Langerhans Cells In Skin Research Articles

Topical calcineurin inhibitors in cutaneous lupus erythematosus

Calcineurin inhibitors are immunomodulator macrolides that block T cell activation in the skin. These molecules bind to macrophilin 12 to form a complex capable of blocking calcineurin, a protein that dephosphorylates, and thereby activates, the cytoplasmic subunit of nuclear factor of activated T cells (NF-AT). Thus, in the presence of calcineurin inhibitors, NF-AT cannot enter the nucleus and interact with the promoter regions of many cytokine genes. In addition to T cells, calcineurin inhibitors have also been shown to inhibit the activation of a number of other cell types in the skin immune system, including eosinophils, basophils, and Langerhans cells. These drugs are highly effective in treating atopic dermatitis, even in monotherapy, and they result in rapid, progressive, and sustained improvement. Additionally, calcineurin inhibitors are well tolerated for long-term treatment, and there are virtually no contraindications when using these medications on the face, eyelids, flexural skin, or mucous membranes. Although calcineurin inhibitors are immunomodulators, no significantly increased incidence of infections can be detected during therapy. Cutaneous lupus erythematosus is a chronic, autoimmune, inflammatory skin condition with a wide spectrum of skin manifestations, which presents a therapeutic challenge for dermatologists. In this review, the current treatment options for the different clinical subtypes of this disease are discussed. Therapy with topical calcineurin inhibitors is emphasized, and advantages and drawbacks, effects and side effects, treatment protocols, dosage, and expected outcome are described in detail.

Local effects of TL01 phototherapy in psoriasis

The mechanisms whereby narrowband ultraviolet B (UVB) (311-313 nm, TL01) phototherapy are effective in psoriasis may differ from those occurring in broadband UVB phototherapy. In the present study, changes in epidermal cells as a result of TL01 therapy were assessed in the skin of patients with psoriasis. The non-lesional skin of five subjects with plaque psoriasis was biopsied before and after a series of 12 whole-body TL01 treatments. Following appropriate staining of skin sections, the numbers of p53-positive keratinocytes, sunburn cells and Langerhans cells in the epidermis were counted. TL01 therapy induced a threefold increase in the number of p53-positive epidermal cells, a 12-fold increase in sunburn cells and a twofold decrease in Langerhans cells. The increase in epidermal p53 expression and apoptosis of keratinocytes together with the depletion of Langerhans cells in the non-lesional skin of psoriasis patients are likely to contribute to the effectiveness of TL01 phototherapy.

IgG regulates the CD1 expression profile and lipid antigen-presenting function in human dendritic cells via FcγRIIa

AbstractDendritic cells (DCs) process and present bacterial and endogenous lipid antigens in complex with CD1 molecules to T cells and invariant natural killer T (NKT) cells. However, different types of DCs, such as blood myeloid DCs and skin Langerhans cells, exhibit distinct patterns of CD1a, CD1b, CD1c, and CD1d expression. The regulation of such differences is incompletely understood. Here, we initially observed that monocyte-derived DCs cultured in an immunoglobulin-rich milieu expressed CD1d but not CD1a, CD1b, and CD1c, whereas DCs cultured in the presence of low levels of immunoglobulins had an opposite CD1 profile. Based on this, we tested the possibility that immunoglobulins play a central role in determining these differences. IgG depletion and intravenous immunoglobulin (IVIg) add-in experiments strongly supported a role for IgG in directing the CD1 expression profile. Blocking experiments indicated that this effect was mediated by FcγRIIa (CD32a), and quantitative polymerase chain reaction data demonstrated that regulation of the CD1 profile occurred at the gene expression level. Finally, the ability of DCs to activate CD1-restricted NKT cells and T cells was determined by this regulatory effect of IgG. Our data demonstrate an important role for FcγRIIa in regulating the CD1 antigen presentation machinery of human DCs.

