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Biomedical subjects

G Girolomoni

Publications and source records attributed to G Girolomoni.

At least 73 records · Page 4Linked to original sources
PubMed1995

Distribution of HLA class II molecules in epidermal Langerhans cells in situ.

We performed immunoelectron microscopic studies to investigate the expression of HLA class II molecules in Langerhans cells (LC). In the epidermis, LC expressed class II molecules on the plasma membrane of the dendrites, resulting in a class II positive reticulo-epithelial network, but not on the surface of the cell body. In contrast, isolated LC as well as activated LC in situ displayed an even and strong expression of class II molecules on their entire cell surface. Therefore, isolation and/or activation results in redistribution and up-regulation of class II molecules on the cell surface. In addition, double-labeling experiments were carried out with monoclonal antibodies to class II antigens and to LAMP-1, CD63 and alpha-glucosidase, specific markers for organelles of the endosomal/lysosomal system. The results show expression of class II molecules on intracellular, electron-dense vesicular structures, and co-localization of class II molecules and the markers for late endosomes and early lysosomes in human LC in situ. Expression of these markers was not found on Birbeck granules, an LC-specific organelle.

Animals↗
PubMed1995

Establishment of a cell line with features of early dendritic cell precursors from fetal mouse skin.

During ontogeny, the skin is progressively populated by major histocompatibility complex class II-negative dendritic cell (DC) precursors that then mature into efficient antigen-presenting cells (APC). To characterize these DC progenitors better, we generated myeloid cell lines from fetal mouse skin by infecting cell suspensions with a retroviral vector carrying an envAKR-mycMH2 fusion gene. These cells, represented by the line FSDC, displayed a dendritic morphology and their proliferation in serum-free medium was promoted by granulocyte/macrophage colony-stimulating factor (GM-CSF), but not macrophage-CSF. FSDC expressed strong surface-membrane ATP/ADPase activity, intracellular staining for 2A1 antigen, and a surface phenotype consistent with a myeloid precursor: H-2d,b+, I-Ad,b+, CD54+, CD11b+, CD11c+, 2.4G2+, F4/80+, CD44+, 2F8+, ER-MP 12-, Sca-1+, Sca-2+, NLDC-145-, B7.2+, B7.1-, J11d-, B220-, Thy-1-, and CD3-. FSDC stimulated poorly allogeneic or syngeneic T cells in the primary mixed-leukocyte reaction, and markedly increased this function after treatment with GM-CSF, GM-CSF and interleukin (IL)-4 or interferon-gamma (IFN-gamma); in contrast, stem cell factor, IL-1 alpha and tumor necrosis factor-alpha had no effect. Preculture with IFN-gamma was required for presentation of haptens to primed T cells in vitro. However, FSDC, even after cytokine activation, were less potent APC than adult epidermal Langerhans cells in both of the above assays. Finally, FSDC derivatized with haptens and injected either intravenously or subcutaneously could efficiently induce contact sensitivity responses in naive syngeneic mice. The results indicate that fetal mouse skin is colonized by myeloid precursors possessing a macrophage/immature DC-like surface phenotype and priming capacity in vivo. These cells need further differentiation and activation signals (e.g. cytokines) to express their antigen presenting potential in vitro.

Animals↗
PubMed1995

Langerhans cells and HIV infection.

Epidermal Langerhans cells (LCs) isolated from individuals infected with human immunodeficiency virus (HIV-1) harbour HIV-1 proviral DNA and RNA, indicating productive infection by the virus in vivo. Furthermore, normal LCs can be infected in vitro by HIV and can present HIV antigens to helper T cells. Here, Giovanna Zambruno and colleagues discuss the possibility that LCs of genital mucosae are among the first targets of HIV infection following sexual contact, and can be involved both in the transmission of the infection to T cells and in T-cell priming to HIV antigens. In addition, epidermal LCs might acquire HIV infection from dermal T cells during transit from blood vessels through the dermis and may, in turn, represent a reservoir of the virus for continued T-cell infection.

Animals↗
PubMed1995

Functional intercellular adhesion molecule-3 is expressed by freshly isolated epidermal Langerhans cells and is not regulated during culture.

