Biomedical subjects
Publications and source records attributed to G Girolomoni.
Congenital dermatofibrosarcoma protuberans occurred in a 16-year-old girl. The lesion was a hard cutaneous plaque on the abdomen. It was present at birth and slowly enlarged during the patient's lifetime. Histologic examination was essential to establish the diagnosis and to differentiate it from other congenital fibrohistiocytic proliferations.
Expression of the adhesion molecules ICAM-1 and LFA-1 (CD11a/CD18) on mouse epidermal Langerhans cells (LC) and on spleen dendritic cells (DC) from BALB/c mice was examined by staining with specific mAb and was evaluated by flow cytometry. LC were shown to express both ICAM-1 and LFA-1, whereas spleen DC expressed only LFA-1. The contribution of these adhesion molecules to LC- or DC-induced activation of keyhole limpet hemocyanin (KLH)-specific, Iad-restricted, Th1 or Th2 clones was investigated in mAb blocking studies. At optimal doses, anti-CD11a or anti-CD18 mAb completely inhibited Th1 proliferation induced by either LC or DC. Anti-ICAM-1 also abrogated Th1 proliferation induced by LC, but only moderately reduced Th1 proliferation induced by DC. Inhibition in these experiments was specific, since isotype-matched control Ab against other Ag constitutively expressed on LC (NLDC 145) or DC (33D1) had no effect on Th1 proliferation. In marked contradistinction, the capacity of LC to present KLH to our Th2 clones was resistant to treatment with the same mAb against ICAM-1, CD11a or CD18. We conclude that interactions between ICAM-1 and LFA-1 on epidermal LC and LFA-1 on spleen DC with their respective ligands on our Th1 clones are required for optimal presentation of protein Ag to Th1. Our results also indicate that neither ICAM-1 nor LFA-1 is required for the analogous activation of our Th2 clones by LC.
Human Langerhans cells (LC) are bone marrow-derived, HLA-DR+, CD1a+, and CD4+ dendritic antigen-presenting cells found in stratified squamous epithelia. As other members of the dendritic leukocyte family, to which they belong, LC have been reported as targets for HIV-1 infection. The aim of the present study was to investigate whether HIV-1 RNA is expressed in epidermal LC of HIV-1-infected patients. Bulk epidermal cell (EC) suspensions were prepared from skin of nine recently deceased AIDS patients and 11 seronegative controls. Purified LC (94 +/- 4% HLA-DR+ cells with no CD3+ cells, as assessed by flow microfluorimetry analysis) and LC-depleted EC were obtained by immunomagnetic separation using an anti-CD1a monoclonal antibody. Samples were analyzed for the presence of HIV-1 RNA by reverse transcription of a spliced mRNA region of the tat gene, followed by polymerase chain reaction amplification. HIV-1-spliced RNA was detected in LC from 6 of 9 patients examined, whereas LC-depleted EC fractions from the same patients were all negative. The results indicate that epidermal LC from HIV-seropositive patients actively transcribe HIV-1 proviral DNA, further supporting the hypothesis that HIV productively infected LC could serve as a reservoir of the virus in the epidermis and as a source for the infection of T lymphocytes.
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We report a case of multinucleate cell angiohistiocytoma occurring in a 50-year-old woman and presenting with a 4-year history of asymptomatic red papules on the face. Histologically, there was a dermal vascular proliferation associated with numerous multinucleate cells. Immunohistochemical studies showed vascular spaces surrounded with mature vascular endothelial cells, and the presence of numerous interstitial factor XIIIa+ cells. Multinucleate cells exhibited intermediate filaments of the vimentin type, but were not stained by endothelial cell or macrophage markers, and were factor XIIIa-. This condition must be addressed in the differential diagnosis of other skin vascular proliferations, such as Kaposi's sarcoma and angiofibromas.
Intracellular acidic compartments serve several functions, including uptake of nutrients, processing and sorting of secreted and membrane-bound proteins, and even entry of viruses into cells. In this study, we examined the distribution of acidic compartments in normal human keratinocytes cultured in serum-free medium. Acridine orange was used to stain acidic organelles (red fluorescence), and adherent cells were evaluated by fluorescence microscopy and by interactive laser cytometry (ILC). Keratinocytes cultured in low [Ca++] (0.15 mM) exhibited morphologic characteristics associated with basal cells; red acidic vesicles in these cells were aggregated around the nucleus, sparing the peripheral cytoplasm. After 24 h of culture in high [Ca++] (1.5 mM) keratinocytes showed morphologic changes associated with differentiated cells, including increased number and dispersal of red vesicles to the periphery of the cytoplasm. Keratinocytes cultured in 0.15 mM [Ca++], but treated with phorbol 12-myristate 13-acetate (PMA, 5-100 ng/ml) to induce terminal differentiation, developed similar features. Incubation in media with either high [Ca++] or PMA also induced radial extension of the microtubule network, suggesting that the distribution of acidic organelles occurs along this network. Finally, crude keratinocyte membranes were evaluated by radioactive assay for the presence of three ion-translocating ATPase activities, plasma membrane Na/K ATPase, mitochondrial ATPase, and vacuolar H+ pump ATPase, the latter being the activity responsible for acidification of intracellular compartments. Both basaloid and differentiated keratinocytes exhibited similar vacuolar H+ pump ATPase activity, as measured by its sensitivity to bafilomycin.