Effect of Niacin and Prostaglandins D and E on Heat Shock Protein Gene Expression in Bovine Mammary Epithelial Cells In Vitro

Niacin induces peripheral vasodilatation via prostaglandin D (PGD) and E (PGE) release by Langerhans cells in skin. We evaluated if niacin alone or in combination with PGD and PGE alters expression of heat shock proteins (HSP) 27 and 70. Bovine mammary epithelial cells (BMEC) were cast in collagen in 24‐well plates containing growth media (GM) composed of DMEM/F‐12, insulin, EGF, IGF‐I, BSA and antibiotics at 37°C, 5% CO2. Cultures grew into ductal structures with media changes at 48 hr intervals. On day 8 cultures were divided into Controls (C) receiving GM, GM with niacin (0.5, 1.0, or 10.0 mM), PGD2 (10 or 24 uM), PGD2 with PGE1 (both at 24uM) alone or in combination with niacin. Half were placed into incubators at 37°C (TN) and the remainder at 42°C (HS) for 8 h. At 0h and 8h, replicates were pooled, placed in TRIzol and stored at −80°C until extracted for RNA. Isolated RNA was reverse transcribed into cDNA. Expression of HSP's‐27 and 70 was measured by q‐ PCR. Addition of PGD increased HSP‐27 and 70 gene expression in HS, (P<.0001). Peak fold increases in HSP‐70 expression at 8 hr over time zero differed between C and PGD, (−2.4 vs. 9.3, P<.0001) and were greater for Hsp‐27 (−115 vs +10, P<.0001). Addition of PGE increased HSP‐27 and 70 expression compared to C and PGD alone (P<.05). We conclude that niacin with PGD or PGD+PGE alters HSP‐27 and Hsp‐70 gene expression in BMEC during HS.

Langerhans cells of human skin are the natural antigen‐presenting cells for CD1c antigen presentation

CD1 proteins are a family of MHC‐like polypeptides that are distinguished from MHC proteins through their presentation of lipids rather than peptides to T cells. Although the expression of CD1 proteins resembles that of MHC class II, i.e. mostly on professional antigen presenting cells, the natural antigen presenting cell population(s) for human CD1c‐restricted T cells have not been identified. CD1c‐restricted T cells derived from leprosy patients were potently activated by LCs derived from the epidermis of healthy donors and their in vitro‐derived counterparts, LC‐like dendritic cells (LC‐like DCs). In contrast, although DCs isolated from the dermis of healthy skin and monocyte‐derived DCs presented antigen to CD1c‐restricted T cells, their level of antigen presentation was greater than two‐fold less effective than LCs. Our data indicate that Langerhans cells from normal human epidermis are natural antigen presenting cells for CD1c‐restricted T cells and suggest that LCs are programmed for CD1c antigen presentation to T cells against cutaneous pathogens such as M. leprae.s

Epithelial dendritic cells in vagina rapidly renew from bone marrow precursors

DCs represent key professional APCs capable of initiating primary immune responses. A specialized subset of DCs, the Langerhans cells (LCs), are located in the stratified squamous epithelial layer of the skin, and within the mucosal epithelial lining of the vaginal and oral cavities. The vaginal mucosa undergoes cyclic changes under the control of sex hormones, and the renewal and phenotypic characteristics of the vaginal epithelial DCs (VEDCs) remain unknown. Here, we examined the origin of VEDCs. In contrast to the skin epidermal LCs, the DCs in the epithelium of the vagina were found to be repopulated mainly by non‐monocyte bone marrow (BM)‐derived precursors, with a half‐life of 13 days under steady‐state conditions. Upon infection with herpes simplex virus 2 (HSV‐2), the Gr‐1hi monocytes were found to give rise to VEDCs. Further, flow cytometric analysis of the VEDCs revealed the presence of at least three distinct populations, namely, CD11b+F4/80hi, CD11b+F4/80int, and CD11b−F4/80neg. Importantly, these VEDC populations expressed CD207 at low levels, and had a constitutively more activated phenotype compared to the skin LCs. Collectively, our results revealed mucosa‐specific features of the VEDCs with respect to their phenotype, activation status and homeostatic renewal potential.

