Ozone depletion and the immune system.
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Animals↗
Biomedical subjects
M L Kripke
Publications and source records attributed to M L Kripke.
High frequency of p53 mutations in ultraviolet radiation-induced murine skin tumors: evidence for strand bias and tumor heterogeneity.
Exposure to UV radiation has long been associated with the development of skin cancers. To identify the molecular targets in UV carcinogenesis, we analyzed 11 UV-induced murine skin cancers for mutations in the p53 tumor suppressor gene and found a 100% incidence rate. Such a high frequency of p53 mutations is unprecedented and suggests that this gene plays an important role in the development of UV-induced skin cancers. The mutations were predominantly "UV-signature" transitions (C-->T and CC-->TT) at pyrimidine-rich sequences located on the nontranscribed strand of the gene. In addition, seven tumors harbored multiple mutant alleles of p53, providing strong evidence for tumor heterogeneity at the molecular level.
Alleles↗
Experimental ultraviolet carcinogenesis.
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Animals↗
Effects of sunscreens and a DNA excision repair enzyme on ultraviolet radiation-induced inflammation, immune suppression, and cyclobutane pyrimidine dimer formation in mice.
Exposure of skin to ultraviolet (UV) radiation inhibits the induction of delayed-type hypersensitivity (DTH) responses initiated at a distant, unirradiated site. Recent studies attributed this form of immune suppression to DNA damage in the form of cyclobutane pyrimidine dimers (CPD). In the present study, we investigated the protective defects of sunscreens on UV-induced systemic suppression of DTH to Candida albicans, inflammation, and DNA damage. The photoprotective effects of sunscreen preparations containing 8% octyl-N-dimethyl-p-aminobenzoate, 7.5% 2-ethylhexyl-p-methoxycinnamate, or 6% benzophenone-3 were studied in C3H mice exposed to a single dose of 500 mJ/cm2 UVB radiation from FS40 sunlamps. Inflammation was determined by the amount of skin edema at the site of UV irradiation, and DNA damage was assessed by measuring the frequency of endonuclease-sensitive sites in the epidermis. Application of the sunscreens before UV irradiation gave 75-97% protection against UV-induced edema, 67-91% protection against formation of CPD, but only 30-54% protection against suppression of DTH. In contrast, the topical application of liposomes containing a CPD-specific DNA repair enzyme immediately after UV irradiation resulted in 82% protection against suppression of DTH, but at best, 39% protection against skin edema. These findings demonstrate that sunscreens give less protection against UV-induced immune suppression than against skin edema and CPD formation. Furthermore, they suggest that less DNA damage is required to cause UV-induced immune suppression than to cause sunburn.
Animals↗
Analysis of the protective effect of different sunscreens on ultraviolet radiation-induced local and systemic suppression of contact hypersensitivity and inflammatory responses in mice.
We investigated the capacity of three sunscreen compounds to protect mice from the inflammatory and immunosuppressive effects of ultraviolet radiation (UVR). The sunscreen preparations contained 7.5% 2-ethylhexyl-p-methoxycinnamate, 8% octyl-N-dimethyl-p-aminobenzoate, or 6% benzophenone-3 in an oil-in-water emulsion. Skin swelling was used as the measure of their effect on UVR-induced inflammation, and immunosuppression was assessed by contact sensitization with 2,4-dinitrofluorobenzene applied to UV-irradiated skin (local suppression) or a distant site (systemic suppression). The sunscreens were applied to the shaved dorsal skin of C3H mice, which were then given a single dose of UVR ranging from 2 to 32 kJ/m2 within the UVB (280-320 nm) region. All three sunscreens gave complete protection against local suppression of contact hypersensitivity caused by a dose of 2 kJ/m2 UVB. They also protected against both inflammation and systemic immunosuppression caused by UVR; however, protection was highly dependent on the UVR dose. Furthermore, the sunscreens were less effective in protecting against systemic immunosuppression than against inflammation. These results indicate that immunosuppression is less sensitive to the protective effects of the sunscreens than inflammation and that protection against UVR-induced inflammation does not necessarily imply prevention of immunologic alterations. In addition, these studies suggest that UVR-induced immunosuppression and inflammation may involve different mechanisms.
Animals↗
Effect of UV irradiation on lethal infection of mice with Candida albicans.
