Effects of chronic ethanol feeding on murine dendritic cell numbers, turnover rate, and dendropoiesis.

BACKGROUND: Chronic alcoholics have increased susceptibility to and severity of infection, which are likely to be a result of impaired immune defense mechanisms. The contribution of dendritic cells (DC) to these immune defense changes is not well understood. Alterations in DC numbers, dendropoiesis, and lifespan have not been specifically studied in vivo in chronic ethanol (EtOH) exposure models. As DC play an essential role in initiating immune responses, alterations in these DC characteristics would help explain changes observed in adaptive immune responses. METHODS: Mice received 20% EtOH (w/v) in the drinking water for up to 28 weeks, with mouse chow ad libitum. In EtOH-fed and water control mice, DC were enumerated by flow cytometry. The effect of EtOH on DC precursor numbers was determined by differentiation in vitro in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4, and the effect of an EtOH environment on untreated DC differentiation was measured following bone marrow transfer to irradiated hosts. DC turnover rate was also examined by bromodeoxyuridine incorporation and loss. RESULTS: The percentage and absolute numbers of DC were decreased in spleen and increased in thymus beginning as early as 4 weeks of EtOH feeding. In addition, the overall cellularity of spleen and thymus were altered by this regimen. However, chronic EtOH consumption did not adversely affect DC precursor numbers, differentiation abilities, or turnover rates. CONCLUSIONS: Decreased splenic DC numbers observed following chronic murine EtOH consumption are not because of altered DC precursor numbers or differentiation, nor increased DC turnover rate. Similarly, increased thymic DC numbers are not the result of alterations in DC precursor differentiation or turnover rate. Compartment size plays a role in determining splenic and thymic DC numbers following chronic EtOH feeding. EtOH-induced alterations in total DC numbers provide several mechanisms to partially explain why chronic alcoholics have increased susceptibility to infections.

Chronic ethanol consumption decreases murine Langerhans cell numbers and delays migration of Langerhans cells as well as dermal dendritic cells.

BACKGROUND: Chronic alcoholics experience increased incidence and severity of infections, the mechanism of which is incompletely understood. Dendritic cells (DC) migrate from peripheral locations to lymph nodes (LN) to initiate adaptive immunity against infection. Little is known about how chronic alcohol exposure affects skin DC numbers or migration. METHODS: Mice received 20% EtOH in the drinking water for up to 35 weeks. Baseline Langerhans cell (LC) and dermal DC (dDC) numbers were enumerated by immunofluorescence (IF). LC repopulation after inflammation was determined following congenic bone marrow (BM) transplant and ultraviolet (UV) irradiation. Net LC loss from epidermis was determined by IF following TNF-alpha or CpG stimulation. LC and dDC migration into LN was assessed by flow cytometry following epicutaneous FITC administration. RESULTS: Chronic EtOH consumption caused a baseline reduction in LC but not dDC numbers. The deficit was not corrected following transplantation with non-EtOH-exposed BM and UV irradiation, supporting the hypothesis that the defect is intrinsic to the skin environment rather than LC precursors. Net loss of LC from epidermis following inflammation was greatly reduced in EtOH-fed mice versus controls. Ethanol consumption for at least 4 weeks led to delayed LC migration into LN, and consumption for at least 8 weeks led to delayed dDC migration into LN following epicutaneous FITC application. CONCLUSIONS: Chronic EtOH consumption causes decreased density of epidermal LC, which likely results in decreased epidermal immunosurveillance. It also results in altered migratory responsiveness and delayed LC and dDC migration into LN, which likely delays activation of adaptive immunity. Decreased LC density at baseline appears to be the result of an alteration in the skin environment rather than an intrinsic LC defect. These findings provide novel mechanisms to at least partially explain why chronic alcoholics are more susceptible to infections, especially those following skin penetration.

Overexpression of protein kinase C-{epsilon} in the mouse epidermis leads to a spontaneous myeloproliferative-like disease.

