Cyclobutane pyrimidine dimer formation is a molecular trigger for solar-simulated ultraviolet radiation-induced suppression of memory immunity in humans.

We tested the hypothesis that DNA is a target for solar-simulated ultraviolet radiation (ssUVR)-induced suppression of the reactivation of memory immunity in humans. T4N5 liposomes contain the DNA repair enzyme T4 endonuclease V. This cleaves DNA at the site of ultraviolet radiation (UVR)-induced cyclobutane pyrimidine dimers (CPD), initiating DNA repair. It has previously been used to show that CPDs are a key molecular trigger for UVR-induced immunosuppression in mice. To determine whether CPD formation is involved in UVR immunosuppression in humans, nickel-allergic volunteers were irradiated with a range of doses of ssUVR. T4N5 or empty liposomes were then applied after irradiation. Nickel-induced recall immunity was assessed by reflectance spectrometry. T4N5 liposomes inhibited immunosuppression and prevented ssUVR from reducing the number of epidermal dendritic cells. T4N5 liposomes also reduced macrophage infiltration into irradiated epidermis. These studies show that enhanced removal of CPDs from human skin protects from immunosuppression, hence demonstrating that these photolesions are an important molecular event in ssUVR-induced immunosuppression in humans. CPDs also triggered loss of dendritic cells and infiltration by macrophages. It is possible that these changes to antigen presenting cells contribute to ssUVR induced suppression of recall immunity to nickel in humans.     

Infiltration by inflammatory cells required for solar-simulated ultraviolet radiation enhancement of skin tumor growth

In this study we compared the effects of subinflammatory and inflammatory doses of solar-simulated ultraviolet (UV) radiation on enhancement of skin tumor growth, sensitization to haptens and cellular changes within the epidermis of C3H/HeN mice. Tumors transplanted into mice 3 days after exposure to inflammatory, but not subinflammatory, doses of UV radiation had a higher growth rate than those tumors inoculated into unirradiated control mice. Both doses of UV radiation suppressed the induction of contact hypersensitivity and induced tolerance when hapten was painted onto the skin 3 days after irradiation. Skin exposed to the higher, but not the lower, dose of UV radiation contained significantly increased numbers of CD11b+, CD45+ MHC class II- and CD45+ MHC class II(hi) inflammatory cells 3 days post-irradiation. The immunosuppression correlated with a reduction in Langerhans cells and dendritic epidermal T cells. Collectively, this suggests that suppression to contact sensitizers is due to the UV radiation effects on Langerhans cells and dendritic epidermal T cells. While these effects may also suppress the induction of anti-tumor immunity, at higher doses of UV radiation inflammatory cells may enhance tumor growth by a non-immunological mechanism.     

The suppression of immunity by ultraviolet radiation: UVA, nitric oxide and DNA damage.

We have examined the mechanism by which solar-simulated ultraviolet radiation (ssUV) suppresses memory immunity to nickel in allergic humans. In initial studies, we used inbred mice to determine the contribution of different wavebands to sunlight-induced immunosuppression. We found that low dose UVA can enhance memory, medium dose UVA (half the amount in one minimum erythemal dose of ssUV) is immunosuppressive, but higher doses protect from UVB. This is genetically dependent, as it is not observed in all mouse strains. UVA caused a similar dose-related change in recall immunity in humans. ssUV dose responses determined the limits of protection provided by sunscreens from immunosuppression in humans. Immune protection factors calculated from these data correlated with UVA protection, but not with sun protection factor, showing that in commercial sunscreens that provide good UVB protection, UVA protection limits prevention of immunosuppression. N(G)-monomethyl-l-arginine acetate (l-NMMA) was used to inhibit nitric oxide (NO) production and T4N5 liposomes containing T4 endonuclease V to enhance DNA repair. Sub-erythemal ssUV caused a dose-related local suppression of recall immunity to nickel in humans. l-NMMA and the liposomes protected the nickel reaction, suggesting that NO and DNA damage are mediators of UV-induced immunosuppression in humans.     