Exposure of mice to UV radiation inhibits the induction and elicitation of the delayed-type hypersensitivity (DTH) response to Candida albicans. To determine whether UV irradiation also affects the pathogenesis of systemic C. albicans infection, C3H mice were exposed to a single dose of 48 kJ/m2 UV-B radiation from FS40 sunlamps 5 days before or 5 days after sensitization with formalin-fixed C. albicans and challenged intravenously (i.v.) with a lethal dose of viable fungi 6 days after sensitization (11 or 1 days after UV irradiation). Exposing unsensitized mice to UV radiation 11 days before lethal challenge had no effect on survival, but the survival time of mice exposed to UV radiation 1 day before challenge was reduced by more than 50%. In the latter group, decreased survival time correlated with persistence of C. albicans in the brain and progressive growth of C. albicans in the kidneys. Sensitization of unirradiated mice with formalin-fixed C. albicans extended their survival time following lethal i.v. challenge with viable C. albicans. Exposing the mice to UV radiation 5 days before sensitization did not abrogate this beneficial effect of sensitization on survival, even though it significantly reduced the DTH response. Thus, immunity to systemic infection did not depend on the ability of the mice to exhibit a DTH response to C. albicans. The beneficial effect of sensitization on survival after lethal infection was abrogated, however, in mice exposed to UV radiation 1 day before lethal challenge with C. albicans. Furthermore, these mice were unable to contain the progressive growth of C. albicans in the kidneys, in contrast to sensitized, unirradiated mice.
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Pyrimidine dimers in DNA initiate systemic immunosuppression in UV-irradiated mice.
Exposing the skin of mice to UV radiation interferes with the induction of delayed and contact hypersensitivity immune responses initiated at nonirradiated sites. The identity of the molecular target in the skin for these immunosuppressive effects of UV radiation remains controversial. To test the hypothesis that DNA is the target for UV-induced systemic immunosuppression, we exposed C3H mice to UV radiation and then used liposomes to deliver a dimer-specific excision repair enzyme into the epidermis in situ. The application of T4 endonuclease V encapsulated in liposomes to UV-irradiated mouse skin decreased the number of cyclobutane pyrimidine dimers in the epidermis and prevented suppression of both delayed and contact hypersensitivity responses. Moreover, the formation of suppressor lymphoid cells was inhibited. Control, heat-inactivated endonuclease encapsulated in liposomes had no effect. These studies demonstrate that DNA is the major target of UV radiation in the generation of systemic immunosuppression and suggest that the primary molecular event mediating these types of immunosuppression by UV radiation is the formation of pyrimidine dimers. Furthermore, they illustrate that the delivery of lesion-specific DNA repair enzymes to living skin after UV irradiation is an effective tool for restoring immune function and suggest that this approach may be broadly applicable to preventing other alterations caused by DNA damage.
Analysis of Variance↗
N-ras mutation in ultraviolet radiation-induced murine skin cancers.
UV radiation is a potent DNA-damaging agent and a known inducer of skin cancer in experimental animals. To elucidate the role of oncogenes in UV carcinogenesis, we analyzed UV-induced murine skin tumors for mutations in codon 12, 13, or 61 of Ha-ras, Ki-ras, and N-ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction followed by dot-blot hybridization to synthetic oligonucleotide probes designed to detect single base-pair mutations. In addition to UV-induced C3H mouse skin tumors, we also analyzed skin tumors induced in the same strain of mice by other carcinogenic agents such as 8-methoxypsoralen + UVA, angelicin + UVA, dimethylbenz-[a]anthracene + UV + croton oil, and 4-nitroquinoline-1-oxide. We found that 4 of 20 UV-induced skin tumors contained either C----A or A----G base substitutions at N-ras codon 61. In addition, 2 of 5 melanomas possessed a G----A transition in N-ras codon 13 and an A----T transversion in N-ras codon 61, respectively. Interestingly, none of the 8-methoxypsoralen + UVA- or angelicin + UVA-induced tumors we analyzed contained mutations in any of the ras genes. However, 1 of 4 4-nitroquinoline-1-oxide-induced tumors exhibited a G----T transversion at Ki-ras codon 12, a potential site for formation of a 4-nitroquinoline-1-oxide adduct with a guanine residue. We also found that 2 nonmelanoma tumors induced by dimethylbenz[a]anthracene + UV + croton oil contained an A----T transversion at Ha-ras codon 61 position 2, which is characteristic of most dimethylbenz[a]anthracene-induced tumors. These results suggest that UV-induced C3H mouse tumors display mutations preferentially in the N-ras oncogene. Since most N-ras mutations in UV-induced tumors occurred opposite dipyrimidine sequences (T-T or C-C), one can infer that these sites are the targets for UV-induced mutation and transformation.