Protein kinase C (PKC)-epsilon, a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mouse lines that overexpress (8- or 18-fold) PKC-epsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent squamous cell carcinoma (SCC) elicited by 7,12-dimethylbenz(a)anthracene initiation and 12-O-tetradecanoylphorbol-13-acetate-promotion or by repeated ultraviolet radiation exposures. The susceptibility to the development of SCC was proportional to the level of expression of the PKC-epsilon transgene. We now report that PKC-epsilon FVB/N transgenic mice (line 215) that overexpress in epidermis approximately 18-fold PKC-epsilon protein more than their wild-type littermates spontaneously develop a myeloproliferative-like disease (MPD) in 100% of PKC-epsilon transgenic mice. The MPD was characterized by an excess of neutrophils and eosinophils, resulting in invasion of almost all vital organs of the mouse by 6 months of age. On gross examination these mice present with splenomegaly, hepatomegaly, and severe lymphadenopathy. Examination of the bone marrow revealed almost complete effacement by neutrophils, eosinophils, and their precursors. Furthermore, the spleen and lymph nodes were enlarged and exhibited marked extramedullary hematopoiesis. Complete pathological analysis of the second PKC-epsilon transgenic mouse (line 224) that expresses approximately eightfold PKC-epsilon protein more than their wild-type littermates revealed no remarkable findings in any of the affected organs as seen in line 215. However, peripheral blood analyses of PKC-epsilon transgenic mice indicated significant increases of neutrophils in the circulating blood in both PKC-epsilon transgenic lines. To determine whether there was an imbalance of cytokines in PKC-epsilon transgenic mice (line 215), resulting in aberrant myelopoiesis, we analyzed 17 cytokines in the peripheral blood. This analysis indicated that interleukin-5, interleukin-6, and granulocyte-colony stimulating factor were up-regulated as a function of age. The transgene PKC-epsilon was not detected in any of the affected organs (bone marrow, liver, spleen, lung) We suggest that overexpression of PKC-epsilon in the epidermis may lead to the induction of specific cytokines that may, in a paracrine mechanism, perturb normal hematopoiesis in bone marrow resulting in a granulocytic skew toward that of neutrophils and eosinophils. The susceptibility of PKC-epsilon transgenic mice to the induction of SCC and the spontaneous development of MPD are unrelated.

Protein kinase C epsilon is an endogenous photosensitizer that enhances ultraviolet radiation-induced cutaneous damage and development of squamous cell carcinomas.

Chronic exposure to UV radiation (UVR), especially in the UVA (315-400 nm) and UVB (280-315 nm) spectrum of sunlight, is the major risk factor for the development of nonmelanoma skin cancer. UVR is a complete carcinogen, which both initiates and promotes carcinogenesis. We found that protein kinase C epsilon (PKCepsilon), a member of the phospholipid-dependent threonine/serine kinase family, is an endogenous photosensitizer, the overexpression of which in the epidermis increases the susceptibility of mice to UVR-induced cutaneous damage and development of squamous cell carcinoma. The PKCepsilon transgenic mouse (FVB/N) lines 224 and 215 overexpressed 8- and 18-fold PKCepsilon protein, respectively, over endogenous levels in basal epidermal cells. UVR exposure (1 kJ/m(2) three times weekly) induced irreparable skin damage in high PKCepsilon-overexpressing mouse line 215. However, the PKCepsilon transgenic mouse line 224, when exposed to UVR (2 kJ/m(2) three times weekly), exhibited minimum cutaneous damage but increased squamous cell carcinoma multiplicity by 3-fold and decreased tumor latency by 12 weeks. UVR exposure of PKCepsilon transgenic mice compared with wild-type littermates (1) elevated the levels of neither cyclobutane pyrimidine dimer nor pyrimidine (6-4) pyrimidone dimer, (2) reduced the appearance of sunburn cells, (3) induced extensive hyperplasia and increased the levels of mouse skin tumor promoter marker ornithine decarboxylase, and (4) elevated the levels of tumor necrosis factor alpha (TNFalpha) and other growth stimulatory cytokines, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. The role of TNFalpha in UVR-induced cutaneous damage was evaluated using PKCepsilon transgenic mice deficient in TNFalpha. UVR treatment three times weekly for 13 weeks at 2 kJ/m(2) induced severe cutaneous damage in PKCepsilon transgenic mice (line 215), which was partially prevented in PKCepsilon-transgenic TNFalpha-knockout mice. Taken together, the results indicate that PKCepsilon signals UVR-induced TNFalpha release that is linked, at least in part, to the photosensitivity of PKCepsilon transgenic mice.

