Targeted disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene.

Microsomal epoxide hydrolase (mEH) is a conserved enzyme that is known to hydrolyze many drugs and carcinogens, and a few endogenous steroids and bile acids. mEH-null mice were produced and found to be fertile and have no phenotypic abnormalities thus indicating that mEH is not critical for reproduction and physiological homeostasis. mEH has also been implicated in participating in the metabolic activation of polycyclic aromatic hydrocarbon carcinogens. Embryonic fibroblast derived from the mEH-null mice were unable to produce the proximate carcinogenic metabolite of 7,12-dimethylbenz[a]anthracene (DMBA), a widely studied experimental prototype for the polycylic aromatic hydrocarbon class of chemical carcinogens. They were also resistant to DMBA-mediated toxicity. Using the two-stage initiation-promotion skin cancer bioassay, the mEH-null mice were found to be highly resistant to DMBA-induced carcinogenesis. In a complete carcinogenesis bioassay, the mEH mice were totally resistant to tumorigenesis. These data establish in an intact animal model that mEH is a key genetic determinant in DMBA carcinogenesis through its role in production of the ultimate carcinogenic metabolite of DMBA, the 3,4-diol-1,2-epoxide.     

Mice deficient in tumor necrosis factor-alpha are resistant to skin carcinogenesis.

Given the associations between chronic inflammation and epithelial cancer, we studied susceptibility to skin carcinogenesis in mice deficient for the pro-inflammatory cytokine TNF-alpha (refs. 5,6). TNF-alpha(-/-) mice were resistant to development of benign and malignant skin tumors, whether induced by initiation with DMBA and promotion with TPA or by repeated dosing with DMBA. TNF-alpha(-/-) mice developed 5-10% the number of tumors developed by wild-type mice during initiation/promotion and 25% of those in wild-type mice after repeated carcinogen treatment. TNF-alpha could influence tumor and stromal cells during tumor development. The early stages of TPA promotion are characterized by keratinocyte hyperproliferation and inflammation. These were diminished in TNF-alpha(-/-) mice. TNF-alpha was extensively induced in the epidermis, but not the dermis, in TPA-treated wild-type skin, indicating that dermal inflammation is controlled by keratinocyte TNF-alpha production. Deletion of a TNF-alpha inducible chemokine also conferred some resistance to skin tumor development. TNF-alpha has little influence on later stages of carcinogenesis, as tumors in wild-type and TNF-alpha(-/-) mice had similar rates of malignant progression. These data provide evidence that a pro-inflammatory cytokine is required for de novo carcinogenesis and that TNF-alpha is important to the early stages of tumor promotion. Strategies that neutralize TNF-alpha production may be useful in cancer treatment and prevention.     

Importance of DNA repair in carcinogenesis: evidence from transgenic and gene targeting studies

We have generated transgenic mice by introducing copies of the E. coli O6-methylguanine-DNA methyltransferase gene, ada. Liver extracts from homozygotes demonstrate about three times the control enzyme activity and increase up to about eight-fold can be induced by treatment with zinc, since the metal-responsive metallothionein promoter is attached to the ada gene. Furthermore, studies of liver carcinogenesis in our transgenic mice demonstrated significantly reduced rates of development of hepatocellular tumors after treatment with dimethylnitrosamine or diethylnitrosamine. It is well known that xeroderma pigmentosum (XP) patients are deficient in DNA repair. The availability of XPA (XP group A complementing) knockout mice has enabled us to investigate the functional role of the XPA nucleotide excision repair gene in carcinogenesis in vivo, first using the mouse skin as a model system. XPA-/- mice demonstrated skin ulcers 5-7 days after 7,12-dimethylbenz[a]anthracene (DMBA) treatment and papilloma development within 4 weeks prior to promotion, skin tumor incidence being also much higher than in heterozygous and wild-type mice. Experiments targeting the lung, liver and tongue have also been conducted to answer the question of whether the internal organs of these mice are also susceptible to chemical carcinogens. For lung carcinogenesis, mice were instilled intratracheally with a small dose of benzo[a]pyrene. The pulmonary tumor incidence in XPA-/- mice was significantly higher than in XPA+/- and XPA+/+ mice. XPA-/- mice were also found to be have enhanced sensitivity to aflatoxin B1 regarding liver tumor induction. In addition, administration of 4-nitroquinoline-1-oxide in drinking water for 50 weeks resulted in tongue tumors only in XPA-/- mice. These studies, thus, provided convincing evidence that XPA mice are also sensitive to carcinogenesis in organs other than the skin.     

