Inhibition of UVB radiation-induced tissue swelling and immune suppression by nicotinamide riboside and pterostilbene.

BACKGROUND: Environmental ultraviolet radiation has deleterious effects on humans, including sunburn and immune perturbations. These immune changes are involved in skin carcinogenesis. OBJECTIVES: To determine whether nicotinamide riboside and/or pterostilbene administered systemically inhibits inflammatory and immune effects of exposure to mid-range ultraviolet radiation. METHODS: To examine UVB radiation-induced inflammatory effects, mice were fed standard chow/water, 0.04% pterostilbene in chow and 0.2% nicotinamide riboside in drinking water, diet with nicotinamide riboside alone, or diet with pterostilbene alone. After 4 weeks, mice were exposed to UVB radiation (3500 J/m2), and 24-/48-h ear swelling was assessed. We also asked if each agent or the combination inhibits UVB radiation suppression of contact hypersensitivity in two models. Mice were fed standard diet/water or chow containing 0.08% pterostilbene, water with 0.4% nicotinamide riboside, or both for 4 weeks. Low-dose: Half the mice in each group were exposed on the depilated dorsum to UVB radiation (1700 J/m2) daily for 4 days, whereas half were mock-irradiated. Mice were immunized on the exposed dorsum to dinitrofluorobenzene 4 h after the last irradiation, challenged 7 days later on the ears with dinitrofluorobenzene, and 24-h ear swelling assessed. High dose: Mice were treated similarly except that a single dose of 10,000 J/m2 of radiation was administered and immunization was performed on the unirradiated shaved abdomen 3 days later. RESULTS: Nicotinamide riboside and pterostilbene together inhibited UVB-induced skin swelling more than either alone. Pterostilbene alone and both given together could inhibit UVB-induced immune suppression in both the low-dose and high-dose models while nicotinamide riboside alone was more effective in the low-dose model than the high-dose model. CONCLUSION: Nicotinamide riboside and pterostilbene have protective effects against UVB radiation-induced tissue swelling and immune suppression.

Soluble adenylyl cyclase contributes to imiquimod-mediated inflammation and is a potential therapeutic target in psoriasis.

Cyclic AMP (cAMP) has a key role in psoriasis pathogenesis, as indicated by the therapeutic efficacy of phosphodiesterase inhibitors that prevent the degradation of cAMP. However, whether soluble adenylate cyclase (sAC) (encoded by the ADCY10 gene), which is an important source for cAMP, is involved in Th17 cell-mediated inflammation or could be an alternative therapeutic target in psoriasis is unknown. We have utilized the imiquimod model of murine psoriasiform dermatitis to address this question. Adcy10-/- mice had reduced erythema, scaling and swelling in the skin and reduced CD4+ IL17+ cell numbers in the draining lymph nodes, compared with wild-type mice after induction of psoriasiform dermatitis with imiquimod. Keratinocyte-specific knock out of Adcy10 had no effect on imiquimod-induced ear swelling suggesting keratinocyte sAC has no role in imiquimod-induced inflammation. During Th17 polarization in vitro, naive T cells from Adcy10-/- mice exhibited reduced IL17 secretion and IL-17+ T-cell proliferation suggesting that differentiation into Th17 cells is suppressed without sAC activity. Interestingly, loss of sAC did not impact the expression of Th17 lineage-defining transcription factors (such as Rorc and cMaf) but rather was required for CREB-dependent gene expression, which is known to support Th17 cell gene expression. Finally, topical application of small molecule sAC inhibitors (sACi) reduced imiquimod-induced psoriasiform dermatitis and Il17 gene expression in the skin. Collectively, these findings demonstrate that sAC is important for psoriasiform dermatitis in mouse skin. sACi may provide an alternative class of topical therapeutics for Th17-mediated skin diseases.

Regulation of Cutaneous Immunity In Vivo by Calcitonin Gene-Related Peptide Signaling through Endothelial Cells.