Suppression of the cutaneous immune response following topical application of the prostaglandin PGE2

UVB irradiation (290-320 nm) and topical applications of arachidonic acid (AA) in mice decrease the number of identifiable Langerhans cells and alter the cutaneous immune response. Application of contact allergens such as dinitrofluorobenzene (DNFB) to irradiated or AA-treated skin induces antigen-specific tolerance. Indomethacin (IM), a cyclooxygenase inhibitor, administered orally to mice prior to UVB irradiation or prior to the topical application of arachidonic acid, abrogates suppression of contact hypersensitivity (CHS) to DNFB. This suggests a byproduct of arachidonic acid generated through the cyclooxygenase pathway may be involved in the immune suppression. Topical application of various prostaglandins (PGE2, PGD2, PGF2 alpha, and CTXA2) did not cause alterations in the population density of the identifiable Ia+ dendritic Langerhans cells. PGE2, but no other tested agent, produced a suppression of the CHS response to DNFB. These observations suggests that of the various prostaglandins, PGE2 might be one of several biochemical signals which mediate the suppression of contact hypersensitivity reactions following ultraviolet radiation exposure. However, the mechanisms by which PGE2 produces its suppressive effects have not been identified.     

Induction and persistence of suppression of contact hypersensitivity against bystander haptens and alloantigens in rats

The shift of suppression from a tolerizing hapten to a so-called bystander antigen was investigated in this study using contact hypersensitivity to trinitrochlorobenzene (TNCB) and dinitrofluorobenzene (DNFB) and delayed type hypersensitivity (DTH) to alloantigens in the rats as experimental models. Primary suppression of contact hypersensitivity was induced by intravenous injection of the water-soluble forms of TNCB and DNFB. A shift of the suppression to the bystander hapten was found if the tolerizing and bystander hapten were mixed and applied to the same area of skin during the sensitization procedure, but not if they were applied to separate areas of skin. With alloantigens, bystander suppression developed only when the sensitizing allogeneic cells were mixed with hapten-modified syngeneic cells. It was not induced by hapten-modified allogeneic cells. Once induced, such bystander suppression of the response to haptens persisted independently of the primarily tolerizing hapten, and it could be adoptively transferred with spleen cells. These results favour the concept that the bystander suppression is mediated by the non-specific action of suppressor cells generated specifically during the mixed sensitization rather than by an antigen bridge.     

Neonatal exposure to UVR alters skin immune system development, and suppresses immunity in adulthood

Neonates have a developing immune response, with a predisposition towards induction of tolerance. As the immune system develops, immunity rather than tolerance is induced, with this development of immunity occurring in response to external factors such as the environment. As ultraviolet radiation (UVR) suppresses immunity, it is likely that the effect of UVR on the neonatal immune system would be augmentation of the suppressive response. In support, childhood exposure to UVR has been linked with an increased incidence of melanoma; consistent with an increase in suppression. To address this, phenotypic and functional immune system studies were undertaken at 8 weeks after one single exposure of solar-simulated UVR to mice, when mice had reached adulthood. Subtle changes were observed in cell populations resident in the skin-draining lymph nodes (LNs) and there also appeared to be a subtle, but not statistically significant, increase in the production of interleukin-10 and interferon-γ. Importantly, these changes also corresponded with significant suppression of the contact hypersensitivity response in irradiated mice compared with their control counterparts. This suppression was apparent when antigen sensitisation occurred during the neonatal or adult period, and thus did not appear to be analogous to UVR-induced suppression in adults. Although the percentage of T regulatory cells was increased in the skin-draining LNs, they were induced in a different manner to those induced following adult UVR exposure, with no increase in function on a per-cell basis. It therefore appears that one single neonatal exposure to UVR alters development of the immune system, leading to long-term implications for induction of immunity.     

Photoimmune protective effect of the phytoestrogenic isoflavonoid equol is partially due to its antioxidant activities

Topical application of lotions containing the phytoestrogenic isoflavonoid equol have been reported to protect mice against UV radiation-induced inflammation, immune suppression and photocarcinogenesis. The photoimmune protective property was shown to depend on equol's activation of oestrogen receptor signalling in the skin. However, isoflavones are also recognised for their antioxidant properties in biological systems. As endogenous cutaneous antioxidant enzymes including the inducible stress protein haem oxygenase (HO)-1, have photoprotective efficacy, this study in the Skh:hr-1 hairless mouse seeks evidence for an antioxidant role for equol in contributing to its photoimmune protection. Oxidative stress has been measured as UVA-induced lipid peroxidation in the mouse skin, and was dose-dependently inhibited by topical equol. Inhibition of the UVA (320-400 nm)-inducible HO activity significantly reduced the level of equol protection against lipid peroxidation, thereby attributing a component of equol's lipid protection capacity to this stress enzyme. It was consistent that topical equol enhanced the level of HO induction by UVA irradiation in both skin and liver. Subsequently, the dose-dependent protection by topical equol lotions against solar simulated UV radiation induced immunosuppression, measured by the contact hypersensitivity reaction, was found also to be partially reduced by the inhibition of HO activity. Therefore, in addition to the activation by equol of oestrogenic signalling pathways for photoprotection, this isoflavonoid also provides UV-protective antioxidant effects that depend partially on HO-1 induction.     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