Epidermal Langerhans cells (LC) possess a plasma membrane Mg(++)- or Ca(++)-dependent ecto-ATPase (mATPase), the function of which is not known. On the other hand, extracellular ATP (ATPo) has been shown to cause a rapid increase in cell-membrane permeability for selected cells, thus inducing the entry of exogenous substances, leading eventually to cell death. ATPo has also been shown to be a mediator of T cell-mediated cytotoxicity. We have observed human LC to be resistant to the permeabilizing effects of ATPo when compared to cultured human keratinocytes and J774 macrophages. In addition, our preliminary evidence in vitro suggests that mATPase on LC may provide protection against the lytic effects of ATPo. Finally, we propose that this latter mechanism may also operate in vivo.
Depletion of Langerhans cells (LC) is known to follow bone marrow transplantation (BMT) and is thought to be mainly related to pretransplant radiation and chemotherapy conditioning regimens. We studied sequential biopsies of clinically normal skin of 22 thalassemic and leukemic patients undergoing allogeneic BMT who had received only chemotherapy (busulfan and cyclophosphamide) as conditioning regimen. LC were identified immunohistochemically using antibodies against CD1a and HLA-DR antigens, and their number expressed per square mm of epidermal vertical section, the latter measured by computerized image analysis. After the preparatory regimen, the number of LC decreased progressively in both leukemic and thalassemic patients. CD1a+ and HLA-DR+ epidermal cells were reduced, respectively, to 68.5% and 64.5% of their original number around Day 2, and to 23.1% and to 18.2% around Day 17. By this time, electron microscopic examination of selected biopsies confirmed the depletion of LC. Variable repopulation was observed between Days 40 and 60. Our results indicate that a conditioning regimen based exclusively on high dose chemotherapy depletes epidermal LC early after BMT, and that such depletion is not related to the development of acute graft-versus-host disease.
Ia-bearing dendritic cells (DC) are a class of bone marrow-derived antigen-presenting cells that appear to possess an increased capacity to stimulate resting T lymphocytes. DC from different tissues share several morphologic, phenotypic and functional attributes. For example, freshly isolated DC from spleen resemble phenotypically and functionally freshly isolated Langerhans cells (LC) from epidermis; in addition, during short-term culture both DC and LC undergo several parallel changes including modifications affecting phenotype, capacity to present protein antigens, and ability to route surface Ia molecules into intracellular acidic compartments (J Immunol 1990: 145: 2820-2826). In the present study we show, using immunoelectron microscopy with anti-Ia and anti-33D1 monoclonal antibodies, freshly isolated DC in suspension to have a smooth cell surface with few and short cytoplasmic projections. By contrast, cultured DC display conspicuous bulbous cytoplasmic protrusions. In addition, spleen DC following culture for 24-48 hours exhibit an increased ability to stimulated allogeneic T lymphocytes in the primary mixed leukocyte reaction. These changes, similar to those described for freshly isolated and cultured LC respectively, further substantiate the close relationship between DC and LC.
Langerhans cells (LC) are the principal antigen-presenting cells (APC) of squamous epithelia. We have previously shown that freshly isolated LC (fLC) are able to deliver endocytosed membrane MHC class II molecules into acidic environments, and that this capacity is lost when LC are placed in culture (cLC). Inasmuch as processing of antigens requires their passage through acidic compartments, we undertook the present study to examine the ability of fLC and cLC to take up acridine orange, and to identify proton-translocating ATPases in these cells. Using flow cytometry and fluorescence microscopy, acridine orange was observed to accumulate in acidic compartments in both fLC and cLC. Using a radioactive ATPase assay, crude membrane preparations from both fLC and cLC were shown to possess three types of ion-translocating ATPase, based on sensitivity to the following inhibitors: ouabain (Na+, K+ ATPase), oligomycin (mitochondrial F1F0 ATPase), and bafilomycin (vacuolar-type proton pump ATPase); the last type is responsible for acidification in vacuolar compartments. cLC displayed markedly less (less than 50%) total ATPase activity compared to fLC; however, the relative proportions of specific ATPases were similar in fLC and cLC. Combined use of the three inhibitors resulted in abrogation of only 25-40% of the total ATPase activity. Finally, treatment of LC with bafilomycin inhibited both acridine orange uptake and acidification of internalized HLA-DR molecules. These results confirm the ability of both fLC and cLC to acidify vacuolar compartments, thereby suggesting that lack of acidification of endocytosed membrane class II molecules in cultured cells is due to alternative routing to non-acidic organelles.