Calcitonin gene-related peptide (CGRP) can bias the outcome of Ag presentation to responsive T cells in vitro away from Th1-type immunity and toward the Th2 and Th17 poles through actions on endothelial cells (ECs). To test the in vivo significance of this observation, we engineered a mouse lacking functional CGRP receptors on ECs (EC receptor activity modifying protein 1 [RAMP1] knockout mice). On percutaneous immunization to 1-fluoro-2,4-dinitrobenzene, stimulated CD4+ T cells from draining lymph nodes showed significantly reduced IL-17A expression with significantly increased IFN-γ, IL-4, and IL-22 expression at the protein and mRNA levels compared with control mice. Retinoic acid receptor-related orphan receptor γ t mRNA was significantly reduced, while mRNAs for T-box expressed in T cells and GATA binding protein 3 were significantly increased. In addition, EC RAMP1 knockout mice had significantly reduced contact hypersensitivity responses, and systemic administration of a CGRP receptor antagonist similarly inhibited contact hypersensitivity in wild-type mice. These observations provide compelling evidence that CGRP is a key regulator of cutaneous immunity through effects on ECs and suggest a novel pathway for potential therapeutic manipulation.

Regulation of T helper cell responses during antigen presentation by norepinephrine-exposed endothelial cells.

Dermal blood vessels and regional lymph nodes are innervated by sympathetic nerves and, under stress, sympathetic nerves release norepinephrine (NE). Exposure of primary murine dermal microvascular endothelial cells (pDMECs) to NE followed by co-culture with Langerhans cells (LCs), responsive CD4+ T-cells and antigen resulted in modulation of CD4+ T-cell responses. NE-treatment of pDMECs induced increased production of interleukin (IL)-6 and IL-17A while down-regulating interferon (IFN)-γ and IL-22 release. This effect did not require contact between pDMECs and LCs or T-cells and depended upon pDMEC production of IL-6. The presence of NE-treated pDMECs increased the proportion of CD4+ T-cells expressing intracellular IL-17A and increased IL-17A mRNA while decreasing the proportion of IFN-γ- or IL-22-expressing CD4+ T-cells and mRNA levels for those cytokines. Retinoic acid receptor-related orphan receptor gamma (ROR-γt) mRNA was significantly increased in CD4+ T-cells while T-box transcription factor (T-bet) mRNA was decreased. Intradermal administration of NE prior to hapten immunization at the injection site produced a similar bias in draining lymph node CD4+ T-cells towards IL-17A and away from IFN-γ and IL-22 production. Under stress, release of NE may have significant regulatory effects on the outcome of antigen presentation through actions on ECs with enhancement of inflammatory skin disorders involving IL-17/T helper type 17 (Th17) cells.

Calcitonin Gene-Related Peptide-Exposed Endothelial Cells Bias Antigen Presentation to CD4+ T Cells toward a Th17 Response.

Calcitonin gene-related peptide (CGRP) is a neuropeptide with well-established immunomodulatory functions. CGRP-containing nerves innervate dermal blood vessels and lymph nodes. We examined whether CGRP regulates the outcome of Ag presentation by Langerhans cells (LCs) to T cells through actions on microvascular endothelial cells (ECs). Exposure of primary murine dermal microvascular ECs (pDMECs) to CGRP followed by coculture with LCs, responsive CD4(+) T cells and Ag resulted in increased production of IL-6 and IL-17A accompanied by inhibition of IFN-γ, IL-4, and IL-22 compared with wells containing pDMECs treated with medium alone. Physical contact between ECs and LCs or T cells was not required for this effect and, except for IL-4, we demonstrated that IL-6 production by CGRP-treated pDMECs was involved in these effects. CD4(+) cells expressing cytoplasmic IL-17A were increased, whereas cells expressing cytoplasmic IFN-γ or IL-4 were decreased by the presence of CGRP-treated pDMECs. In addition, the level of retinoic acid receptor-related orphan receptor γt mRNA was significantly increased, whereas T-bet and GATA3 expression was inhibited. Immunization at the site of intradermally administered CGRP led to a similar bias in CD4(+) T cells from draining lymph node cells toward IL-17A and away from IFN-γ. Actions of nerve-derived CGRP on ECs may have important regulatory effects on the outcome of Ag presentation with consequences for the expression of inflammatory skin disorders involving Th17 cells.