Therapeutic Efficacy and Immunological Response of CCL5 Antagonists in Models of Contact Skin Reaction

Skin-infiltrating T-cells play a predominant role in allergic and inflammatory skin diseases such as atopic dermatitis, psoriasis and allergic contact dermatitis. These T-cells are attracted by several chemotactic factors including the chemokine CCL5/RANTES, a CC chemokine inducing both the migration and activation of specific leukocyte subsets. CCL5 has been found to be associated with various cell-mediated hypersensitive disorders such as psoriasis, atopic dermatitis and irritant contact dermatitis. We have used two antagonists, the first, Met-CCL5, a dual CCR1/CCR5 antagonist and the second, a variant in which GAG binding is abrogated, ⁴⁴AANA⁴⁷-CCL5, which acts as a dominant negative inhibitor of CCL5. The antagonists were tested in two models of contact skin reaction. The first, irritant contact dermatitis (ICD) is a pathological non-specific inflammatory skin condition arising from the release of pro-inflammatory cytokines by keratinocytes in response to haptens, usually chemicals. The second, contact hypersensitivity (CHS) is a T-cell dependent model, mimicking in part the T-cell-mediated skin diseases such as psoriasis. In both models, the CCL5 antagonists showed therapeutic efficacy by reducing swelling by 50% as well as the reduction of soluble mediators in homogenates derived from challenged ears. These results demonstrate that blocking the receptor or the ligand are both effective strategies to inhibit skin inflammation.     

Measurement of ultraviolet radiation-induced suppression of recall contact and delayed-type hypersensitivity in humans

This article describes methodology used for assessment of ultraviolet radiation-induced suppression of recall responses in humans. Nickel allergy is common in the general population and patch testing of nickel-allergic volunteers provides a convenient model of contact hypersensitivity. Similarly, Mantoux-positive volunteers, recruited from within hospital staff, are used as a model for delayed-type hypersensitivity. Use of secondary rather than primary immune responses allows placement of multiple test sites on each volunteer. Further, each volunteer acts as his or her own unirradiated control. This enables UV immunosuppression to be studied with relatively few human volunteers, and makes determination of UV immunosuppression dose responses feasible in human subjects. The method can also be used for assessment of the level of immune protection afforded by agents such as sunscreens or biologically active substances.     

Nickel allergy in mice: enhanced sensitization capacity of nickel at higher oxidation states.

Attempts to induce contact hypersensitivity to nickel in mice using, e.g., Ni(II)Cl2 often failed. Here, we report that sensitization was achieved by injecting Ni(II)Cl2 in combination with either CFA or an irritant, such as SDS and PMA, or IL-12, or by administering nickel at higher oxidation states, i.e., Ni(III) and Ni(IV). Although Ni(II), given alone, was ineffective in T cell priming, it sufficed for eliciting recall responses in vivo and in vitro, suggesting that Ni(II) is able to provide an effective signal 1 for T cell activation, but is unable to provide an adequate signal 2 for priming. Immunization of mice with nickel-binding proteins pretreated with Ni(IV), but not with Ni(II), allowed them to generate nickel-specific CD4+ T cell hybridomas. Ni(II) sufficed for restimulation of T cell hybridomas; in this and other aspects as well, the hybridomas resembled the nickel-specific human T cell clones reported in the literature. Interestingly, restimulation of hybridomas did not require the original Ni(IV)-protein complex used for priming, suggesting either that the nickel ions underwent ligand exchange toward unknown self proteins or peptides or that nickel recognition by the TCR is carrier-independent. In conclusion, we found that Ni(III) and Ni(IV), but not Ni(II) alone, were able to sensitize naive T cells. Since both Ni(III) and Ni(IV) can be generated from Ni(II) by reactive oxygen species, released during inflammation, our findings might explain why in humans nickel contact dermatitis develops much more readily in irritated than in normal skin.     