Local defense systems in the prepuce

Urinary tract infection (UTI) is a common bacterial infection in children. Circumcision reduces the incidence of UTI, and prevents preputial colonization with uropathic bacteria in childhood. However, there is insufficient information about the reason(s) why such colonization tends to occur in prepucium skin. We evaluated some of the humoral and cellular defense systems in the prepucium skins of 30 boys (age range 6 months to 5 years). We measured the expressions of Langerhans cells, mast cells, T lymphocytes, IgA, IgG and IgM in the prepuce and compared them with those in normal skin. Tissues obtained during circumcision were divided into two groups. In the first group, sections were stained with hematoxylin-eosin and toluidine blue. Immunohistochemical reactions were performed on paraffin-embedded tissue by means of the avidin-biotin-peroxidase complex method. The antibodies used were S-100 for Langerhans cells and CD8 and CD4 for T lymphocytes. In the second group, frozen sections were stained for IgA, IgG and IgM using an immunofluorescence method. The distribution of mast cells and IgG positivity was similar to that of normal skin. Langerhans cells were increased in prepucium skin (p<0.05), whilst only a few CD4 T lymphocytes were observed around the perivascular area and no expression of CD8 was observed in the prepucium and normal skin. The increase in Langerhans cells in prepucium skin may be the result of continuous stimulation of bacteria found in the periurethral area, and the absence of CD8 may help the colonization of uropathic bacteria.

Vaginal epithelial dendritic cells renew from bone marrow precursors

Dendritic cells (DCs) represent key professional antigen-presenting cells capable of initiating primary immune responses. A specialized subset of DCs, the Langerhans cells (LCs), are located in the stratified squamous epithelial layer of the skin and within the mucosal epithelial lining of the vaginal and oral cavities. The vaginal mucosa undergoes cyclic changes under the control of sex hormones, and the renewal characteristics of the vaginal epithelial DCs (VEDCs) remain unknown. Here, we examined the origin of VEDCs. In contrast to the skin epidermal LCs, the DCs in the epithelium of the vagina were found to be repopulated mainly by nonmonocyte bone-marrow-derived precursors, with a half-life of 13 days under steady-state conditions. Upon infection with HSV-2, the Gr-1(hi) monocytes were found to give rise to VEDCs. Furthermore, flow cytometric analysis of the VEDCs revealed the presence of at least three distinct populations, namely, CD11b(+)F4/80(hi), CD11b(+)F4/80(int), and CD11b(-)F4/80(-). Importantly, these VEDC populations expressed CD207 at low levels and had a constitutively more activated phenotype compared with the skin LCs. Collectively, our results revealed mucosa-specific features of the VEDCs with respect to their phenotype, activation status, and homeostatic renewal potential.

Cutting Edge: Lack of Evidence for Connexin-43 Expression in Human Epidermal Langerhans Cells

A provocative study has shown that viral peptides may be transferred in vitro from epithelial cells to APC through connexin-43 gap junction channels. In support of this cross-presentation pathway, the study also reported that human dendritic cells, including Langerhans cells of skin, express connexin-43. In this report we show that if this was the case, the levels of connexin-43 are below those detectable by immunofluorescence, flow cytometry, quantitative PCR of purified CD1a+ cells, and electron microscopy, raising questions about the relevance of the connexin-43-dependent mechanism for Langerhans cells of noninflamed human skin.

Niacin in cardiovascular prevention: mechanisms, efficacy, and safety

This review describes niacin's mechanism of action, efficacy in cardiovascular prevention, and safety. A G-protein-coupled receptor [GPR109A/HM74A, mouse PUMA-G (protein upregulated in macrophages by interferon-gamma)] was found to mediate the antilipolytic effect of niacin via inhibition of adenylyl cyclase in adipocytes. The same receptor in skin Langerhans cells mediates the common flushing side effect. The endogenous ligand for the receptor may be beta-hydroxybutyrate. Among nine controlled clinical trials using niacin, mostly combined with other drugs, statistically significant positive impact on clinical or anatomic cardiovascular end-points was found in seven, which represents a remarkably consistent record of benefit. Although niacin induces insulin resistance, deterioration of glycemic control in diabetes is usually minor, and there is no evidence of increased incidence of new onset diabetes. Hepatic toxicity is common with higher doses of sustained-release niacin but rare with immediate-release and extended-release niacin at doses up to 2000 mg/day. Extended-release and immediate-release niacin do not substantially potentiate myopathic effects when given in combination with statins. Recently developed understanding of the mechanisms, efficacy, and safety of niacin, along with progress in reducing the chief side effect of flushing, should enhance the use of this valuable agent for cardiovascular prevention.