Involvement of prostaglandins in the immunosuppression occurring during experimental infection by Paracoccidioides brasiliensis

We investigated whether PGE2 mediates the immunosuppression observed during Paracoccidioides brasilensis infection. Con-A-stimulated splenocytes, isolated from mice on days 15 and 60 of infection, release high amounts of PGE2, this release was inhibited by the treatment of animals with indomethacin, sodium salicylate or meloxicam. The treatment of the animals with salicylate or meloxicam, but not indomethacin, enhanced the release of IL-2 by splenocytes from animals on day 15, but not on day 60 of infection. Furthermore, we demonstrated that the productions of TNF-alpha, IFN-gamma, IL-4, and IL-10 by Con-A-stimulated splenocytes from mice at 15 days of infection were inhibited by treatment with salicylate or meloxicam. Indomethacin inhibited only TNF-alpha and IFN-gamma production. The three treatments caused reduction of granuloma areas in the liver and lungs of infected mice. In conclusion, results suggest that the PGE2 released by COX-2 mediates the immunosuppression early on (day 15), but not during the later phase (60 days) of P. brasiliensis infection by a mechanism dependent upon IL-4 and IL-10.     

Yersinia enterocolitica evasion of the host innate immune response by V antigen-induced IL-10 production of macrophages is abrogated in IL-10-deficient mice.

The virulence-associated V Ag (LcrV) of pathogenic Yersinia species is part of the translocation apparatus, required to deliver antihost effector proteins (Yersinia outer proteins) into host cells. An orthologous protein (denoted as PcrV) has also been identified in the ExoS regulon of Pseudomonas aeruginosa. Additionally, it is known that LcrV is released by yersiniae into the environment and that LcrV causes an immunosuppressive effect when injected into mice. In this study, we demonstrate for the first time that rLcrV, but not PcrV, is capable of suppressing TNF-alpha production in zymosan A-stimulated mouse macrophages and the human monocytic Mono-Mac-6 cell line. The underlying mechanism of TNF-alpha suppression could be assigned to LcrV-mediated IL (IL)-10 production, because 1) LcrV induces IL-10 release in macrophages, 2) anti-IL-10 Ab treatment completely abrogated TNF-alpha suppression, and 3) TNF-alpha suppression was absent in LcrV-treated macrophages of IL-10-deficient (IL-10-/-) mice. The relevance of LcrV-mediated immunosuppression for the pathogenicity of yersiniae became evident by experimental infection of mice; in contrast to wild-type mice, IL-10-/- mice were highly resistant against Yersinia infection, as shown by lower bacterial load in spleen and liver, absent abscess formation in these organs, and survival.     

Failure of Immunological Memory in DMBA-treated Mice

SEVERAL chemical carcinogens show immunosuppressive activity in various experimental systems^1–4. Stjernswärd⁵ reported a remarkable correlation between carcinogenic activity and immunosuppressive capacity of chemicals, that is, potent carcinogens such as 3-methylcholanthrene (MCA), benz(a)pyrene and 7,12-dimethylbenzanthracene (DMBA) suppressed the immune response to sheep red blood cells (SRBC) but noncarcinogenic analogues did not. Stutman² showed that administration of MCA depressed the immune response to SRBC in mice (C3Hf/Bi strain) sensitive to oncogenic effect of the drug, but the drug was not immunosuppressive in the I strain, which is relatively resistant to its oncogenic effect. These suggest an important role of immunosuppressive activity of the carcinogens on the process of carcinogenesis, perhaps by weakening the immune surveillance capacity of the host. On the other hand, many potent carcinogens, such as urethane, do not seem to have an immunosuppressive effect⁶. Here we have analysed the effect of DMBA on the antibody response of C3H/He mice to soluble protein antigen, bacterial α-amylase (BαA). Our findings suggest a marked effect of DMBA on the immunological memory.     

