Dat X. Nghiem

Immune suppression and skin cancer development: regulation by NKT cells.

Ultraviolet (UV) radiation is carcinogenic and immunosuppressive. UV-induced immune suppression is mediated by antigen-specific T cells, which can transfer suppression to normal recipients. These cells are essential for controlling skin cancer development in the UV-irradiated host and in suppressing other immune responses, such as delayed-type hypersensitivity. Despite their importance in skin cancer development, their exact identity has remained elusive. We show here that natural killer T cells from UV-irradiated donor mice function as suppressor T cells and play a critical role in regulating the growth of UV-induced skin cancers and suppressing adaptive immune responses in vivo.

A Role for Inflammatory Mediators in the Induction of Immunoregulatory B Cells

UV exposure suppresses the immune response to a variety of microbial, fungal, and viral Ags. In addition, UV radiation is a complete carcinogen and the immune suppression induced by UV radiation is a major risk factor for skin cancer induction. In this study, we examined the mechanisms underlying the induction of immune suppression and tolerance induction by UV radiation. Transferring lymph nodes cells from UV-irradiated, FITC-sensitized mice into normal recipients transferred immune tolerance. Contrary to expectations, the cell responsible was an FITC+, IL-10-secreting, CD19+, B220+ B cell. Because the lipid mediator of inflammation, platelet-activating factor (PAF) is released by UV-irradiated keratinocytes and is essential for the induction of immune suppression, we determined its role in tolerance induction. When UV-irradiated mice were injected with PCA 4248, a selective PAF receptor (PAFR) antagonist, transfer of tolerance was suppressed. However, immune suppression was not transferred when FITC+ cells from the draining lymph nodes of UV-irradiated, PAFR-deficient donor mice were injected into the recipients. Because PCA 4248 also blocks serotonin receptor binding, we measured the effect that blocking both serotonin and PAFR binding has on the transfer of immune suppression. Only when both PAF and serotonin binding were blocked could we inhibit tolerance induction. These data identify a novel function for PAF and serotonin in modulating immune function, the activation of immunoregulatory B cells.

Suppression of an Established Immune Response by UVA— A Critical Role for Mast Cells

Exposing experimental animals or human volunteers to UVA II (320–340 nm) radiation after immunization suppresses immunologic memory and the elicitation of delayed‐in‐time hypersensitivity reactions. Previous studies indicated that the mechanisms underlying UVA‐induced immune suppression are similar to those described for UVB‐induced immune suppression, i.e. transferred by T regulatory cells, overcome by repairing DNA damage, neutralizing interleukin (IL)‐10 activity, or injecting recombinant IL‐12. Here we continued our examination of the mechanisms involved in UVA II‐induced suppression. Antibodies to cis‐urocanic acid blocked UVA‐induced immune suppression. Treating UVA‐irradiated mice with histamine receptor antagonists, calcitonin gene‐related peptide (CGRP) receptor antagonists or platelet activating factor receptor antagonists blocked immune suppression in UVA‐irradiated mice. In light of the fact that cis‐urocanic acid and CGRP target mast cells, which can then release platelet activating factor and histamine, we measured UVA‐induced immune suppression in mast cell‐deficient mice. No immune suppression was noted in UVA‐irradiated mast cell‐deficient mice. These findings indicate that exposure to UVA II activates many of the same immune regulatory factors activated by UVB to induce immune suppression. Moreover, they indicate that mast cells play a critical role in UVA‐induced suppression of secondary immune reactions.

Determining the role of cytokines in UV-induced immunomodulation

Ultraviolet radiation exposure damages DNA and promotes the development of skin cancer. In addition, UV exposure suppresses the immune response. Although the mechanism by which epidermal exposure to UV induces systemic immune suppression is not fully understood, it is clear that cytokines are involved. Therefore, quantitative measurement of cytokines is a critical aspect of modern research techniques. Determining the level of synthesis and secretion of cytokines in vivo or in vitro can be achieved through several possible techniques, depending on the sampling size, its physical state, and the type of answers required to test the hypothesis. When studying transcriptional activation, the level of cytokine mRNA is often determined using reverse transcription polymerase chain reaction (RT-PCR), ribonuclease protection assay (RPA), or Northern blot. Quantitative determinations of specific protein levels require a capture ELISA. As with any analytical technique, there are compromises among expense of sensitivity, labor, and time. These methods are discussed as they pertain to surveying cytokine induction and their relative usefulness to the laboratory scientist.