Activation of T lymphocytes by antigen-presenting cells requires the interaction of major histocompatibility complex/antigen complexes with the T-cell receptor as well as the binding of co-stimulatory molecules to receptors on T cells. Freshly isolated epidermal Langerhans cells (LC) do not display a significant number of co-stimulatory molecules. After short-term culture, LC express and then upregulate intercellular adhesion molecule-1 (ICAM-1) (CD54), leukocyte function-associated antigen (LFA)-3 (CD58), and B7-1 (CD80) accessory molecules and exhibit an enhanced antigen-presenting function. The present study examined the presence on human LC of the LFA-1 ligands ICAM-2 (CD102) and ICAM-3 (CD50) and their functional role in the activation of allogeneic T cells. Immunohistochemistry of skin sections and flow-cytometry analysis of freshly procured epidermal cell suspensions showed that LC (CD1a+ or HLA-DR+) expressed ICAM-3 but not ICAM-2. After 48-72-h culture in the presence of granulocyte/macrophage colony-stimulating factor, LC did not stain for ICAM-2 but expressed ICAM-3 at the same level as fresh cells. Incubation of both freshly isolated and cultured LC with monoclonal antibodies directed against ICAM-3 reduced T-cell proliferation (25-75% inhibition) in the primary allogeneic mixed leukocyte reaction assay; incubation of cultured LC with anti-ICAM-1 and anti-ICAM-3 synergistically reduced T-cell response. The results indicate that ICAM-3 is constitutively expressed and represents an important costimulatory molecule on freshly isolated LC but, in contrast to other accessory molecules, is not subjected to regulation during LC culture.

Antigens↗
PubMed1995

In vitro infection of human epidermal Langerhans' cells with HIV-1.

Epidermal Langerhans' cells (LC) from human immunodeficiency virus type-1 (HIV-1)-infected patients harbour HIV-1 proviral DNA and RNA. In the present study, we investigated whether LC from epidermis of normal, HIV-seronegative subjects could be infected in vitro with HIV-1. Epidermal cells (EC) spontaneously detached from epidermal sheet cultures were enriched for LC (10-25% of CD1a+/CD4+ cells), deprived of contaminating T cells and then incubated with HIV-1IIIB. After 24 hr, purified LC and LC-depleted EC fractions were obtained by immunomagnetic separation. Polymerase chain reaction (PCR) analysis showed the presence of HIV-1 proviral DNA (gag) only in purified LC. In addition, LC-enriched EC, purified LC, LC-depleted EC or the non-permissive cell line, TF-1, the latter having being previously challenged with HIV-1IIIB for the same length of time as the EC, were co-cultivated with C8166 cells, and the co-cultures assessed for the presence of HIV DNA by PCR. Co-cultures of C8166 cells with purified LC or LC-enriched EC previously exposed to HIV-1IIIB exhibited a time-dependent increase in HIV proviral DNA. In contrast, PCR analysis of C8166 cells co-cultured with either LC-depleted EC or TF-1 cells gave negative results. Finally, C8166 cells co-cultured with HIV-infected LC formed syncytia, showed membrane budding and released numerous retroviral particles. The results indicate that LC from normal subjects can be infected in vitro with HIV and can transmit infection to myeloid cells. This in vitro model may help in understanding the regulation of HIV infection of LC.

Cell Movement↗
PubMed1994

Modulation of cytokine expression in mouse dendritic cell clones.

Dendritic cells (DC) play an essential role in the induction of primary immune responses; however, very little information is available on cytokine production by DC. Here we determined the cytokine gene expression profile of two immortalized DC clones, CB1 and D2SC/1, both generated from mouse spleen but differing in their activation requirements. Among the cytokines tested, only transforming growth factor-beta 1 was transcribed constitutively, but its production was detected only in D2SC/1 cells after treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF). GM-CSF also promoted transcription and synthesis of interleukin (IL)-1 beta in CB1 cells that need pretreatment with GM-CSF to present major histocompatibility complex class II-restricted antigens efficiently in vitro. Lipopolysaccharide (LPS) up-regulated gene expression and induced release of tumor necrosis factor-alpha in both DC clones. In addition, LPS induced transcription of IL-1 alpha and both gene expression and synthesis of IL-1 beta in D2SC/1 cells. Interferon-gamma was ineffective in inducing cytokine gene expression, although it augmented the antigen-presentation capacity of DC, IL-4, IL-10 and IL-12 mRNA were not induced by any of the tested stimuli. The results suggest that DC have a limited cytokine gene expression pattern compared to macrophages and are heterogenous in some functional properties.

Animals↗
PubMed1994

Expression of B7 costimulatory molecule in cultured human epidermal Langerhans cells is regulated at the mRNA level.