Primary defect in UVB-induced systemic immunomodulation does not relate to immature or functionally impaired APCs in regional lymph nodes

UVB irradiation of the shaved dorsal skin of mice can cause both local and systemic suppression of contact hypersensitivity responses; the former demonstrated by administration of the sensitizing Ag/hapten to the irradiated site and the latter by its administration at least 72 h later to distal unirradiated sites. The immunological basis of systemic immunomodulation is not clear. When haptens (trinitrochlorobenzene, FITC) were administered to the shaved ventral skin 4 days after irradiation (8 kJ/m(2)) to the shaved dorsum of BALB/c mice, CD11c(+)/FITC(+) cells in the skin-draining lymph nodes from control and irradiated mice produced on a per cell basis similar levels of IL-12 and PGE(2) were phenotypically mature and efficient at presenting FITC to lymphocytes from FITC-sensitized mice. Ag presentation by FACS-sorted CD11c(+) lymph node cells isolated 4 days after UVB irradiation was as efficient as were cells from unirradiated mice at presentation in vitro of an OVA peptide (OVA(323-339)) to CD4(+) cells from OVA-TCR-transgenic DO11.10 mice. Further, IFN-gamma levels were increased in the cultures containing CD11c(+) cells from UVB-irradiated mice, suggesting that inflammation may precede downstream immunosuppression. These results suggest that the primary cause of reduced contact hypersensitivity responses in mice in which UV irradiation and the sensitizing Ag are applied to different sites several days apart must originate from cells other than CD11c(+) APCs that directly or by production of soluble mediators (IL-12, PGE(2)) affect cellular responses in the nodes of UVB-irradiated mice.     

Mast cells,neuropeptides, histamine,and prostaglandins in UV-induced systemic immunosuppression

There is a direct correlation between dermal mast cell prevalence in dorsal skin of different mouse strains and susceptibility to UVB-induced systemic immunosuppression; highly UV-susceptible C57BL/6 mice have a high dermal mast cell prevalence while BALB/c mice, which require considerable UV radiation for 50% immunosuppression, have a low mast cell prevalence. There is also a functional link between the prevalence of dermal mast cells and susceptibility to UVB- and cis-urocanic acid (UCA)-induced systemic immunosuppression. Mast cell-depleted mice are unresponsive to UVB or cis-UCA for systemic immunosuppression unless they are previously reconstituted at the irradiated or cis-UCA-administered site with bone marrow-derived mast cell precursors. cis-UCA does not stimulate mast cell degranulation directly. Instead, in support of studies showing that neither UVB nor cis-UCA was immunosuppressive in capsaicin-treated, neuropeptide-depleted mice, cis-UCA-stimulated neuropeptide release from sensory c-fibers which, in turn, could efficiently degranulate mast cells. Studies in mice suggested that histamine, and not tumor necrosis factor alpha (TNF-alpha), was the product from mast cells that stimulated downstream immunosuppression. Histamine receptor antagonists reduced by approximately 60% UVB and cis-UCA-induced systemic immunosuppression. Indomethacin administration to mice had a similar effect which was not cumulative with the histamine receptor antagonists. Histamine can stimulate keratinocyte prostanoid production. We propose that both histamine and prostaglandin E(2) are important in downstream immunosuppression; both are regulatory molecules supporting the development of T helper 2 cells and reduced expression of type 1 immune responses such as a contact hypersensitivity reaction.     

Epstein-Barr virus Zta-induced immunomodulators from nasopharyngeal carcinoma cells upregulate interleukin-10 production from monocytes