Tobacco smoke-induced immunologic changes may contribute to oral carcinogenesis.

OBJECTIVE: The objective of this study was to determine if tobacco smoke (TS), a risk factor for cancers of the aerodigestive tract, may contribute to oral carcinogenesis, in part, by suppressing local immunity. METHODS: Mice were placed in Plexiglas holders in which they breathed TS through the nose and mouth for 1 hour daily for 21 days. Control mice breathed room air in the same manner. One day after the last exposure, mice were immunized by application of oxazolone to each buccal mucosa. Control mice were mock immunized by application of vehicle alone. Five days later, all mice were challenged on the ears with oxazolone, and 24-hour ear swelling assessed as contact hypersensitivity. RESULTS: Mice exposed to TS had a significantly smaller contact hypersensitivity response compared with controls. When subsequently reimmunized on the glabrous skin, mice originally primed through TS-exposed mucosa could not be fully immunized, indicating induction of immunologic tolerance by exposure to hapten through TS-perturbed mucosa. Immunocompetent mice exposed to TS in this manner and challenged by submucosal placement of a syngeneic malignant tumor had significantly increased tumor growth over time compared with controls. No difference in growth rate was observed when the experiment was performed with natural killer cell-deficient, SCID (severe combined immunodeficiency) mice. In addition, exposure of epidermal Langerhans cells in vitro to an aqueous extract of TS impaired their ability to undergo maturation and to present antigen to responsive T cells. CONCLUSIONS: Immunologic changes induced in the oral cavity by exposure to TS may play a role in the development of oral cancers.

Norepinephrine and adenosine-5'-triphosphate synergize in inducing IL-6 production by human dermal microvascular endothelial cells.

Endothelial cells (ECs) play important roles in cutaneous inflammation, in part, by release of inflammatory chemokines/cytokines. Because dermal blood vessels are innervated by sympathetic nerves, the sympathetic neurotransmitter norepinephrine (NE) and the co-transmitter adenosine-5'-triphosphate (ATP) may regulate expression of EC inflammatory factors. We focused on IL-6 regulation because it has many inflammatory and immune functions, including participation in Th17 cell differentiation. Strikingly, NE and ATP synergistically induced release of IL-6 by a human dermal microvascular endothelial cell line (HMEC-1). Adrenergic antagonist and agonist studies indicated that the effect of NE on induced IL-6 release is primarily mediated by ß2-adrenergic receptors (ARs). By real-time PCR IL-6 mRNA was also synergistically induced in HMEC-1 cells. This synergistic effect of NE and ATP was reproduced in primary human dermal endothelial cells (pHDMECs) and is also primarily mediated by ß2-ARs. Under conditions of stress, activation of the symphathetic nervous system may lead to release of ATP and NE by sympathetic nerves surrounding dermal blood vessels with induction of IL-6 production by ECs. IL-6 may then participate in immune and inflammatory processes including generation of Th17 cells. Production of IL-6 in this manner might explain stress-induced exacerbation of psoriasis, and perhaps, other skin disorders involving Th17-type immunity.

N-acetyl-S-farnesyl-l-cysteine suppresses chemokine production by human dermal microvascular endothelial cells.

Isoprenylcysteine (IPC) molecules modulate G-protein-coupled receptor signalling. The archetype of this class is N-acetyl-S-farnesyl-l-cysteine (AFC). Topical application of AFC locally inhibits skin inflammation and elicitation of contact hypersensitivity in vivo. However, the mechanism of these anti-inflammatory effects is not well understood. Dermal microvascular endothelial cells (ECs) are involved in inflammation, in part, by secreting cytokines that recruit inflammatory cells. We have previously shown that the sympathetic nerve cotransmitter adenosine-5'-triphosphate (ATP) and adenosine-5'-O-(3-thio) triphosphate (ATPγS), an ATP analogue that is resistant to hydrolysis, increase secretion of the chemokines CXCL8 (interleukin-8), CCL2 (monocyte chemotactic protein-1) and CXCL1 (growth-regulated oncogene α) by dermal microvascular ECs. Production of these chemokines can also be induced by the exposure to the proinflammatory cytokine TNFα. We have now demonstrated that AFC dose-dependently inhibits ATP-, ATPγS- and TNFα-induced production of CXCL1, CXCL8 and CCL2 by a human dermal microvascular EC line (HMEC-1) in vitro under conditions that do not affect cell viability. Inhibition of ATPγS- or TNFα-stimulated release of these chemokines was associated with reduced mRNA levels. N-acetyl-S-geranyl-l-cysteine, an IPC analogue that is inactive in inhibiting G-protein-coupled signalling, had greatly reduced ability to suppress stimulated chemokine production. AFC may exert its anti-inflammatory effects through the inhibition of chemokine production by stimulated ECs.