Ultraviolet radiation-induced immunosuppression of delayed-type hypersensitivity in mice

Ultraviolet (UV) radiation present in sunlight plays a critical role in the initiation and promotion of nonmelanoma skin carcinogenesis and immune suppression. The immune suppressive effects of UV have been identified as a risk factor for skin cancer induction. For these reasons, scientists have focused on elucidating the mechanisms of UV-induced immune suppression to better understand the pathogenesis of skin cancer induction. A hallmark of UV-induced immune suppression is the generation of antigen-specific suppressor T cells. These suppressor cells have been shown to suppress antitumor immunity as well as other cell-mediated responses such as delayed-type hypersensitivity (DTH) reactions. Due to the excessive cost and time involved in traditional UV carcinogenic experiments, scientists have opted to use UV-induced suppression of DTH reactions as a surrogate model. DTH has been, and continues to be, a widely used assay system to measure in vivo immune function. Although somewhat unsophisticated by todays standards, this assay has great advantages because it presents a fast, inexpensive, and reliable model system to help dissect the mechanisms involved in UV-induced immune suppression. Furthermore, the murine model of DTH enables scientists to perform additional procedures, such as adoptive transfer studies with suppressor T cells, which are currently unavailable with human subjects.

In vivo expression of interleukin‐8, and regulated on activation, normal, T‐cell expressed, and secreted, by human germinal centre B lymphocytes

T‐cell homing within germinal centres (GCs) is required for humoral B‐cell responses. However, the mechanisms implicated in the recruitment of T cells into the GC are not completely understood. Here we show, by immunohistology, and Northern and Western blots, that in vivo human GC B lymphocytes can express CxC and CC chemokines. Moreover, B‐cell subset‐specific experiments reveal that interleukin (IL)‐8 and regulated on activation, normal, T‐cell expressed, and secreted (RANTES) are predominantly expressed by GC centroblast and centrocytes, suggesting that chemokine expression is essential at stages in which B‐lymphocytes engage in active antigen‐dependent interactions with T lymphocytes. In keeping with this hypothesis, we show that the T cells recruited into the GC correlatively express the receptors for IL‐8 and RANTES. We propose that chemokine expression is a key B‐cell function that facilitates T‐lymphocyte recruitment into the GCs and supports cognate B‐cell : T‐cell encounters. Moreover, our data are consistent with the impaired homing of T cells to secondary lymphoid organs in mice that are either deficient in CC and CxC chemokines or their receptors.

Resistance of CD1d-/- mice to ultraviolet-induced skin cancer is associated with increased apoptosis

Inhibition of p53-induced epidermal apoptosis, generation of p53 mutations, and suppressor T cells are the critical events responsible for the induction and development of UV-induced skin cancers. Recently, we demonstrated that CD1d knockout mice were resistant to UV-induced immunosuppression, prompting us to further address the role of CD1d in regulating UV carcinogenesis. We, therefore, investigated the response of wild-type (WT) and CD1d-/- mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d-/- mice developed skin tumors. Surprisingly, keratinocytes and fibroblasts from CD1d-/- mice were more sensitive to UV-induced apoptosis and persisted longer than cells derived from WT mice. In addition, epidermis and dermis taken from chronically UV-irradiated CD1d-/- mice harbored significantly fewer p53 mutations than WT mice. Our findings identify an unexpected and novel function for CD1d as a critical molecule regulating UV carcinogenesis, by inhibiting apoptosis to prevent elimination of potentially malignant keratinocytes and fibroblasts.

Inverse Relationship Between Increased Apoptosis and Decreased Skin Cancer in UV-irradiated CD1d-/- Mice¶

Abstract We previously demonstrated that CD1d knockout mice were resistant to ultraviolet (UV)–induced immunosuppression. Because immune suppression is a critical factor in the development of UV-induced skin cancers, we investigated the response of wild type (WT) and CD1d−/− mice to UV carcinogenesis. We found that although 100% of WT mice developed skin tumors after 45 weeks of UV irradiation, only 60% of CD1d−/− mice developed skin tumors. To investigate the mechanisms involved in the resistance of CD1d−/− mice to UV-induced carcinogenesis, we determined the time course and kinetics of keratinocyte cell death after UV irradiation. After acute UV exposure, the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL)-positive keratinocytes were eliminated from the skin of WT mice by 72 h post-UV, but they still persisted until 96 h in CD1d−/− mice. The kinetics of p53 protein expression closely followed the kinetics of apoptotic cell death. Chronic UV irradiation resulted in induction of a significantly higher number of apoptotic keratinocytes in CD1d−/− than WT mice. In addition, epidermis and dermis from chronically UV-irradiated CD1d−/− mice harbored significantly fewer p53 mutations than WT mice. These results indicate that the resistance of CD1d−/− mice to UV carcinogenesis may be due to increased cell death and elimination of keratinocytes and fibroblasts containing DNA damage and p53 mutations.