During lytic infection with Epstein-Barr virus (EBV), several viral lytic proteins function to evade immune recognition or to actively suppress immune cells. An EBV lytic transactivator, Zta, induces an immunosuppressive cytokine interleukin 10 (IL-10) in B cells, but whether it regulates IL-10 in the context of epithelial cells is unclear. In this study, we tested nasopharyngeal carcinoma (NPC) cell lines and found that Zta did not induce IL-10 in these epithelial cells. Interestingly, the conditioned medium of Zta-expressing NPC cells enhanced IL-10 production from monocytes. We further revealed that the IL-10-inducing effect involved at least two immunomodulators that were upregulated by Zta and secreted from NPC cells: granulocyte-macrophage colony-stimulating factor (GM-CSF) and prostaglandin E(2) (PGE(2)). Zta was recruited to and activated the GM-CSF promoter, thus upregulating GM-CSF expression. Zta also activated the promoter of cyclooxygenase-2 (COX-2), and Zta-induced COX-2 increased downstream PGE(2) production. Cotreatment with GM-CSF and PGE(2) synergistically induced IL-10 production from monocytes. The IL-10-inducing effect of the Zta-conditioned medium was reduced when GM-CSF or the COX-2/PGE(2) pathway was blocked. The conditioned medium of NPC cells with EBV lytic infection showed a similar increase of GM-CSF and PGE(2) levels as well as the IL-10-inducing effect on monocytes, and knockdown of Zta abolished all the effects. Therefore, through Zta-induced immunomodulators, EBV lytic infection in NPC cells can direct bystander monocytes to produce IL-10, which may be a novel way of EBV to promote local immunosuppression.     

Differential expression and tissue compartmentalization of the inflammatory response following thermal injury

Studies have shown that both animal tissue-fixed immune cells and human peripheral blood mononuclear cell (PBMC) functions are altered after burn injury. Additional studies suggest that the burn injury-induced alterations in these divergent cell populations from different species are similar. It remains unknown, however, whether the observed changes in animal tissue-fixed immune cell function following thermal injury also occurs to a similar extent in the PBMC population. The aim of our study was to compare PBMC and tissue-fixed immune cell functions from the same animal using a murine burn model. At 7 days post-burn, mice were more susceptible to sepsis and delayed type hypersensitivity responses were suppressed. Splenocytes isolated from injured mice displayed suppressed proliferation and increased IL-10 production. In contrast, PBMC from injured mice displayed suppressed proliferation, IL-2 and IFN-gamma production. Splenic macrophage nitric oxide, PGE(2), TNF-alpha, IL-6 and IL-10 production was enhanced post-burn and IL-12 production was suppressed. PBMC from such animals displayed enhanced PGE(2) production and suppressed IL-6 and IL-12 production. These results indicate that while an immunosuppressive Th(2) phenotype (increased IL-10 and/or suppressed IL-2, IFN-gamma) was induced in both the splenic and PBMC compartments post-injury, differential expression and dimorphism in the response also exists. Thus, the assessment of only PBMC function in burn patients may not accurately reflect the patient's actual immune status at the tissue level.     

Anti-tumor activity of rosmarinic acid in 7,12-dimethylbenz(a)anthracene (DMBA) induced skin carcinogenesis in Swiss albino mice

Aim of the present study was to evaluate the anti-tumor effect of orally administered rosmarinic acid in 7,12-dimethylbenz(a)anthracene (DMBA) induced skin carcinogenesis in Swiss albino mice. Phase I and II detoxication agents, lipid peroxidation byproducts, antioxidants and apoptotic biomarkers were used to assess chemopreventive efficacy of rosmarinic acid in DMBA induced skin carcinogenesis. Skin squamous cell carcinoma was induced at the shaved back of mice by applying DMBA (20 microg in 0.1 mL acetone) twice weekly for 8 weeks. Tumor formation (100%) was observed within 15 weeks of treatment in DMBA alone. Marked alterations in the status of above mentioned biomarkers were observed in tumor bearing mice. Oral administration of rosmarinic acid completely prevented the formation of skin tumors during DMBA-induced mouse skin carcinogenesis. Also, oral administration of rosmarinic acid brought back the status of phase I and phase II detoxication agents, lipid peroxidation byproducts, antioxidants and apoptotic markers (p53, Bcl-2, caspase-3 and caspase-9) in DMBA treated mice. Results of the present study suggested that rosmarinic acid had potent anticancer, anti-lipid peroxidative and apoptotic effect in DMBA-induced skin carcinogenesis.     