Effect of inhibitors of oxygen radical and nitric oxide formation on UV radiation-induced erythema, immunosuppression and carcinogenesis

We investigated whether supplementation of a sunscreen containing the UVB absorber 2-ethyl-hexyl-methoxycinnamate (cinnamate) with oxygen radical inhibitors (ORI) would improve protection from sunburn, immunosuppression and carcinogenesis. Mice were exposed to solar-simulated UV radiation (ssUV) containing a mixture of UVB and UVA. In initial studies, the ORI 2,2'-dipyridyl and N(G)-monomethyl-L-arginine acetate (L-NMMA) were shown to prevent UVA-induced suppression of contact sensitivity (CS) in mice. Addition of these inhibitors to the sunscreen did not affect the sun protection factor (SPF), but lowered the level of edema when mice were exposed to ssUV. Combination of both inhibitors with the sunscreen, however, increased the SPF from 5 to 5.5. The immune protection factor (IPF) of the sunscreen was only 1.18, but addition of neither dipyridyl nor L-NMMA singly or in combination measurably improved immune protection. However, the ORI improved the ability of the sunscreen to prevent carcinogenesis. The results indicate that reactive oxygen or nitrogen species produced in response to UV radiation are important for erythema, immunosuppression and carcinogenesis, and addition of inhibitors improves the protective capacity of sunscreens.     

Effect of inhibitors of oxygen radical and nitric oxide formation on UV radiation-induced erythema,immunosuppression and carcinogenesis

Abstract

We investigated whether supplementation of a sunscreen containing the UVB absorber 2-ethyl-hexyl-methoxycinnamate (cinnamate) with oxygen radical inhibitors (ORI) would improve protection from sunburn, immunosuppression and carcinogenesis. Mice were exposed to solar-simulated UV radiation (ssUV) containing a mixture of UVB and UVA. In initial studies, the ORI 2,2′-dipyridyl and N^G-monomethyl-L-arginine acetate (L-NMMA) were shown to prevent UVA-induced suppression of contact sensitivity (CS) in mice. Addition of these inhibitors to the sunscreen did not affect the sun protection factor (SPF), but lowered the level of edema when mice were exposed to ssUV. Combination of both inhibitors with the sunscreen, however, increased the SPF from 5 to 5.5. The immune protection factor (IPF) of the sunscreen was only 1.18, but addition of neither dipyridyl nor L-NMMA singly or in combination measurably improved immune protection. However, the ORI improved the ability of the sunscreen to prevent carcinogenesis. The results indicate that reactive oxygen or nitrogen species produced in response to UV radiation are important for erythema, immunosuppression and carcinogenesis, and addition of inhibitors improves the protective capacity of sunscreens.     

L-ascorbic acid microencapsulated with polyacylglycerol monostearate for milk fortification

Efficiency was examined of microencapsulating L-ascorbic acid by polyglycerol monostearate (PGMS), and changes in the chemical and sensorial aspects of L-ascorbic acid and/or iron-fortified milk during storage were evaluated. The selected core materials were ferric ammonium sulfate and L-ascorbic acid. The highest efficiency (94.2%) of microencapsulation was found with the ratio of 5:1 as the coating to core material. The release of ascorbic acid from the microcapsules increased sharply from 1.6 to 6.7% up to 5 d of storage. The TBA value was the lowest in the milk sample with added encapsulated iron and unencapsulated L-ascorbic acid up to 5 d of storage in comparison with the other treated samples. A sensory analysis showed that most aspects were not significantly different between the control and fortified samples encapsulated with ascorbic acid after 5 d of storage. The results indicate that L-ascorbic acid microencapsulated with PGMS can be applied to fortify milk and acceptable milk products can be prepared with microencapsulated L-ascorbic acid and iron.     

Studies on the role of antigen-presenting cells in the systemic suppression of contact hypersensitivity by UVB radiation

Exposure of mice to UVB radiation produces a highly selective, systemic immunosuppression associated with the appearance of suppressor T lymphocytes. Suppression of delayed hypersensitivity to hapten-coupled syngeneic cells has been shown to result from an altered distribution of antigen-presenting cells. The purpose of this study was to determine whether an alteration in the activity of antigen-presenting cells could account for the systemic suppression of contact hypersensitivity (CHS) by UVB radiation. Fluorescein isothiocyanate (FITC) was used for contact sensitization because it uses different antigen-presenting cells than does oxazolone to induce CHS. Our previous studies demonstrated that CHS to oxazolone was suppressed by UVB irradiation. In these studies, we show that exposure of mice to UVB radiation before epicutaneous application of FITC onto unirradiated skin markedly decreased the CHS response to FITC painted on unexposed ears. Cyclophosphamide-sensitive suppressor T cells were detectable in the spleens of mice exhibiting decreased CHS. The antigen-presenting activity of cells in lymph nodes draining the site of epicutaneous sensitization (DLN cells) was assessed by injecting them into the hind footpads of syngeneic recipients and measuring the CHS response to FITC 6 days later. Viable DLN cells from UVB-irradiated, FITC-sensitized mice were equal to those from unirradiated, FITC-sensitized mice in their ability to induce CHS in normal recipients. No sensitization resulted when killed DLN cells were used for immunization, indicating that sensitization was not caused by reprocessing of antigen by host cells. We conclude that impairment of the CHS reaction in UVB-irradiated mice does not appear to be blocked at an initial step of antigen uptake, processing, or presentation, but must be impaired at some other step in the immunologic pathway.     