Pituitary adenylate cyclase-activating peptide and vasoactive intestinal polypeptide bias Langerhans cell Ag presentation toward Th17 cells.

Epidermal Langerhans cells (LCs) are dendritic APCs that play an important role in cutaneous immune responses. LCs are associated with epidermal nerves and the neuropeptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) inhibit LC Ag presentation for Th1-type immune responses. Here, we examined whether PACAP or VIP modulates LC Ag presentation for induction of IL-17A-producing CD4(+) T cells. Treatment with VIP or PACAP prior to in vitro LC Ag presentation to CD4(+) T cells enhanced IL-17A, IL-6, and IL-4 production, decreased interferon (IFN)-γ and interleukin (IL)-22 release, and increased RORγt and Gata3 mRNA expression while decreasing T-bet expression. The CD4(+) T-cell population was increased in IL-17A- and IL-4-expressing cells and decreased in IFN-γ-expressing cells. Addition of anti-IL-6 mAb blocked the enhanced IL-17A production seen with LC preexposure to VIP or PACAP. Intradermal administration of VIP or PACAP prior to application of a contact sensitizer at the injection site, followed by harvesting of draining lymph node CD4(+) T cells and stimulation with anti-CD3/anti-CD28 mAbs, enhanced IL-17A and IL-4 production but reduced production of IL-22 and IFN-γ. PACAP and VIP are endogenous mediators that likely regulate immunity and immune-mediated diseases within the skin.

Calcitonin gene-related peptide inhibits chemokine production by human dermal microvascular endothelial cells.

This study examined whether the sensory neuropeptide calcitonin gene-related peptide (CGRP) inhibits release of chemokines by dermal microvascular endothelial cells. Dermal blood vessels are associated with nerves containing CGRP, suggesting that CGRP-containing nerves may regulate cutaneous inflammation through effects on vessels. We examined CGRP effects on stimulated chemokine production by a human dermal microvascular endothelial cell line (HMEC-1) and primary human dermal microvascular endothelial cells (pHDMECs). HMEC-1 cells and pHDMECs expressed mRNA for components of the CGRP and adrenomedullin receptors and CGRP inhibited LPS-induced production of the chemokines CXCL8, CCL2, and CXCL1 by both HMEC-1 cells and pHDMECs. The receptor activity-modifying protein (RAMP)1/calcitonin receptor-like receptor (CL)-specific antagonists CGRP8-37 and BIBN4096BS, blocked this effect of CGRP in a dose-dependent manner. CGRP prevented LPS-induced IκBα degradation and NF-κB binding to the promoters of CXCL1, CXCL8 and CCL2 in HMEC-1 cells and Bay 11-7085, an inhibitor of NF-κB activation, suppressed LPS-induced production of CXCL1, CXCL8 and CCL2. Thus, the NF-κB pathway appears to be involved in CGRP-mediated suppression of chemokine production. Accordingly, CGRP treatment of LPS-stimulated HMEC-1 cells inhibited their ability to chemoattract human neutrophils and mononuclear cells. Elucidation of this pathway may suggest new avenues for therapeutic manipulation of cutaneous inflammation.

Muramyl dipeptide induces Th17 polarization through activation of endothelial cells.