Prostaglandins regulate melanoma-induced cytokine production in macrophages

Tumor-secreted products can affect macrophage cytokine expression and in that way alter the immune response. Prostaglandins (PGs) are found in the tumor microenvironment and have been associated with local and regional immunosuppression. We investigated whether tumor-secreted factors could induce PG synthesis in macrophages and whether these PGs could alter macrophage production of immunoregulatory cytokines. In both murine and human models, melanoma conditioned medium (MCM) induced macrophage production of PGE(2), IL-6, and TNF-alpha. PGE(2) production increased over 24 h and was accompanied by an increase in cyclooxygenase-2 (COX-2) expression, while COX-1 expression remained unchanged. In the presence of 10 microM NS398, a selective COX-2 inhibitor, MCM-stimulated PGE(2) synthesis was almost completely suppressed, while production of IL-6 and TNF-alpha proteins and mRNA also was partially abrogated. In the murine model, 200 microM NS398 resulted in more significant inhibition of cytokine protein and mRNA production. Although MCM induced NFkappaB and NF-IL-6 activation, neither dose of NS398 altered this effect. We conclude that melanoma-secreted products stimulate COX-2 expression and PGE(2) synthesis in macrophages and that inhibition of COX-2-derived PG synthesis results in partial abrogation of macrophage cytokine production.     

Soybean isoflavones inhibit tumor necrosis factor-alpha-induced apoptosis and the production of interleukin-6 and prostaglandin E2 in osteoblastic cells

The effects of individual soybean isoflavones, genistein (4',5,7-trihydroxyisoflavone) and daidzein (4',7-dihydroxyisoflavone), on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis and the production of local factors in osteoblastic cells has been investigated. Soybean isoflavones increased DNA synthesis and the number of viable cells. When cells were treated with TNF-alpha, the number of viable cells dose-dependently decreased. The decrease in cell number caused by TNF-alpha treatment was due to apoptosis, which was confirmed by TUNEL and cell death ELISA analyses. Soybean isoflavones inhibited apoptosis of osteoblastic cells subjected to TNF-alpha treatment. MC3T3-E1 osteoblastic cells secrete interleukin-6 (IL-6), interleukin-1beta (IL-1beta), nitric oxide (NO) and prostaglandin E(2) (PGE(2)) constitutively, but at low levels. Soybean isoflavones had no effect on the constitutive production of these local factors. When cells were treated with TNF-alpha (10(-10)M), the production of IL-6 and PGE(2), but not that of IL-1beta and NO, significantly increased. Treatment with soybean isoflavones (10(-5)M), in the presence of TNF-alpha (10(-10)M), for 48 h inhibited production of IL-6 and PGE(2), suggesting the antiresorptive action of soy phytoestrogen may be mediated by decreases in these local factors. The findings of this study thus suggest that soybean isoflavones may promote the function of osteoblastic cells and play an important role in bone remodeling.     

Inhibition of IL-6, TNF-alpha, and cyclooxygenase-2 protein expression by prostaglandin E2-induced IL-10 in bone marrow-derived dendritic cells

Several endogenously produced mediators, including cytokines such as IL-6, IL-10, and TNF-alpha and prostanoids such as prostaglandin E(2) (PGE(2)), regulate dendritic cell (DC) function and contribute to immune homeostasis. In this study, we report that exogenous PGE(2) enhances the production of IL-10 from bone marrow-derived DC (BM-DC). IL-6, but not TNF-alpha, release is enhanced by PGE(2) in the presence of anti-IL-10, suggesting that endogenous IL-10 masks PGE(2)-induced IL-6. Furthermore, both exogenous IL-10 and PGE(2) inhibit LPS-induced IL-6 and TNF-alpha, whereas selective inhibition of cyclooxygenase-2 (COX-2) or addition of anti-IL-10 causes the reverse effects. Exogenous IL-10, but not IL-6, dose-dependently suppresses COX-2 protein expression and PGE(2) production, and TNF-alpha does not reverse this effect. In contrast, anti-IL-10 up-regulates prostanoid production by LPS-stimulated BM-DC. Taken together, our results show that in response to PGE(2), BM-DC produce IL-10, which in turn down-regulates their own production of IL-6-, TNF-alpha-, and COX-2-derived prostanoids, and plays crucial roles in determining the BM-DC pro-inflammatory phenotype.     