Selective 5-Lipoxygenase Expression in Langerhans Cells and Impaired Dendritic Cell Migration in 5-LO-Deficient Mice Reveal Leukotriene Action in Skin

5-Lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes (LTs), which are implicated in immune reactions. Recently, it was shown that FITC-triggered epidermal Langerhans cell (LC) emigration to draining lymph nodes (LNs) is impaired in LTC4 export pump (multidrug resistance-associated protein 1)-deficient mice. Here, we sought genetic evidence for a role of endogenous LTs in dendritic cell function through the study of 5-LO-deficient mice. Though DC numbers in skin, spleen, and peripheral LNs were similar in both 5-LO-deficient and wild-type (WT) mice, DC homing from skin to draining LNs induced by FITC was reduced by 75% in 5-LO-deficient mice. Moreover, in WT mice, all epidermal LCs, dermal langerin+ LCs, and subsets of dermal macrophages and langerin+ LCs in T-cell areas of skin-draining LNs markedly expressed 5-LO. However, the enzyme was noticeably absent in all DC subsets of the dermis, thymus, spleen, Peyer's patches, mesenteric LNs, and mucosal surfaces of lung and intestine. As all epidermal cells other than LCs lacked 5-LO and because differentiation and activation of DCs generated from 5-LO-deficient mice in vitro were normal, these data support a selective role of endogenous LTs in DC homing following skin sensitization.

Runx3 regulates dendritic epidermal T cell development

The Runx3 transcription factor regulates development of T cells during thymopoiesis and TrkC sensory neurons during dorsal root ganglia neurogenesis. It also mediates transforming growth factor-β signaling in dendritic cells and is essential for development of skin Langerhans cells. Here, we report that Runx3 is involved in the development of skin dendritic epidermal T cells (DETCs); an important component of tissue immunoregulation. In developing DETCs, Runx3 regulates expression of the αEβ7 integrin CD103, known to affect migration and epithelial retention of DETCs. It also regulates expression of IL-2 receptor β (IL-2Rβ) that mediates cell proliferation in response to IL-2 or IL-15. In the absence of Runx3, the reduction in CD103 and IL-2Rβ expression on Runx3 −/− DETC precursors resulted in impaired cell proliferation and maturation, leading to complete lack of skin DETCs in Runx3 −/− mice. The data demonstrate the requirement of Runx3 for DETCs development and underscore the importance of CD103 and IL-2Rβ in this process. Of note, while Runx3 −/− mice lack both DETCs and Langerhans cells, the two most important components of skin immune surveillance, the mice did not develop skin lesions under pathogen-free (SPF) conditions.

Langerhans Cells Release Prostaglandin D2 in Response to Nicotinic Acid

Nicotinic acid, used for atherosclerosis treatment, has an adverse effect of skin flushing. The flushing mechanism, thought to be caused by the release of prostaglandin D(2) (PGD(2)), is not well understood. We aimed to identify which cells mediate the flushing effect. Nicotinic acid receptor (GPR109A) gene expression was assessed in various tissues and cell lines. Cells expressing GPR109A mRNA were further assayed for PGD(2) release in response to nicotinic acid. Of all samples, only skin was able to release PGD(2) upon stimulation with nicotinic acid. The responsive cells were localized to the epidermis, and immunocytochemical studies revealed the presence of GPR109A on epidermal Langerhans cells. CD34+ cells isolated from human blood and differentiated into Langerhans cells (hLC-L) also showed GPR109A expression. IFNgamma treatment increased both mRNA and plasma membrane expression of GPR109A. IFNgamma-stimulated hLC-Ls released PGD(2) in response to nicotinic acid in a dose-dependant manner (effector concentration for half-maximum response=1.2 mM+/-0.7). Acifran, a structurally distinct GPR109A ligand, also increased PGD(2) release, whereas isonicotinic acid, a nicotinic acid analog with low affinity for GPR109A, had no effect. These results suggest that nicotinic acid mediates its flushing side effect by interacting with GPR109A on skin Langerhans cells, resulting in release of PGD(2).