Endothelial cells (ECs) express the nucleotide-binding oligomerization domain (Nod) receptor 2, which recognizes the bacterial derivate muramyl dipeptide (MDP). MDP stimulation of these cells enhances their IL-6 production and may thus contribute to the immune and inflammatory activities in the skin. However, whether ECs are capable of influencing the development of T cell priming and its polarization remains unknown. We report that in vitro the murine bEnd.3 EC line induces, following MDP stimulation, a Th17 polarization at the expense of Th1 and Th2 polarization in the setting of Langerhans cell (LC) Ag presentation to responsive T cells as assessed by IL-17, IL-6, IFN-γ, and IL-4 production. Interestingly, IL-22 production, which has been associated with Th17 priming, was not influenced by MDP-treated bEnd.3 cells, illustrating differential regulation of this cytokine from IL-17. Additional analysis confirmed a significantly increased percentage of IL-17(+)CD4(+) T cells by flow cytometry and an increased mRNA level of the specific Th17 transcription factor retinoic acid-related orphan receptor γt in cocultures of LCs and responsive T cells in the presence of activated bEnd.3 cells. Experiments using the RNA interference technique to knockdown IL-6 in bEnd.3 cells confirmed that IL-6 produced by bEnd.3 cells stimulated by MDP is at least partially involved in Th17 polarization. Our data suggest that activated ECs are capable of influencing LC Ag processing and presentation to T cells and induce a Th17 polarization. These results are important for the understanding of Th17-related disorders of the skin such as psoriasis.

Calcitonin gene-related peptide biases Langerhans cells toward Th2-type immunity.

Langerhans cells (LC) are epidermal dendritic cells capable, in several experimental systems, of Ag-presentation for stimulation of cell-mediated immunity. LC have been considered to play a key role in initiation of cutaneous immune responses. Additionally, administration of donor T cells to bone marrow chimeric mice with persistent host LC, but not mice whose LC have been replaced by donor cells, exhibit marked skin graft-vs-host disease, demonstrating that LC can trigger graft-vs-host disease. However, experiments with transgenic mice in which regulatory elements from human langerin were used to drive expression of diphtheria toxin, resulting in absence of LC, suggest that LC may serve to down-regulate cutaneous immunity. LC are associated with nerves containing the neuropeptide calcitonin gene-related peptide (CGRP), and CGRP inhibits LC Ag-presentation in several models including presentation to a Th1 clone. We now report that CGRP enhances LC function for stimulation of Th2 responses. CGRP exposure enhanced LC Ag presentation to a Th2 clone. Upon presentation of chicken OVA by LC to T cells from DO11.10 chicken OVA TCR transgenic mice, pretreatment with CGRP resulted in increased IL-4 production and decreased IFN-gamma production. CGRP also inhibited stimulated production of the Th1 chemokines CXCL9 and CXCL10 but induced production of the Th2 chemokines CCL17 and CCL22 by a dendritic cell line and by freshly obtained LC. Changes in production of these chemokines correlated with the effect of CGRP on mRNA levels for these factors. Exposure of LC to nerve-derived CGRP in situ may polarize them toward favoring Th2-type immunity.

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/m(2) 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/m(2) 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.

Tetracycline suppresses ATP gamma S-induced CXCL8 and CXCL1 production by the human dermal microvascular endothelial cell-1 (HMEC-1) cell line and primary human dermal microvascular endothelial cells.

Tetracyclines (TCN) have powerful anti-inflammatory properties in addition to their anti-microbial effects. These anti-inflammatory effects are thought to play a role in inhibiting cutaneous inflammation in patients with rosacea and acne; however, the mechanism(s) of this action remains poorly understood. We have previously shown that adenosine-5'-triphosphate (ATP)gamma S, a hydrolysis-resistant ATP analogue, augments secretion of pro-inflammatory messengers by a human dermal microvascular endothelial cell line (HMEC-1). ATP released by the sympathetic nerves during stress may stimulate release of pro-inflammatory chemokines by dermal vessel endothelial cells, resulting in recruitment of inflammatory cells and exacerbation of inflammatory skin disease. Here we demonstrate that TCN inhibits ATP gamma S-induced release of pro-inflammatory mediators by HMEC-1 cells and primary human dermal microvascular endothelial cells. TCN dose-dependently inhibited ATP gamma S-induced augmentation of CXCL8 (interleukin-8) and CXCL1 (growth-regulated oncogene-alpha) production by HMEC-1 cells and primary human dermal endothelial cells in vitro. TCN and ATP gamma S did not affect HMEC-1 cell viability as determined by trypan-blue exclusion and cell counts. Inhibition of production of inflammatory mediators by endothelial cells may be one mechanism by which TCN improves inflammatory skin diseases. The ability to inhibit release of inflammatory mediators induced in HMEC-1 cells by purinergic agonists may be a useful way to screen for potential therapeutic agents for cutaneous inflammation.