The essential role of the intestinal microbiota in facilitating acute inflammatory responses

The restoration of blood flow, i.e., reperfusion, is the treatment of choice to save viable tissue following acute ischemia of a vascular territory. Nevertheless, reperfusion can be accompanied by significant inflammatory events that limit the beneficial effects of blood flow restoration. To evaluate the potential role of the intestinal microbiota in facilitating the development of tissue injury and systemic inflammation, germ-free and conventional mice were compared in their ability to respond to ischemia and reperfusion injury. In conventional mice, there was marked local (intestine) and remote (lung) edema formation, neutrophil influx, hemorrhage, and production of TNF-alpha, KC, MIP-2, and MCP-1. Moreover, there was an increase in the concentration of serum TNF-alpha and 100% lethality. In germ-free mice, there was no local, remote, or systemic inflammatory response or lethality after intestinal ischemia and reperfusion and, in contrast to conventional mice, germ-free animals produced greater amounts of IL-10. Similar results were obtained after administration of LPS, i.e., little production of TNF-alpha or lethality and production of IL-10 after LPS in germ-free mice. Blockade of IL-10 with Abs induced marked inflammation and lethality in germ-free mice after ischemia and reperfusion or LPS administration, demonstrating that the ability of these mice to produce IL-10 was largely responsible for their "no inflammation" phenotype. This was consistent with the prevention of reperfusion-associated injury by the exogenous administration of IL-10 to conventional mice. Thus, the lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness.     

Mutual modulation between interleukin-10 and interleukin-6 induced by Rhodococcus aurantiacus infection in mice

The interaction between interleukin-10 (IL-10) and interleukin-6 (IL-6) was investigated in the inflammatory response to Rhodococcus aurantiacus (R. aurantiacus) infection, in which both cytokines act as anti-inflammatory cytokines. Compared with wild-type (WT) counterparts, IL-6 gene-deficient (IL-6(-)/(-)) mice mounted a more robust production of IL-10 and tumor necrosis factor-alpha (TNF-alpha) during the initial phase of infection. Administration of anti-IL-10 antibody resulted in all the mice dying within 3 days post-infection as well as a further elevated TNF-alpha release. In vitro challenge of the macrophages from IL-6(-)/(-) and WT mice with heat-killed R. aurantiacus also showed similar results. Addition of exogenous IL-6 depressed IL-10 and TNF-alpha production by either IL-6(-)/(-) mice or IL-6(-)/(-) mouse macrophages. Likewise, WT mouse macrophages pretreated with anti-IL-10 or anti-IL-6 antibody exhibited increased production of TNF-alpha and IL-6 or IL-10 respectively. Moreover, neutralization of both IL-10 and IL-6 induced a further increase in TNF-alpha production by WT mouse cells. Overall, we conclude that IL-10 is a key element in protecting mice against mortality, and that IL-10 and IL-6 production are negatively regulated by each other although they are additive in suppressing TNF-alpha release in R. aurantiacus-infected mouse model.     

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.     

Role of COX-1 and COX-2 on skin PGs biosynthesis by mechanical scratching in mice

We examined the involvement of cyclooxygenase (COX)-1 and COX-2 on mechanical scratching-induced prostaglandins (PGs) production in the skin of mice. The dorsal regions of mice were scratched using a stainless brush. COXs expressions in the skin were analyzed using real-time PCR and Western blotting. The effect of acetylsalicylic acid (ASA) on the ability of PGs production were determined based on skin PGs level induced by arachidonic acid (AA) application. Mechanical scratching increased PGD2, PGE2, PGI2 and PGF(2 alpha). COX-1 was constitutively expressed and COX-2 expression was enhanced by scratching. Intravenous administration of ASA inhibited PGs biosynthesis in the normal skin. PGs levels of the skin 6h after ASA administration (ASA 6 h) were almost equal to those of the skin 10 min after ASA administration (ASA 10 min). In the scratched skin, AA-induced PGE2 and PGI2 of ASA 6 h were significantly higher than those of ASA 10 min. The skin PGD2 and PGF(2 alpha) of ASA 10 min were almost same to those of ASA 6 h. In the normal skin of COX-1-deficient mice, skin PGD2 level was lower than that of wild-type mice, although PGE2, PGI2 and PGF(2 alpha) levels were almost equal to those of wild type. In the scratched skin of COX-1-deficient mice, PGD2, PGE2, PGI2 and PGF(2 alpha) levels were lower than those of wild-type mice. These results suggested that cutaneous PGD2 could be mainly produced by COX-1, and PGE2 and PGI2 could be produced by COX-1 and COX-2, respectively, in mice.     

