of 8-methoxypsoralen followed by exposure to UVA, 320-400 nm, radiation) up to that which can systemically suppress the induction of CHS failed to affect the elicitation of CHS. Irritation of the sites of elicitation of CHS by applications of turpentine also failed to affect the CHS elicitation reaction. Enhancement of the CHS elicitation response by local ultraviolet radiation exposure prior to challenge appears to be a specific photobiologic event.     

Systemic suppression of contact hypersensitivity in mice by psoralen plus UVA radiation (PUVA)

Treatment of mice with 8-methoxypsoralen plus longwave UV radiation (UVA, 320-400 nm) decreased their response to contact sensitizers applied subsequently to unirradiated skin. This decreased reactivity exhibited a delayed time course, it affected the afferent but not the efferent phase of the reaction, and it was associated with the development of splenic suppressor cells. These suppressor cells were antigen-specific T lymphocytes, and they prevented the induction, but not the elicitation, of contact hypersensitivity in recipient mice. In all of these characteristics, the decreased reactivity induced by treatment with psoralen plus UVA radiation (PUVA) resembled that produced by UV radiation of shorter wavelengths (less than 320 nm). These studies suggest that PUVA treatment may initiate the same sequence of cellular events as does exposure to sunlamp (UVB, 280-320 nm) radiation, leading to preferential activation of the suppressor cell pathway.     

UVB irradiation decreases the magnitude of the Th1 response to hapten but does not increase the Th2 response

Exposure of murine skin to low doses of ultraviolet-B (UVB) radiation before sensitization with hapten reduces the ability of antigen presenting cells (APC) in the draining lymph nodes to initiate contact hypersensitivity responses in vivo and results in the induction of hapten-specific suppressor T cells. In the present study, we tested the hypothesis that exposure of skin to UVB radiation suppresses T cell responses to hapten in vivo by altering the functions of APC, resulting in decreased stimulation of Th1 lymphocytes, which mediate contact hypersensitivity responses, and preferential activation of Th2 cells. C3H/HeN mice were exposed to either a single 2 kJ/m² dose of UVB or to 400 J/m² of UVB daily from FS40 sunlamps for four consecutive days and sensitized with fluorescein isothiocyanate on UV-irradiated skin. Draining lymph node cells were collected 18 h after sensitization and co-cultured with nylon wool-purified T cells from naive or fluorescein-immunized mice. Unseparated lymph node cells or sorter-purified fluorescein-bearing APC from UV-irradiated mice induced less T cell proliferation than APC from non-UV-exposed mice. Lymph node cells produced less Th1 and Th2-associated cytokines, interferon-gamma and interleukin-4, respectively, in response to APC from UV-irradiated animals compared with APC from unirradiated, fluorescein-sensitized mice. Thus, low doses of UV radiation do not result in preferential stimulation of Th2 response in lymph nodes, and results from cloned cell lines may incompletely reflect T cell responses in vivo.     

The role of UVB radiation in the induction and elicitation of photocontact hypersensitivity to TCSA in the mouse

Photocontact hypersensitivity (PHS) to 3,3',4',5 tetrachlorosalicylanilide (TCSA) can be induced in mice by using cyclophosphamide as an immunopotentiator. Only UVA (320-400 nm) radiation was required for both sensitization and elicitation of PHS. The reaction was successfully transferred to syngeneic mice by injecting them with lymph node cells from sensitized donors, a finding that demonstrates the immunologic nature of PHS. The presence of UVB (280-320 nm) radiation was not necessary for sensitization and did not increase PHS beyond the levels observed with UVA radiation alone. Ultraviolet radiation in the UVB range (plus a small amount of UVA radiation) from FS40 sunlamps in the dose employed did not induce statistically significant PHS to TCSA, nor did it elicit a significant response in mice sensitized with TCSA plus UVA radiation. However, treatment of mice with UVB radiation at a distant site 6 days before sensitization suppressed the induction of PHS. This suppression appeared to be analogous to the systemic suppression of ordinary contact hypersensitivity in mice by UVB radiation.     