Groucho/transducin-like Enhancer-of-split (TLE)-dependent and -independent transcriptional regulation by Runx3

Regulation of gene expression by tissue-specific transcription factors involves both turning on and turning off transcription of target genes. Runx3, a runt-domain transcription factor, regulates cell-intrinsic functions by activating and repressing gene expression in sensory neurons, dendritic cells (DC), and T cells. To investigate the mechanism of Runx3-mediated repression in an in vivo context, we generated mice expressing a mutant Runx3 lacking the C-terminal VWRPY, a motif required for Runx3 interaction with the corepressor Groucho/transducin-like Enhancer-of-split (TLE). In contrast with Runx3(-/-) mice, which displayed ataxia due to the death of dorsal root ganglia TrkC neurons, Runx3(VWRPY-/-) mice were not ataxic and had intact dorsal root ganglia neurons, indicating that ability of Runx3 to tether Groucho/TLE is not essential for neurogenesis. In the DC compartment, the mutant protein Runx3(VWRPY-) promoted normally developed skin Langerhans cells but failed to restrain DC spontaneous maturation, indicating that this latter process involves Runx3-mediated repression through recruitment of Groucho/TLE. Moreover, in CD8(+) thymocytes, Runx3(VWRPY-) up-regulated alphaE/CD103-like WT Runx3, whereas unlike wild type, it failed to repress alphaE/CD103 in CD8(+) splenocytes. Thus, in CD8-lineage T cells, Runx3 regulates alphaE/CD103 in opposing regulatory modes and recruits Groucho/TLE to facilitate the transition from activation to repression. Runx3(VWRPY-) also failed to mediate the epigenetic silencing of CD4 gene in CD8(+) T cells, but normally regulated other pan-CD8(+) T cell genes. These data provide evidence for the requirement of Groucho/TLE for Runx3-mediated epigenetic silencing of CD4 and pertain to the mechanism through which other Runx3-regulated genes are epigenetically silenced.

The Class 6 Semaphorin SEMA6A Is Induced by Interferon-γ and Defines an Activation Status of Langerhans Cells Observed in Pathological Situations

Originally implicated in axon guidance, semaphorins represent a large family of molecules that are now known to be expressed in the immune system. Among different semaphorins tested by reverse transcriptase-polymerase chain reaction in human immune cells, the expression of class 6 transmembrane semaphorin SEMA6A was restricted to dendritic cells (DCs). Using in-house generated monoclonal antibodies, SEMA6A expression appeared further restricted to Langerhans cells (LCs). In vivo, SEMA6A mRNA was expressed in freshly isolated skin LCs but SEMA6A protein was not detectable on normal skin and tonsillar epithelium. Of interest, SEMA6A protein was strongly expressed on skin and bone LCs and on LCs in draining lymph nodes from patients with LC histiocytosis or dermatopathic lymphadenitis, respectively, representing two inflammatory conditions in which LCs display an immature DC-LAMP(low), CD83(low), and CCR7+ phenotype. SEMA6A expression was low in resting LCs generated in vitro and was enhanced by interferon (IFN)-gamma but not by interleukin-4, interleukin-10, IFN-alpha/beta, or lipopolysaccharide. Most IFN-gamma-induced SEMA6A-positive cells remained immature with low CD83 and DC-LAMP/CD208 expression, but they expressed CCR7 and responded to macrophage inflammatory protein-3beta (MIP-3beta/CCL19). The expression of SEMA6A, for which the ligand and function remain unknown, may therefore identify an alternative IFN-gamma-dependent activation status of LCs in vivo.

Use of interleukin 7 receptor-α knockout donor cells demonstrates the lymphoid independence of dendritic cells

The precise lineage of dendritic cells (DCs), including skin Langerhans cells (LCs), is unclear. Interleukin 7 (IL-7) and its receptor (IL-7R alpha) are known to mediate lymphopoiesis, and IL-7 is also known to be essential for the generation of DCs from lymphoid-committed precursors in vitro. Thus, to determine the developmental lymphoid (or IL-7R alpha) dependency of various DCs and to examine the importance of IL-7/IL-7R alpha for DC development in vivo, we used IL-7R alpha knockout (KO) donor cells to reconstitute DCs/LCs in sublethally irradiated recipients and compared the results to those obtained using wild-type (WT) donor cells. We found that lymphoid lineage cells (except natural killer [NK] cells), including thymocytes, were less efficiently reconstituted by IL-7R alpha KO donor cells, whereas myeloid lineage cells and DCs/LCs were equally well reconstituted by both the IL-7R alpha KO and WT donor cells. Overall, we conclude that IL-7R alpha is not required for the development of DCs/LC in vivo.