Langerhans cells (LC) belong to the dendritic cell lineage and are the principal antigen-presenting cells of squamous epithelia. Short-term cultured LC (cLC) exhibit a marked augmented capacity to stimulate allogeneic T cells and acquire the ability to activate naive T cells, probably in relation to enhanced expression of accessory signals. In this study, we evaluated the expression of B7 costimulatory molecule (CD80) in human freshly isolated (fLC) and cLC at both the protein and mRNA level. Staining of frozen skin sections did not reveal any epidermal dendritic cell reactive with either of two different anti-B7 monoclonal antibodies. fLC in suspension did not exhibit any B7 staining as evaluated by two-color flow-cytometry analysis and immunoelectron microscopy. In contrast, LC that were cultured for 24-72 h displayed strong surface B7 reactivity with a characteristic patchy pattern. Treatment with dispase and trypsin did not reduce B7 staining of cLC. Following warming to 37 degrees C, cLC tagged with anti-B7 monoclonal antibody and gold-conjugated secondary antibody could internalize surface B7 by using the organelles of receptor-mediated endocytosis. B7 mRNA, detected by the reverse-transcriptase polymerase chain reaction technique, was expressed at a low level in purified (> 90% HLA-DR+) fLC but not in LC-depleted epidermal cells, and was markedly upregulated in purified cLC. The results indicate that 1) fLC do not express B7 protein on their surface, but acquire B7 during culture, 2) surface B7 is not sensitive to trypsin, 3) B7 expression is regulated primarily at the mRNA level, and 4) membrane B7 can be internalized within cLC. B7 molecule on CLC may be relevant to their increased antigen-presenting cell potency and ability to stimulate naive T lymphocytes.

Antibodies, Monoclonal↗
PubMed1994

Integrin expression in middle ear cholesteatoma.

Cholesteatoma is lined by a squamous keratinizing epithelium exhibiting most of the features of normal epidermis. In this study, we investigated by immunohistochemistry the expression of integrin adhesion molecules in primary acquired and recurrent cholesteatomas, and compared it with common epidermal cysts and normal human skin. The results showed that cholesteatoma epithelium exhibited a markedly augmented expression of alpha v integrin subunit and a corresponding increased deposition of vitronectin (alpha v ligand) in the surrounding stroma as compared to epidermal cyst and normal human skin. In contrast, the expression pattern of alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 integrins as well as the distribution of laminin, collagen IV and fibronectin were similar in cholesteatomas, epidermal cysts and normal human skin. Similar staining pattern was observed in primary acquired and recurrent cholesteatoma.

Antigens, CD↗
PubMed1994

Quantitation by competitive PCR of HIV-1 proviral DNA in epidermal Langerhans cells of HIV-infected patients.

Langerhans cells (LC) belong to the dendritic cell family and represent the principal antigen presenting cells populating squamous epithelia. We have reported the presence of human immunodeficiency virus Type 1 (HIV-1) proviral DNA and RNA in purified LC from the epidermis of seropositive patients. The aim of this study was to quantify HIV-1 proviral DNA in LC of infected patients using a competitive polymerase chain reaction (PCR) assay. Bulk epidermal cell (EC) suspensions were obtained from the skin of nine AIDS patients and six seronegative subjects. Purified LC and LC-depleted EC were prepared by immunomagnetic separation using an anti-CD1a monoclonal antibody. LC preparations did not contain T cells, as assessed by reverse transcription PCR analysis of the T cell receptor beta-chain gene (C region). In addition, no CD14+ cells could be detected in LC fractions by immunostaining of cytospin preparations. To quantify HIV-1 DNA, a new competitive PCR system was devised using SK145/150 as primers (gag) and a competitor plasmid DNA with a modified sequence (209 instead of 142 bp). The number of HIV-1 DNA copies found in the LC of AIDS patients ranged from 107 to 3,645/10(5) LC. In contrast, LC-depleted EC from the same subjects were all negative. The results indicate that in AIDS patients the frequency of infected LC is comparable to that reported for peripheral blood CD4+ T cells.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Immortalized dendritic cell line fully competent in antigen presentation initiates primary T cell responses in vivo.

Dendritic cells (DC) can provide all the known costimulatory signals required for activation of unprimed T cells and are the most efficient and perhaps the critical antigen presenting cells in the induction of primary T cell-mediated immune responses. It is now shown that mouse cell lines with many of the features of DC can be generated using the MIB phi 2-N11 retroviral vector transducing a novel envAKR-mycMH2 fusion gene. The immortalized dendritic cell line (CB1) displays most of the morphologic, immunophenotypic, and functional attributes of DC, including constitutive expression of major histocompatibility complex (MHC) class II molecules, costimulatory molecules B7/BB1, heat stable antigen, intracellular adhesion molecule 1, and efficient antigen-presenting ability. Granulocyte/macrophage colony-stimulating factor (GM-CSF) proved to be effective in increasing MHC class II molecule expression and in enhancing presentation of native protein antigens. In comparison with macrophages, CB1 dendritic cells did not exhibit phagocytic and chemotactic activity in response to various stimuli and lipopolysaccharide activation was ineffective in inducing tumor necrosis factor alpha or interleukin 1 beta production. CB1 cells, pulsed with haptens in vitro and injected into naive mice were able to induce delayed-type hypersensitivity responses, further increased with pretreatment with GM-CSF, indicating that these cells may represent an immature, rather than a mature DC. The ability of CB1 to prime T cells in vivo could provide a tool to design novel immunization strategies.