Dietary lutein reduces ultraviolet radiation-induced inflammation and immunosuppression

Ultraviolet radiation (UVR) promotes skin cancer development by mutagenic, immunosuppressive, and oxidative-stress-inducing mechanisms; however, certain antioxidants may counteract and prevent UVR-induced photodamage. Lutein is a xanthophyll carotenoid with potent antioxidant activity. Because reactive oxygen species (ROS) are believed to have a role in UVR-induced skin damage, we investigated whether lutein can modify UVR effects including the tissue swelling response to midrange UVR (280-320 nm, ultraviolet B (UVB) radiation) and UVB suppression of contact hypersensitivity (CHS) in both the local and the systemic models of UV-induced immunosuppression. We found that compared to mice fed the standard laboratory diet, mice fed dietary lutein demonstrated significant inhibition of ear swelling owing to UVB radiation. Mice exposed to 1700 J per m2 UVB radiation four times at daily intervals and then sensitized to dinitrofluorobenzene at the site of irradiation showed a decreased CHS response upon challenge. This suppression by UVB radiation was significantly inhibited by lutein feeding. When UVB radiation was given at a single dose of 10,000 J per m2 to inhibit the induction of CHS at a distant, nonirradiated site, no effect of lutein was seen. Finally, lutein accumulated in the skin of mice following diet supplementation and was shown to decrease ROS generation following UVR exposure. Thus, lutein modulates the skin's response to UVR and may contribute to the defense against some of the deleterious effects of solar radiation.     

Failure of coconut oil to accelerate psoriasis clearance in narrow-band UVB phototherapy or photochemotherapy

Despite a widely held belief that the use of emollients prior to broad-band UVB irradiation accelerates clearance of psoriasis, only one single-blind controlled study exists in support of this. No similar study has been carried out with photochemotherapy (PUVA) or narrow-band UVB (311–313 nm) phototherapy. As some emollients absorb UV radiation, and thereby inhibit psoriasis clearance, there is a need to identify emollients suitable for pre-irradiation use. Coconut oil may be useful in this respect. In two randomized groups of patients with chronic plaque psoriasis undergoing either routine PUVA(n=14) or narrow-band UVB phototherapy (n=15), a single-blind controlled (half-body) study was undertaken to assess the died of pre-irradiation application of coconut oil. Patients were given PUVA twice weekly, or TL-01 therapy thrice weekly. The initial UV dose was 70% of previously determined minimal phototoxic (MPD) or minimal erythema doses (MED), with 40% incremental steps at each visit (reduced if adverse effects occurred). Psoriasis severity was scored on each side after every three treatments. No significant acceleration of psoriasis clearance was seen in either group. We do not, therefore, recommend the routine use of emollients prior to PUVA or TL-01 therapy when using near erythemogenic irradiation regimens.     

Polypodium leucotomos inhibits ultraviolet B radiation-induced immunosuppression

Background: An extract of the tropical fern Polypodium leucotomos (PL) administered orally to mice inhibits ultraviolet B (UVB) radiation-induced skin cancer formation. UVB-induced murine skin cancers occur, in part, because of UVB-induced immunosuppression. Thus, we examined whether PL inhibits UVB-suppression of the induction of contact hypersensitivity (CHS) locally or systemically.
Methods: C57BL/6 mice received standard drinking water or water-containing PL. In the local model, mice were shaved on the dorsum and exposed to 3500 J/m² of UVB radiation daily for 4 days. Control mice were not irradiated. After the last irradiation they were sensitized to oxazolone topically at the irradiated site. To examine the ability of PL to inhibit systemic UVB-induced immunosuppression, mice were given 10 000 J/m² of UVB radiation once and immunized at a non-exposed site 3 days later. Six days after immunization (in both models), mice were challenged on the ears with oxazolone and 24/48 h ear swelling assessed.
Results: PL in drinking water significantly reduced the inhibition of CHS observed with exposure to UVB radiation in both the local and systemic models.
Conclusions: The ability of PL to inhibit UVB radiation-induced immune suppression may explain, in part, its ability to inhibit UVR-induced skin cancer induction in mice.     

A comparison of the dose-response relationship for psoralen-UVA erythema and UVB erythema

Twenty patients with psoriasis were phototested to determine their erythemal responses to UVB and psoralen-UVA (PUVA) (oral 8-methoxypsoralen). The smallest ultraviolet radiation doses to produce erythema (minimal erythema dose and minimal phototoxic dose, respectively) were recorded and dose-response curves were constructed for UVB (24 hours after irradiation) and PUVA (48 hours) using objective measurement. The choice of a 48-hour measurement was validated by phototesting an additional 11 subjects to determine the time course of PUVA erythema. No correlation was demonstrated between minimal erythema dose for UVB, minimal phototoxic dose for PUVA, and sun-reactive skin type. The mean slope of the dose-response curve for UVB erythema was four times steeper than that for PUVA. Psoralen-UVA erythema reached a broad maximum between 48 and 96 hours after irradiation. Using objective methods we have demonstrated that the commonly accepted view of a steep dose-response relationship for PUVA erythema is not valid.     

Doses of ultraviolet radiation that modulate accessory cell activity and ICAM-1 expression are ultimately cytotoxic for murine epidermal Langerhans cells.