A role for parasite-induced PGE2 in IL-10-mediated host immunoregulation by skin stage schistosomula of Schistosoma mansoni

Significant quantities of PGE(2) were produced by cercariae of Schistosoma mansoni following incubation with linoleic acid, a free fatty acid found on the surface of the skin. Cyclooxygenase (COX) 2 inhibitors failed to block this PGE(2) production, suggesting that a different biochemical pathway may be involved in the production of PGE(2) by the parasite. In addition, the parasites were also able to induce PGE(2) and IL-10 from human and mouse keratinocytes. Analysis of mouse skin during skin migratory phases of infection confirmed these in vitro observations. COX2 inhibitors blocked the parasite-induced PGE(2) and IL-10 from keratinocytes. Further analysis of the parasite secretions showed that the PGE(2)/IL-10-inducing effect was associated with a fraction <30 kDa molecular size. Addition of this fraction or parasite-stimulated keratinocyte culture supernatant to Con A-stimulated spleen cells resulted in the suppression of cell proliferation. This effect could be blocked by anti-IL-10 treatment. In sharp contrast, attenuation of the parasites with gamma-irradiation significantly abrogated their ability to induce PGE(2) or IL-10 from skin cells. Significance of IL-10 in host immunoregulation by skin stage schistosomula of S. mansoni was further confirmed by using IL-10-deficient mice. In these mice the normal subdued cutaneous reaction to the parasite was absent. Instead, a prominent cellular reaction occurred around the parasite, and there was considerable delay in parasitic migration through the skin. Thus these results suggest a key role for parasite-induced PGE(2) in IL-10-dependent down-regulation of host immune responses in the skin.     

Mechanisms of dimethylbenzanthracene-induced immunotoxicity

Traditional methods for toxicological assessment have implicated the immune system as a frequent target organ of toxic insult following chronic exposure to certain environmental chemicals, radiation or therapeutic drugs (xenobiotics). Immunotoxicity is expressed as autoimmunity, chemical hypersensitivity or immunosuppression. A tiered approach for characterizing chemical and drug-induced immunomodulation has been developed and validated in laboratory animals. Polycyclic aromatic hydrocarbons (PAH) have been studied because of their ubiquitous presence in the environment and carcinogenic potential. Since immunosuppression induced by PAH carcinogens has been implicated as an epigenetic mechanism in the outgrowth of initiated cells, this tiered approach was used to characterize the mechanism of PAH immunosuppressive capacity. Previously, studies in this laboratory have demonstrated that subchronic exposure of B6C3F1 mice to PAH carcinogens suppresses both humoral immunity (HI) and cell-mediated immunity (CMI), concurrently with decreased resistance to tumor challenge. The potent carcinogenic PAH, 7,12-dimethylbenz[a]anthracene (DMBA) was subchronically administered subcutaneously at 5, 50, or 100 micrograms/g of body weight. Natural killer (NK) cell tumor cytolysis, generation of cytotoxic T-cells (CTL), and lymphoproliferation to mitogens and allogeneic splenocytes in mixed leukocyte cultures (MLC) were quantitated 3-5 days after exposure to assess CMI. Mitogen and alloantigen-induced proliferation (MLC) of splenocytes was suppressed up to 90%. CTL and NK tumor cytolysis of radiolabelled target cells were similarly depressed up to 88 and 82%, respectively. Impairment of MLC or CTL responses correlated with increased susceptibility to challenge with PYB6 sarcoma cells. HI was measured by quantitating the number of antibody (IgM) plaque-forming cells (PFC) produced in response to T-cell dependent antigen challenge (sheep erythrocytes) and was similarly suppressed up to 95%. To understand the mechanism of PAH-induced immunotoxicity, splenocytes from DMBA-exposed mice were sensitized to alloantigens in the presence of interleukin-2 (IL-2) because there were indications that T-helper cell function was suppressed. In these preliminary studies, CTL suppression could be completely restored by the addition of the T-cell growth supporting lymphokine (IL-2) during the inductive phase of CTL generation, suggesting that DMBA exposure directly or indirectly induced deficits in T-helper cell function.