Identification of the Circulating Langerhans Cell Precursor.

We have recently shown that by contrast to other dendritic cell populations, epidermal dendritic cells also called Langerhans cells (LCs) are maintained by a pool of local radio-resistant precursor cells that persist in quiescent skin throughout life (Merad et al. Nat Immunol (3): 1135. 2002). However, skin LC precursors are replaced by circulating LC precursors after major skin lesions such as exposure to ultraviolet light (UV) but not after minor injuries such as skin sensitization (Merad et al. Nat Immunol (3): 1135. 2002). To identify the developmental stage at which LC precursors seeds quiescent skin, we reconstituted sub-lethally irradiated mice between day 0 and day 2 of life with fetal liver cells isolated from E14.5 congenic mice embryos. No donor LCs were detected over a period of one year despite high level (80%) of blood leukocyte chimerim, suggesting that a first wave of LC precursors seeds the skin during embryonic life and lose their capacity to infiltrate quiescent skin at birth or soon after birth. To explore the homeostasis of LCs recruited in inflamed skin, we devised a model of tandem transplant in which (CD45.1+ BM >CD45.2+ mice) chimeric mice were exposed to UV light to induce major skin lesions and the recruitment of blood-borne CD45.1+ LCs to the skin. Six weeks later, chimeric mice were lethally irradiated again and injected with GFP+ CD45.2+ BM cells. Although more than 90% of B cells, monocytes and granulocytes were GFP+ CD45.2+ CD45.1--, 85% of LCs in the skin was GFP- CD45.2− CD45.1+ up to 6 months after the second transplant. Using Ki-67 staining, BrdU labeling studies and cell cycle analysis, we found that CD45.1+ LCs proliferate in situ at the same rate as LCs in naïve mice. We also found that after minor injury such as skin sensitization, 40% of LCs are lost and are replaced locally by CD45.1+ LC precursors. These results suggest that a second wave of LC precursors is recruited during adult life in inflamed skin and behave as their embryonic counterpart. Indeed, adult LC precursors are radio-resistant, self renew in situ and can repopulate and maintain the LC pool during steady state or minor injuries. To explore the nature of the circulating LC precursor, we labeled monocytes in vivo using intravenous injection of fluorescent latex beads and followed the capacity of beads+ monocytes to differentiate into LC precursors in inflamed skin. We found that a subset of monocytes (CCR2+ Ly6Chi) infiltrate the inflamed skin, proliferate in situ and differentiate into LC precursors in 10 days. Similarly, we found that purified Ly6Chi monocytes injected into a UV treated animal infiltrate inflamed skin and differentiate into LC precursors. Using transfer experiment of cells deficient in the chemokine receptor CCR2 or CCR6 into wild type animals exposed to UV light, we found that recruitment of Ly6Chi monocytes to the skin was dependent on CCR2 and independent of CCR6 but CCR2+ Ly6Chi monocytes require CCR6 to differentiate into LC precursors. To our knowledge this is the first time that circulating monocytes are shown to proliferate in inflamed skin and differentiate into a LC precursor that self-renew in situ throughout life.