Mechanisms that underlie immunomodulatory properties of ultraviolet (UV) radiation remain incompletely characterized. Recently, we have studied effects of UV on the functional activity of epidermal Langerhans cells (LC) and have attempted to relate inhibitory effects of UV on LC function to modulatory effects of UV on adhesion molecule expression by LC. Exposure of LC in vitro to amounts of UVB, UVC, or psoralen+UVA (PUVA) radiation that inhibited LC function also prevented increased expression of intercellular adhesion molecule-1 by LC in vitro. Subsequent studies revealed that amounts of UV radiation that inhibited LC function and modulated ICAM-1 expression also decreased LC survival in vitro, although UV-induced LC cytotoxicity did not become apparent until 48-72 h after UV exposure. Our results are consistent with those of previous studies that suggested that low doses of UV radiation were cytotoxic for LC in situ. The potential cytotoxicity of UV radiation for LC should be considered when studies of effects of UV radiation on immune responses in skin are interpreted.     

Effect of cutaneous hypoxia upon erythema and pigment responses to UVA, UVB, and PUVA (8-MOP + UVA) in human skin

The effect of oxygen deprivation upon UVA-, UVB-, and PUVA-induced pigment and erythema responses in normal human skin was examined. Before exposure, varying degrees of hypoxia in the skin of the forearm were achieved by inflating a sphygmomanometer cuff applied to the upper arm. After the transcutaneously measured pO2 had stabilized, sites on the inner forearm were exposed to UVA, UVB, or 8-MOP + UVA radiation, to determine dose thresholds for the induction of erythema and pigmentation at different cuff pressures. Inflation of the cuff to greater than systolic pressure completely inhibited immediate and delayed pigment responses (IPD, DT) to UVA doses greater than 10 times the normal pigmentation threshold dose. UVA-induced delayed erythema responses were partially inhibited by cuff inflation: 2.7 times the minimal erythema dose of UVA was necessary to cause an erythema response when exposure occurred during vascular occlusion. In contrast, erythema and pigment responses to UVB and PUVA were unaltered by cuff pressures exceeding systolic pressure during exposure. Inhibition of UVA-induced erythema and pigment responses by vascular occlusion were reversed by the transcutaneous diffusion of 100% O2. These findings indicate that the cutaneous responses to UVA and UVB occur by separate pathways differing with respect to O2 dependence. Our findings agree with those of other studies which indicate that PUVA-induced phototoxicity and melanogenesis are not O2-dependent.     

Unscheduled DNA synthesis in normal human skin after single and combined doses of V-A, UV-B and UV-A with methoxsalen (PUVA)

The aim of this study was to measure unscheduled DNA synthesis (UDS) by autoradiography in normal htiman skin (1) after high dose UV-A, (2) after low dose UV-A applied before or after erythemogenic doses of UV-B, (3) after high dose PUVA and (4) after therapeutic doses of PUVA applied before and after erythemogenic doses of UV-B. Single high dose UV-A exposure induced rougbly 60% of the amount of UDS induced by equally erythemogenic doses of UV-B. Single low dose UV-A exposure did not induce UDS, nor did it significantly alter the amount of UV-B induced UDS when combined with UV-B exposure. Single high dose PUVA did not lead to UDS and had no influence on UV-B induced UDS when combined with UV-B exposure. Our findings indicate: (1) erythemogenic doses of UV-A induce a considerable DNA excision repair; (2) low dose UV-A neither augments UV-B induced DNA repair nor does it inhibit the repair process; (3) no UDS was shown to occur after either high or therapeutic doses of PUVA. This was unexpected since psoralen-DNA monoadducts have been shown to be repairable by a mechanism similar to excision repair of pyrimidine dimers. It is therefore assumed tbat PUVA as performed for therapeutic purposes either preferentially induced interstrand crosslinks not repairable via the classical repair mechanism or the repair of monoadducts was below resolution in this study; (4) therapeutic PUVA doses apparently do not interfere with excision repair of UV-B induced DNA lesions.     

Exposure to low-dose ultraviolet radiation suppresses delayed-type hypersensitivity to herpes simplex virus in mice

Ultraviolet B (UVB) radiation is reported to induce a defect in epidermal antigen presentation which leads to specific suppression of the delayed-type hypersensitivity (DTH) response to trinitrochlorobenzene. We have used a similar system to examine the murine DTH response to herpes simplex virus type 1 (HSV-1). Mice irradiated with 96 mJ/cm2 UVB on shaved dorsal skin 3 days before s.c. injection of live HSV-1 in the flank showed 54-92% suppressed DTH responses to challenge with inactivated virus compared with nonirradiated control animals. If irradiation took place 7 days before inoculation with virus, some suppression of DTH occurred; if 14 days before, no suppression was found. The transient nature of the UVB response is further illustrated by the observation that irradiation with the same dose of UVB 5 h before, or 3 days after, inoculation with virus had no effect on DTH. Once induced, some degree of UVB suppression was found to persist for at least 3 months after irradiation.     