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Studies on the mechanism of immunologic tolerance induction by murine dendritic epidermal Thy-1+ cell lines.
We are investigating the functions of the Thy-1+ dendritic cells present in murine epidermis. Dendritic epidermal Thy-1+ cell (DETC) lines conjugated in vitro with hapten induce specific immunologic tolerance upon intravenous (i.v.) or subcutaneous injection into the footpad of normal mice. In these studies, we demonstrate that hapten-conjugated cells of other long-term, interleukin-2 (IL-2)-dependent, T cell lines are unable to induce tolerance upon footpad injection, indicating that the ability of DETC lines to induce tolerance is not a function of long-term cell culture or IL-2 dependence. Suppressor T cells were not found in mice made tolerant by footpad injection of hapten-conjugated cells of the DETC line AU16, although they could be demonstrated in mice made tolerant by i.v. injection. Studies of lymphocyte proliferation in vitro suggested that hapten-conjugated AU16 cells may induce tolerance by inhibiting the proliferation of activated T lymphocytes.
Animals↗
Immunobiology of primary murine melanomas.
Primary cutaneous melanomas can be induced in inbred mice by applying a dose of dimethylbenz[a]anthracene to the skin of 4-day-old mice, and then applying repeated doses of a tumor promoter to the same site over a long period of time. Preliminary experiments suggest that the final incidence of melanomas is strongly influenced by the age at which the initiating dose of carcinogen is applied. Melanomas induced by this method in C3H mice are immunogenic and exhibit a high degree of cross-reactivity when tested by immunization and challenge in vivo. Exposing the mice to ultraviolet (UV) radiation during carcinogenesis dramatically accelerates the appearance of melanoma. We are attempting to determine how UV radiation potentiates melanoma induction by studying the growth of melanoma cells transplanted into UV-irradiated skin. Our studies suggest that UV irradiation accelerates the outgrowth of melanoma cells by means of a local, immunosuppressive effect on the skin. However, this effect is distinct from the ability of UV irradiation to alter epidermal Langerhans cells and interfere with the induction of contact hypersensitivity responses. We postulate that UV irradiation augments melanoma development by interfering with the efferent arm of the immune response in the UV-irradiated site.
9,10-Dimethyl-1,2-benzanthracene↗
Effect of local ultraviolet irradiation on infections of mice with Candida albicans, Mycobacterium bovis BCG, and Schistosoma mansoni.
In this study, we investigated whether mice given ultraviolet (UV)-B (280-320 nm) radiation in doses sufficient to alter cutaneous immune cells and impair the induction of contact hypersensitivity would also have impaired resistance to infectious agents administered at the site of UV irradiation. C3H mice were exposed to 400 J/m2 UVR from FS40 sunlamps on four consecutive days. Immediately after the last UV treatment, groups of mice were injected subcutaneously with Candida albicans, injected intradermally (ID) with Mycobacterium bovis bacillus Calmette-Guerin (BCG), or infected percutaneously with Schistosoma mansoni in UV-irradiated skin. The induction of the delayed hypersensitivity response to C. albicans and BCG, as assessed by footpad swelling, was unaffected by UV irradiation. However, the number of viable mycobacteria recovered from the lymphoid organs of BCG-infected mice was increased significantly in the UV-irradiated animals for a period of more than 2 months. Low-dose UV irradiation of the skin at the site of infection did not influence the number of S. mansoni parasites recoverable from the internal organs of mice that had been infected with cercariae percutaneously 6 weeks earlier. We conclude that the ability of UV radiation to impair the development of cell-mediated immunity to antigens introduced in a UV-irradiated site is not universal and depends on the particular antigen administered. We hypothesize that the involvement of epidermal Langerhans cells as the primary antigen-presenting cells in the induction of cell-mediated immunity may be the critical factor in determining whether a particular immune response will be affected by local UV irradiation.