CGRP, PACAP, and VIP modulate Langerhans cell function by inhibiting NF-kappaB activation.

The neuropeptides calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP), and vasoactive intestinal peptide (VIP) suppress Langerhans cell (LC) antigen presentation and modulate cytokine production. We have tested the hypothesis that these neuropeptides (NP) inhibit LC function by modulating activation of NF-kappaB. Lipopolysaccharide (LPS) activates NF-kappaB in both a LC-like cell line (XS52) and epidermal LC enriched to approximately 95% and this effect is inhibited by each of the NP. Furthermore, CGRP, PACAP, and VIP suppress phosphorylation of IkappaB kinase beta (P-IKKbeta), prevent degradation of the IkappaB alpha, and inhibit activation of NF-kappaB. Thus, these NP modulate LC function by reducing NF-kappaB activation. Bay 11-7085, an inhibitor of IKK, reduced tumor necrosis factor-alpha (TNFalpha) production from LPS-stimulated XS52 cells and inhibited the ability of LC to present antigen to a T-cell clone in vitro. Each NP also inhibited LPS-induced secretion of TNFalpha by XS52 cells and LC enriched to approximately 95% homogeneity. We suggest that the inhibitory activities of CGRP, PACAP, and VIP on LC function are mediated, at least in part, by inhibition of P-IKKbeta, which prevents IkappaB alpha degradation and activation of NF-kappaB. Modulation of this signaling pathway may be useful for therapeutic modulation of immunity in the skin.

Vaccinia virus infection attenuates innate immune responses and antigen presentation by epidermal dendritic cells.

Langerhans cells (LCs) are antigen-presenting cells in the skin that play sentinel roles in host immune defense by secreting proinflammatory molecules and activating T cells. Here we studied the interaction of vaccinia virus with XS52 cells, a murine epidermis-derived dendritic cell line that serves as a surrogate model for LCs. We found that vaccinia virus productively infects XS52 cells, yet this infection displays an atypical response to anti-poxvirus agents. Whereas adenosine N1-oxide blocked virus production and viral protein synthesis during a synchronous infection, cytosine arabinoside had no effect at concentrations sufficient to prevent virus replication in BSC40 monkey kidney cells. Vaccinia virus infection of XS52 cells not only failed to elicit the production of various cytokines, including tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), IL-6, IL-10, IL-12 p40, alpha interferon (IFN-alpha), and IFN-gamma, it actively inhibited the production of proinflammatory cytokines TNF-alpha and IL-6 by XS52 cells in response to exogenous lipopolysaccharide (LPS) or poly(I:C). Infection with a vaccinia virus mutant lacking the E3L gene resulted in TNF-alpha secretion in the absence of applied stimuli. Infection of XS52 cells or BSC40 cells with the DeltaE3L virus, but not wild-type vaccinia virus, triggered proteolytic decay of IkappaBalpha. These results suggest a novel role for the E3L protein as an antagonist of the NF-kappaB signaling pathway. DeltaE3L-infected XS52 cells secreted higher levels of TNF-alpha and IL-6 in response to LPS and poly(I:C) than did cells infected with the wild-type virus. XS52 cells were productively infected by a vaccinia virus mutant lacking the K1L gene. DeltaK1L-infected cells secreted higher levels of TNF-alpha and IL-6 in response to LPS than wild-type virus-infected cells. Vaccinia virus infection of primary LCs harvested from mouse epidermis was nonpermissive, although a viral reporter protein was expressed in the infected LCs. Vaccinia virus infection of primary LCs strongly inhibited their capacity for antigen-specific activation of T cells. Our results highlight suppression of the skin immune response as a feature of orthopoxvirus infection.