Protein kinase Cepsilon is linked to 12-O-tetradecanoylphorbol-13-acetate-induced tumor necrosis factor-alpha ectodomain shedding and the development of metastatic squamous cell carcinoma in protein kinase Cepsilon transgenic mice.

Protein kinase Cepsilon (PKCepsilon), a Ca(2+)-independent, phospholipid-dependent serine/threonine kinase, is among the PKC isoforms expressed in mouse epidermis. We reported that FVB/N transgenic mice that overexpress ( approximately 18-fold) PKCepsilon protein in basal epidermal cells and cells of the hair follicle develop papilloma-independent metastatic squamous cell carcinoma (mSCC) elicited by 7,12-dimethylbenz(a)anthracene-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion protocol. We now present that PKCepsilon transgenic mice elicit elevated serum tumor necrosis factor (TNF)alpha levels during skin tumor promotion by TPA, and this increase may be linked to the development of mSCC. A single topical application of TPA (5 nmol) to the skin, as early as 2.5 h after treatment, resulted in a significant (P < 0.01) increase (2-fold) in epidermal TNFalpha and more than a 6-fold increase in ectodomain shedding of TNFalpha into the serum of PKCepsilon transgenic mice relative to their wild-type littermates. Furthermore, this TPA-stimulated TNFalpha shedding was proportional to the level of expression of PKCepsilon in the epidermis. Using the TNF-alpha converting enzyme (TACE) inhibitor, TAPI-1, TPA-stimulated TNFalpha shedding could be completely prevented in PKCepsilon transgenic mice and isolated keratinocytes. These results indicate that PKCepsilon signal transduction pathways to TPA-stimulated TNFalpha ectodomain shedding are mediated by TACE, a transmembrane metalloprotease. Using the superoxide dismutase mimetic CuDIPs and the glutathione reductase mimetic ebselen, TPA-stimulated TNFalpha shedding from PKCepsilon transgenic mice could be completely attenuated, implying the role of reactive oxygen species. Finally, i.p. injection of a TNFalpha synthesis inhibitor, pentoxifylline, during skin tumor promotion completely prevented the development of mSCC in PKCepsilon transgenic mice. Taken together, these results indicate that: (a) PKCepsilon activation is an initial signal in TPA-induced shedding of TNFalpha from epidermal keratinocytes; (b) PKCepsilon-mediated signals to TACE are possibly mediated through reactive oxygen species; and (c) TPA-induced TNFalpha shedding may play a role in the development of mSCC in PKCepsilon transgenic mice.

Inhibition of the development of metastatic squamous cell carcinoma in protein kinase C epsilon transgenic mice by alpha-difluoromethylornithine accompanied by marked hair follicle degeneration and hair loss.

The role of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated polyamine biosynthesis in the development of metastatic squamous cell carcinoma (mSCC) in protein kinase C epsilon (PKC epsilon) transgenic mice was determined. TPA treatment induced epidermal ornithine decarboxylase (ODC) activity and putrescine levels approximately 3-4-fold more in PKC epsilon transgenic mice than their wild-type littermates. Development of mSCC by the 7,12-dimethylbenz(a)anthracene (100 nmol)-TPA (5 nmol) protocol in PKC epsilon transgenic mice was completely prevented by administration of the suicide inhibitor of ODC alpha-difluoromethylornithine (DFMO, 0.5% w/v) in the drinking water during TPA promotion. However, DFMO treatment led to marked hair loss in PKC epsilon transgenic mice. DFMO treatment-associated hair loss in PKC epsilon transgenic mice was accompanied by a decrease in the number of intact hair follicles. These results indicate that TPA-induced ODC activity and the resultant accumulation of putrescine in PKC epsilon transgenic mice are linked to growth and maintenance of hair follicles, and the development of mSCC. Severe hair loss observed in PKC epsilon transgenic mice on DFMO during skin tumor promotion has not been reported before in the prevention of cancer in other animal models or in human cancer prevention trials.