Animals↗
Internalization of Ia molecules into Birbeck granule-like structures in murine dendritic cells.
Dendritic cells isolated from the draining lymph nodes of mice sensitized epicutaneously with hapten are potent antigen-presenting cells and contain Birbeck granules and cored tubules characteristic of antigen-activated epidermal Langerhans cells. We used immunogold labeling and transmission electron microscopy to follow the internalization of Ia molecules in these antigen-presenting cells. We found that Ia molecules were internalized into Birbeck granule-like structures in the antigen-activated dendritic cells. Computer reconstruction of serial sections of the dendritic cells demonstrated that these structures span the cytoplasm from the cell membrane to the nuclear membrane and are associated with lysosomes. The internalization of Ia molecules into these structures supports the hypothesis that the Birbeck granule-like structures are derived from the cell membrane and are involved in the antigen-processing/presenting function of the dendritic cells.
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Characteristics of antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice.
Exposure of mice to ultraviolet (UV)-B (280-320 nm) radiation alters their immune response to contact-sensitizing haptens applied to UV-irradiated skin. Under these conditions, the contact-hypersensitivity response is reduced, and hapten-specific suppressor T lymphocytes can be found in the spleen. Considerable evidence suggests that the epidermal Langerhans cell is one of the principal targets of this effect of UV irradiation that leads, ultimately, to suppressor cell formation. We are using a combination of cell-surface markers and light and electron microscopy to determine whether differences can be detected in the antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice.
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Effects of ultraviolet radiation on the pathogenesis of Mycobacterium lepraemurium infection in mice.
The purpose of this study was to determine whether exposing mice to ultraviolet radiation (UVR) would alter the pathogenesis of infection with Mycobacterium lepraemurium (MLM), which causes a chronic, progressive, lethal disease in susceptible mouse strains. BALB/c mice were irradiated on dorsal skin with various doses of UVR from FS40 sunlamps 3 days before infection with MLM in the hind footpad. The course of disease was followed by assessing the number of acid-fast bacteria in the footpad, regional lymph node and spleen, and measuring the size of the lesion at the site of MLM infection at various times after infection. Mice were also tested periodically for a delayed-type hypersensitivity (DTH) response by injecting MLM antigen into the uninfected footpad and measuring footpad swelling 24 hours later. Mice treated with a single high dose of UVR (45 kJ/m2) had significantly more bacteria in the infected footpad, lymph node and spleen than unirradiated control animals. They also had larger lesions at the site of MLM infection and exhibited significant suppression of the DTH response at 3 and 6 months after infection. Injection of mice s.c. in the footpad with MLM 3d after 45 kJ/m2 UVR reduced the median survival time from 391 to 305 d and after i.v. infection from 171 to 139 d. Dose-response studies indicated that exposing mice to 2.3 kJ/m2 of UVR, which is approximately 1 minimal erythemal dose for this strain, suppressed the DTH response by 50% at 3 months after infection.(ABSTRACT TRUNCATED AT 250 WORDS)
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Lack of correlation between UV-induced enhancement of melanoma development and local suppression of contact hypersensitivity.
Injection of melanoma cells into the UV-irradiated ear skin of syngeneic mice results in an increased incidence of melanomas compared with that in nonirradiated ear skin. This effect of UV is localized to the site of irradiation and appears to be immunologically mediated. In these studies we test the hypothesis that the effect of UV irradiation on melanoma development is related to its ability to alter epidermal Langerhans cells and impair the induction of contact hypersensitivity. A regimen of UV irradiation that altered epidermal immune cells and interfered with the generation of contact hypersensitivity was tested for its ability to increase the incidence of melanoma. Conversely, the ear skin of C3H mice treated with a regimen of UV radiation that enhanced melanoma development was examined for the number of appearance of ATPase+ and Thy-1+ dendritic epidermal cells and tested for the ability to initiate a contact hypersensitivity response. No correlation between these effects of UV irradiation could be detected. Furthermore, implantation of melanoma cells into UV-irradiated ear skin resulted in the generation of systemic immunity against subsequent tumor challenge. Therefore, we conclude that the ability of UV irradiation to modify melanoma development is unrelated to its effects on the afferent arm of the contact hypersensitivity response and that enhanced melanoma development is not due to an impairment in the induction of tumor immunity.