Tumor growth and immune function in mice during hind-limb unloading.

INTRODUCTION: Spaceflight is associated with changes in several immune parameters. Studies in rodents and humans have shown a decrease in resistance to bacterial and viral infections. However, the effect of spaceflight conditions on tumor immunity has not been explored. METHODS: The hindlimb unloading (HU) murine model of spaceflight was used to assess growth and immune reactivity to the S1 509a tumor cell line during HU as a model of microgravity. Changes in splenic mass of mice in the HU model were compared with mice in orthostatic suspension and standard housing controls. Furthermore, the role of host immunity in these changes was confirmed using mice with the severe combined immunodeficiency (SCID) mutation. RESULTS: Mice in the HU model demonstrated significantly increased tumor growth (p < 0.01), greater splenic atrophy, and a significantly diminished delayed-type hypersensitivity response to tumor antigens (p < 0.05) compared with controls. However, when immunodeficient mice were employed, no difference in tumor growth was observed. DISCUSSION: Our findings suggest antitumor immunity is inhibited in antiorthostatic suspension. The lack of a difference in mean tumor size in SCID mice in antiorthostatic suspension compared with standard housing controls supports the concept that HU alters host immunity against the S1 509a tumor. Further studies are warrranted to delineate the precise effects of spaceflight on host immunity, carcinogenesis, and tumor progression.

Augmentation of cutaneous immune responses by ATP gamma S: purinergic agonists define a novel class of immunologic adjuvants.

Extracellular nucleotides activate ligand-gated P2XR ion channels and G protein-coupled P2YRs. In this study we report that intradermal administration of ATPgammaS, a hydrolysis-resistant P2 agonist, results in an enhanced contact hypersensitivity response in mice. Furthermore, ATPgammaS enhanced the induction of delayed-type hypersensitivity to a model tumor vaccine in mice and enhanced the Ag-presenting function of Langerhans cells (LCs) in vitro. Exposure of a LC-like cell line to ATPgammaS in the presence of LPS and GM-CSF augmented the induction of I-A, CD80, CD86, IL-1beta, and IL-12 p40 while inhibiting the expression of IL-10, suggesting that the immunostimulatory activities of purinergic agonists in the skin are mediated at least in part by P2Rs on APCs. In this regard, an LC-like cell line was found to express mRNA for P2X(1), P2X(7), P2Y(1), P2Y(2), P2Y(4), P2Y(9), and P2Y(11) receptors. We suggest that ATP, when released after trauma or infection, may act as an endogenous adjuvant to enhance the immune response, and that P2 agonists may augment the efficacy of vaccines.

Vasoactive intestinal peptide modulates Langerhans cell immune function.

Epidermal nerves lie in close proximity to Langerhans cells (LC) and are capable of releasing peptides that modulate LC function, including calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide. The neuropeptide vasoactive intestinal peptide (VIP) has also been found in cutaneous nerves and mRNA, for the VIP receptor vasoactive intestinal peptide receptor type 1, and vasoactive intestinal peptide receptor type 2 have been found in murine LC and the LC-like cell line XS106. We examined the effects of VIP on LC function and cutaneous immunity. VIP inhibited elicitation of a delayed-type hypersensitivity response in previously immunized mice by epidermal cells enriched for LC content pulsed with Ag in vitro. VIP also inhibited the ability of unseparated epidermal cells to present Ag to a T cell clone and hybridoma and the ability of highly enriched LCs to present to the T cell clone. Inhibition of presentation to the hybridoma was observed with an antigenic peptide that does not require processing, suggesting that VIP is active at a step independent of Ag processing. To elucidate the mechanism(s) by which VIP may mediate these effects, we determined the effects of VIP on LC cytokine production using the XS106 cell line as a surrogate for LC. VIP augmented the production of the IL-10 in LPS-stimulated XS106 cells while down-regulating IL-12 and IL-1beta production. Thus, VIP, like pituitary adenylate cyclase-activating polypeptide and calcitonin gene-related peptide, down-regulates LC function and the associated immune response.

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.