Triptolide, a constituent of immunosuppressive Chinese herbal medicine, is a potent suppressor of dendritic-cell maturation and trafficking

AbstractTriptolide (TPT) is a chemically defined, potent immunosuppressive compound isolated from an anti-inflammatory Chinese herbal medicine. TPT has been reported to inhibit autoimmunity, allograft rejection, and graft-versus-host disease (GVHD), and its efficacy was previously attributed to the suppression of T cells. Since dendritic cells (DCs) play a major role in the initiation of T-cell–mediated immunity, we studied the effects of TPT on the phenotype, function, and migration of human monocyte–derived DCs. TPT treatment, over a pharmacologic concentration range, inhibited the lipopolysaccharide (LPS)–induced phenotypic changes, characteristic of mature DCs and the production of interleukin-12p70 (IL-12p70). Consequently, the allostimulatory functions of DCs were impaired by TPT treatment. Furthermore, the calcium mobilization and chemotactic responses of LPS-stimulated DCs to secondary lymphoid tissue chemokine (SLC)/CC chemokine ligand 21 (CCL21) were significantly lower in TPT-treated than untreated DCs, in association with lower chemokine receptor 7 (CCR7) and higher CCR5 expression. Egress of Langerhans cells (LCs) from explanted mouse skin in response to macrophage inflammatory protein-3β (MIP-3β)/CCL19 was arrested by TPT. In vivo administration of TPT markedly inhibited hapten (fluorescein isothiocyanate [FITC])–stimulated migration of mouse skin LCs to the draining lymph nodes. These data provide new insight into the mechanism of action of TPT and indicate that the inhibition of maturation and trafficking of DCs by TPT contributes to its immunosuppressive effects.

S100B Increases Proliferation in PC12 Neuronal Cells and Reduces Their Responsiveness to Nerve Growth Factor via Akt Activation

S100B is a Ca2+-modulated protein of the EF-hand type expressed in high abundance in a restricted set of cell types including certain neuronal populations. S100B has been suggested to participate in cell cycle progression, and S100B levels are high in tumor cells, compared with normal parental cells. We expressed S100B in the neuronal cell line PC12, which normally does not express the protein, by the Tet-Off technique, and found the following: (i) proliferation was higher in S100B+ PC12 cells than in S100B- PC12 cells; (ii) nerve growth factor (NGF), which decreased the proliferation of S100B- PC12 cells, was less effective in the case of S100B+ PC12 cells; (iii) expression of S100B made PC12 cells resistant to the differentiating effect of NGF; and (iv) interruption of S100B expression did not result in an immediate restoration of PC12 cell sensitivity to the differentiating effect of NGF. Expression of S100B in PC12 cells resulted in activation of Akt; increased levels of p21WAF1, an inhibitor of cyclin-dependent kinase (cdk) 2 and a positive regulator of cdk4; increased p21WAF1-cyclin D1 complex formation; and increased phosphorylation of the retinoblastoma suppressor protein, Rb. These S100B-induced effects, as well as the reduced ability of S100B+ PC12 cells to respond to NGF, were dependent on Akt activation because they were remarkably reduced or abrogated in the presence of LY294002, an inhibitor of the Akt upstream kinase phosphatidylinositol 3-kinase. Thus, S100B might promote cell proliferation and interfere with NGF-induced PC12 cell differentiation by stimulating a p21WAF1/cyclin D1/cdk4/Rb/E2F pathway in an Akt-mediated manner.

Phenotype and functional identity of GM-CSF–independent dendritic cells generated by long-term propagation of DC progenitor cells in bone marrow cells and skin langerhans cells

Evidence is provided that dendritic cells (DC) generated by either long-term bone marrow cell (BMC) culture with Flt3L and interleukin-6 (IL-6), or after short-term BMC culture with granulocyte macrophage–colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), contain heterogeneous cell populations of admixed DC and Mϕ, regardless of the cytokine source. By employing GM-CSF–independent culture systems with the aid of Flt3/Flk-2 ligand and IL-6 and phenotypic characterization of BMC-derived DC and skin Langerhans cells (LC), revealed similar phenotypes. Furthermore, CD103 (OX62), which is widely used for rat DC separation, was found to be insufficient to enrich DC, due to downregulation of the marker. In this regard, the most efficient selection of rat DC, was obtained by CD161a (NKR-P1A), a member of the C-type lectin family. Despite the phenotypic similarity with BMC-derived DC, the nucleus of LC showed a distinct morphology. A large population of DC generated by Flt3L/IL-6 from GM-CSF receptor-deficient mice by do not express NK1.1 (NKR-P1B and NKR-P1C). The profiles for BMC-derived DC were the same as for skin Langerhans cells.