Adenosine Triphosphatases↗
Antigen presentation in the skin: modulation by u.v. radiation and chemical carcinogens.
The skin provides an excellent model to investigate antigen presenting cells (APC) particularly using contact hypersensitivity responses. The skin has its own distinct APC of which Langerhans cells (LC) are the best characterized. Fluorescein isothiocyanate (FITC) has proved a useful reagent with which to follow antigen-labeled APC to draining lymph nodes, where they bind to T lymphocytes. Cluster formation appears to be a necessary component of the APC-T cell interaction. Both u.v. radiation and chemical carcinogens are able to alter LC in the skin, resulting in immunological tolerance when antigen is presented through such treated sites. The changes in APC function are linked mainly to the tumor promoting action of carcinogens. The nature of the APC that trigger suppressor circuits and the potential chemical mediators involved are discussed. These studies have important implications for the immunobiology of the skin and for cutaneous carcinogenesis.
9,10-Dimethyl-1,2-benzanthracene↗
Effect of psoralen plus ultraviolet A radiation on in vivo growth of melanoma cells.
Exposure of murine skin to UVB (280-320 nm) radiation accelerates the outgrowth of melanoma cells implanted into the irradiated site. Because many of the biological effects of psoralen plus UVA (320-400 nm) radiation (PUVA) resemble those of UVB radiation and because PUVA therapy is used extensively in the treatment of cutaneous diseases in humans, we determined the effect of PUVA on the growth of transplanted murine melanoma cells. Unshaved C3H/HeN(MTV-) mice were treated twice each week for 3 weeks with 0.4 mg 8-methoxypsoralen i.p. plus 4.25 kJ/m2 UVA radiation; syngeneic K1735 melanoma cells were then injected s.c. into the external ear. This treatment stimulated the outgrowth of the melanomas compared to that in untreated mice and mice treated with 8-methoxypsoralen or UVA alone. The use of a nonphototoxic, monofunctional psoralen plus UVA was equally effective, indicating that neither phototoxicity nor the ability to form DNA crosslinks was required for this effect. In vitro treatment of a murine keratinocyte cell line PAM 212 with PUVA caused the release of soluble factors that, when mixed with K1735 melanoma cells prior to injection, stimulated their outgrowth in vivo. These studies demonstrate that PUVA treatment can contribute to the pathogenesis of melanoma by exerting a stimulatory effect on the outgrowth of melanoma cells. Furthermore, they suggest that this effect may result from the ability of PUVA to cause the release of stimulatory factors from keratinocytes.
Animals↗
Evidence that the local effect of ultraviolet radiation on the growth of murine melanomas is immunologically mediated.
We had reported previously that the outgrowth of melanoma is enhanced when melanoma cells are injected into UV-irradiated skin of syngeneic mice. To determine whether this effect was specific for melanomas, we compared the growth of 13 different tumor cell lines (3 melanomas, 6 fibrosarcomas, 2 undifferentiated skin tumors, a squamous cell carcinoma, and a spontaneous hepatocarcinoma) in UV-irradiated and nonirradiated syngeneic mice. C3H/HeN(MTV-) mice were exposed to 4.8 kJ/m2 UV-B (280-320 nm) radiation twice a week for 3 weeks; the tumor cells were injected into the UV-irradiated pinna 24 h after the final UV irradiation. The growth of all the melanomas and 4 of the fibrosarcomas was enhanced in UV-irradiated mice, indicating that the effect of UV radiation was not specific for melanomas or tumors of a particular etiology. Using an in vivo immunization and challenge assay, we found that the 7 tumors exhibiting enhanced development in UV-irradiated skin were highly immunogenic, whereas the remaining 6 tumors were not. This suggested that enhanced tumor outgrowth resulted from an immunosuppressive effect of the UV radiation. When tested further, we found that UV-B radiation had no effect on melanoma outgrowth in congenitally athymic mice, sublethally X-irradiated mice, or mice depleted of Thy1+ cells in vivo. These results indicate that immunological mechanisms play a role in the effect of UV radiation on the growth of murine melanomas.
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