Ultraviolet radiation and the contact hypersensitivity reaction in mice

The article reviews the application of the contact hypersensitivity assay in mice to the science of photoimmunology. The contact hypersensitivity (CHS) reaction, which is suppressed by UV irradiation in mice similarly to their ability to respond immunologically to skin tumors, has been used very profitably to reveal many of the regulating factors that control photoimmunosuppression, such as the identity of the photoreceptors that initiate immunosuppression, the defects induced in the cutaneous antigen presenting pathway, the local cytokine imbalance, and the protective intervention by various molecules, drugs, or interacting UV wavebands. Technical hints to optimize the measurement of the CHS response are suggested, including information on UV radiation wavebands and dosages and sensitivities of different mouse strains.     

Sensitivity of human diploid fibroblast cell strains from various genetic disorders to acute and protracted radiation exposure

The cytotoxic effect of acute X irradiation was studied by a colony formation assay in 114 human skin fibroblast cell strains from 31 apparently normal individuals and 83 patients with a variety of genetic disorders possibly associated with in vitro hypersensitivity to ionizing radiation. The effect of protracted exposure to beta radiation from tritiated water (HTO) was examined in parallel experiments in 65 of these strains. The disorders included neurological diseases and syndromes characterized by an increased susceptibility to spontaneous and radiation-induced cancer. Homozygous ataxia telangiectasia and Nijmegen break syndrome cells were highly sensitive to both types of radiation. However, the response of cells from the other genetic disorders fell within the broad range characteristic of normal cell strains. While HTO may be useful as a quantitative method for determining the cytotoxic response of human diploid cells to ionizing radiation, the present results indicate that it does not offer a more sensitive assay than acute X irradiation for detecting minor degrees of hypersensitivity.     

Influence of antioxidant (L- ascorbic acid) on tolbutamide induced hypoglycaemia/antihyperglycaemia in normal and diabetic rats

BACKGROUND: Diabetes mellitus is a chronic metabolic disorder characterized by hyperglycaemia. Increased oxidative stress and decreased antioxidant levels are the leading cause of diabetes and diabetic complications. So it is felt that supplementation of antioxidants may be useful in controlling the glucose levels and to postpone the occurrence of diabetic complications. The objective of our study is to find the influence of antioxidant supplementation (L-ascorbic acid) on tolbutamide activity in normal and diabetic rats. METHODS: L- ascorbic acid/tolbutamide/L-ascorbic acid + tolbutamide were administered orally to 3 different groups of albino rats of either sex in normal and diabetic condition. Blood samples were collected from retro-orbital puncture at different time intervals and were analyzed for blood glucose by GOD-POD method. Diabetes was induced by alloxan 100 mg/kg body weight administered by I.P route. RESULTS: L-ascorbic acid/ tolbutamide produced hypoglycaemic activity in a dose dependant manner in normal and diabetic condition. In the presence of L-ascorbic acid, tolbuatmide produced early onset of action and maintained for longer period compared to tolbutamide matching control. CONCLUSION: Supplementation of antioxidants like L-ascorbic acid was found to improve tolbutamide response in normal and diabetic rats.     

Conditioned suppression of contact sensitivity is independent of sympathetic splenic innervation

The present study investigated the role of sympathetic innervation of the spleen in conditioned suppression of a contact hypersensitivity (CHS) reaction. Behavioral conditioning was achieved by pairing saccharin drinking solution (conditioned stimulus, CS) with injection of cyclosporin A (CsA, 20 mg/kg; unconditioned stimulus, UCS). Four days after sensitization of the animals by application of a 5% 2,4-dinitrochlorobenzene (DNCB) to abdominal skin, the animals were challenged by applying a 1% DNCB solution to the ear. The CHS response was monitored by measuring the degree of ear swelling. Saccharin re-presentation reduced ear swelling to a magnitude that approached that achieved by CsA treatment. Histological examination demonstrated that the conditioned reduction of ear swelling was produced by a reduced leukocyte infiltration of the ear. Prior sympathetic denervation of the spleen did not alter the conditioned suppression of the CHS response. These data indicate that behavioral conditioning using CsA produces alterations of CHS that, unlike conditioned prolongation of heart allograft survival, are independent of sympathetically regulated conditioned alterations in the spleen.