A possible role of prostaglandins in PUVA-induced inflammation: implication by organ cultured skin

The role of arachidonate metabolites in UV-induced inflammation was analyzed by measuring the amounts of prostaglandins or leukotrienes released into a culture medium in the organ culture of biopsied guinea pig skin following UV exposure. Increased concentrations of prostaglandin E2, F2 alpha, and 6-oxo-F1 alpha were found in the medium subjected to UVB radiation followed by 24 h incubation in the organ culture, compared to medium from non-exposed skin, whereas LTC4 was at an undetectable level in both. This release occurred in UVB dose-dependent fashion and was completely inhibited by the addition of indomethacin to the medium. Exposure to monochromatic UV light of 250, 300, and 350 nm released prostaglandin E2, F2 alpha, and 6-oxo-F1 alpha, in increasing order of 250, 300, and 350 nm, but not LTC4. This order of amount released almost paralleled the intensity of inflammation that occurred with each wavelength. Exposure of skin to UVA following intraperitoneal injection of 8-methoxypsoralen (PUVA) was also found to stimulate the synthesis of prostaglandin E2 in UVA dose-dependent fashion, and the stimulation through an increasing dose of UVA occurred more significantly than UVB exposure. The time course of prostaglandin release following UVB and PUVA exposures was found to have a pattern similar to their dynamics in association with erythema and edema, conditions which could be significantly diminished by the topical application of indomethacin. The present studies demonstrate that prostaglandins play an important role in the production of inflammation by UVB as well as UVA and PUVA.     

The influence of ultraviolet radiation on allergic contact dermatitis in the guinea-pig. I. UVB radiation

Guinea-pigs were sensitized by percutaneous application of dinitrochlorobenzene and exposed to UVB (280–320 nm) radiation. The exposure to radiation diminished the response to an elicitation dose of the hapten administered 14 days later within the site of irradiation. The exposure dose of radiation required to produce this effect resulted in a marked erythemal response, but this response did not conceal the contact allergic reaction. The site of elicitation of the allergic response had to be included in the exposure field.     

Prophylactic PUVA and UVB therapy in polymorphic light eruption—a controlled trial

A double-blind controlled trial of low-dose prophylactic oral psoralen photochemotherapy (PUVA) and ultraviolet-B (UVB) irradiation therapy was undertaken from April to September 1983 in 42 patients with polymorphic light eruption (PLE). Patients were randomly allocated to three groups, PUVA with oral 8-methoxypsoralen (8-MOP), UVB with oral placebo, and control low-dose UVA with oral placebo. The initial dose given to each active treatment group was a third of the predetermined minimal phototoxic or erythema dose, followed three times weekly for 6 weeks by doses incremented by an eighth on each occasion in the PUVA group and by a seventh in the UVB group. Ultraviolet radiation exposure was monitored throughout with polysulphone film lapel badges. Patients recorded their symptoms on a visual analogue scale. Symptoms of rash and itch in patients treated with PUVA and UVB were significantly less affected by increasing exposure to ultraviolet radiation than were these symptoms in control patients.     

The effect of ultraviolet B irradiation, cis-urocanic acid and tumour necrosis factor-alpha on delayed hypersensitivity to herpes simplex virus.

Previously it has been shown that ultraviolet-B (UVB) irradiation or cis-urocanic acid (cis-UCA) treatment of mice before infection with herpes simplex virus (HSV) suppressed the delayed hypersensitivity (DH) response when the mice were subsequently challenged with inactivated virus. In the present study, the time course of the elicitation phase of the DH was examined, and it was found to be biphasic with one peak 1 h following challenge and a second at 24 h. Both UVB irradiation and cis-UCA treatment of mice before infection with HSV significantly suppressed the DH at 1 h as well as at 24 h. The role of tumour necrosis factor-alpha (TNF-alpha) in the suppression was tested by injecting mice intraperitoneally with neutralizing TNF-alpha antibodies 2 h before UVB irradiation or cis-UCA treatment followed by infection with HSV. This had no effect on the suppression of DH to HSV induced by cis-UCA but significantly reduced that generated by UVB exposure. Thus, the mechanism of suppression of DH induced by UVB irradiation or cis-UCA may be different.