Animals↗
PubMed1993

Phosphatidylserine enhances the ability of epidermal Langerhans cells to induce contact hypersensitivity.

Phosphatidylserine (PS) modulates several immune functions in vitro, including T cell activation, antibody and cytokine production, and macrophage growth. In the present work we studied the effects of PS on the induction of contact hypersensitivity (CH) in mice. BALB/c mice painted with PS (9.4-75 mg/kg) and with a sensitizing dose of DNFB or oxazolone on the same skin site exhibited a dose-dependent augmentation of CH reactions to either DNFB (> 60%) or oxazolone (> 35%), respectively. Bovine brain PS-enriched phospholipid mixture, lyso-PS, and dipalmitoyl-PS also induced similar enhanced CH responses, whereas phosphatidylglycerols had no effect. Increased CH was observed only when PS was applied from 2 days before to 12 h after DNFB. Immunization of naive syngeneic mice with skin grafts that were treated with PS and DNFB also led to enhanced (> 50%) CH responses. In addition, immunization by iv injection of epidermal cell suspensions enriched for Langerhans cells (LC) or of purified LC that were treated with PS (1-100 microM, 30 min, 37 degrees C), and then modified in vitro with DNBS (1 mg/ml, 30 min, 37 degrees C) led to increased (> 30-75%) CH responses in recipient syngeneic animals. Finally, adoptive transfer of DNFB-immune lymph node cells obtained from mice that were treated with PS induced augmented CH responses in recipient animals. The results suggest that PS is capable of up-regulating the induction of CH in mice by stimulating the APC function of epidermal LC.

Animals↗
PubMed1993

Prolactin stimulates proliferation of cultured human keratinocytes.

The effect of the pituitary hormone prolactin on in vitro proliferation of human keratinocytes has been studied. Cell proliferation was determined by [3H] thymidine incorporation and cell enumeration in culture. Physiologic concentrations of prolactin markedly stimulated proliferation of newborn foreskin keratinocytes in serum-free medium. In addition, it was able to replace almost completely the growth-promoting effects of bovine pituitary extract, a commonly added supplement for keratinocyte culture. This activity was also evident in the absence of epidermal growth factor, but required the presence of insulin. Radioligand-binding studies confirmed the expression of specific prolactin binding sites (Kd 8.9 nM; 1350 sites per cell) on freshly procured keratinocyte membranes. These results extend its hormonal influences to include regulation of in vitro proliferation of human keratinocytes, and suggest the possibility of a completely defined growth medium for keratinocytes.

Binding Sites↗
PubMed1993

Epidermal Langerhans cells are resistant to the permeabilizing effects of extracellular ATP: in vitro evidence supporting a protective role of membrane ATPase.

Extracellular adenosine 5'-triphosphate (ATPo) can induce pore formation in cell membranes, leading to cell permeabilization and eventual cell death. In this study, we examined the sensitivity of human epidermal Langerhans cells to ATP-induced permeabilization and tested the possibility that the Mg(++)- or Ca(++)-dependent plasma membrane ectonucleotidase (mATPase) on Langerhans cells provides protection against the cytotoxic effects of ATPo. Membrane permeability was assessed by using the fluorescent tracer propidium iodide, which confers red nuclear fluorescence to permeabilized cells. Langerhans cells were identified within human epidermal cell suspensions with fluorescein isothiocyanate-conjugated MoAb against CD1a or human leukocyte antigen-DR (HLA-DR) antigens. Cultured human keratinocytes and J774 macrophages were both highly sensitive to permeabilization induced by incubation with ATP (0.5 to 20 mM at 37 degrees C), whereas Langerhans cells were relatively resistant. The non-hydrolyzable ATP analog, adenosine 5'-(beta,gamma-imido) triphosphate, but not other nucleotides such as ADP, AMP, GTP, or UTP, was also able to induce permeabilization comparable to that of ATP, thereby suggesting that ATP hydrolysis is not required for this effect. ATP4- is the moiety most likely responsible for permeabilization, because propidium iodide uptake occurred only when the pH of the medium was > or = 7.4. Permeabilization induced by ATP was augmented by chelation of divalent cations with ethylene-diamine-tetraacetic acid and by the addition of lanthanum or cerium (0.01 to 1 mM). Finally, incubation with the adenosine analog, 5'-p-fluorosulfonylbenzoyl-adenosine (1 mM), inhibited mATPase staining of Langerhans cells in human epidermal sheets, but markedly augmented ATP-induced permeabilization of Langerhans cells. The results indicate that epidermal LC are resistant to the lytic effects of ATPo and that mATPase is involved in such resistance.

Adenosine Triphosphatases↗