Graft-versus-host disease (GVHD) is an immunologically mediated disease occurring most frequently after allogeneic bone marrow transplantation. The aim of this study was to evaluate the contribution of immunohistochemistry in the diagnosis of cutaneous GVHD. Patients transplanted for either leukemia or beta-thalassemia were included in the study. Skin lesions of acute and chronic GVHD were examined both by direct immunofluorescence to detect immunoglobulin deposits and by an avidin-biotin-peroxidase complex technique to evaluate the inflammatory cell infiltrate. Epidermal and dermal fluorescent bodies (IgG and IgM) were frequently found in both acute and chronic GVHD. Most of the infiltrating cells were CD3+ T lymphocytes, with CD8+ cells representing the major cell population invading the epidermis both in acute GVHD and in chronic lichenoid GVHD. A small proportion of the dermal cells were CD14+ macrophages; no B cells were detected. HLA-DR, but not HLA-DQ antigens, were variably expressed by keratinocytes in all cases of acute GVHD and in chronic lichenoid GVHD. KL-1, a monoclonal antikeratin antibody specific for the 56.5 KD acidic polypeptide usually present in suprabasal keratinocytes, stained all epidermal layers, including the basal layer. Langerhans cells were dramatically reduced in number in the epidermis of both acute and chronic lichenoid GVHD. It is concluded that immunohistologic analysis may be supportive in the diagnosis of acute and early chronic lichenoid cutaneous GVHD.
Spleen dendritic cells (DC) and epidermal Langerhans cells (LC) belong to the same family of dendritic leukocytes and are considered to be prototypes of lymphoid DC and nonlymphoid DC, respectively. These cells are active APC in vitro and play a key role in the induction of primary T cell dependent immune responses in vivo. Two functional states of LC have been characterized in vitro, freshly isolated LC and cultured LC (cLC). That cLC closely resemble spleen DC in phenotype and function, has led to the hypothesis that LC undergo maturation toward DC while in culture, an event that has been correlated with the emigration of LC from skin into lymphoid organs. To date, however, DC have been studied only after overnight culture. To better understand the relationship between LC and DC, we examined DC shortly after their isolation from spleen, and after 24 h of culture. Freshly isolated DC (fDC) express high levels of MHC molecules and low levels of Fc gamma RII and C3biR; fDC also uniformly express the Ag recognized by the mAb 33D1, NLDC-145, and J11d. After culture, DC display a marked increase in the expression of MHC molecules, and they are induced to express the low affinity receptor for IL-2. By contrast, the expression of Fc gamma RII and F4/80 decreases with culture. With respect to function, fDC can efficiently present keyhole limpet hemocyanin to Ag-specific T cells, whereas cultured DC exhibit a marked reduction in this capacity. Finally, both fDC and cultured DC are capable of endocytosing surface Ia molecules, but only fDC are able to deliver them into acidic compartments. Our data indicate that fDC from spleen resemble freshly isolated LC from epidermis and that both cells undergo parallel changes during culture. These results suggest that LC and DC possess analogous attributes in vivo and respond similarly to external influences.
Capsaicin, a neurotoxin that depletes primary sensory neurons (polymodal nociceptors) of neuropeptides, was used to explore the role of such neurons on the expression of delayed-type hypersensitivity reactions in mice. BALB/c mice received s.c. injections with capsaicin (100 mg/kg) and tested 1 to 2 wk later exhibited insensitivity to chemically induced irritation (greater than 80% reduction in the eye-wiping response for more than 15 wk) as well as loss (greater than 95% reduction) of the ear swelling response to topical capsaicin. Early (less than or equal to 4 h) ear swelling to topical DNFB and oxazolone was also markedly reduced by capsaicin pretreatment, suggesting neurogenic inflammation as a major component of the early irritant reaction to haptens. In contrast, capsaicin-pretreated mice exhibited enhanced contact sensitivity (CS) reactions to oxazolone (greater than 90%) and DNFB (greater than 50%) and enhanced delayed-type hypersensitivity reactions to SRBC (greater than 20%). Adoptive transfer experiments revealed that CS augmentation was not due to generation of increased numbers and/or activity of effector T cells. Histologic studies as well as experiments measuring migration of 51Cr-labeled, Ag-nonspecific cells showed increased edema and enhanced cell localization in CS elicitation sites in capsaicin-pretreated mice. These results indicate that peptidergic neurons, via neuropeptide release, regulate the expression of T cell-mediated, delayed-in-time, cutaneous inflammatory reactions. The net effect of these neurons on the late (cellular) phase of such responses seems to be suppressive, because their impairment results in augmented reactions.
Epidermal growth factor receptors (EGFR) and transferrin receptors (TFR) are known to be involved in cell proliferation and to be expressed in normal human epidermis. To date little is known about EGFR and TRF expression in human skin during embryonic and fetal development. In the present work, we studied skin specimens from 30 aborted embryos and fetuses ranging from 7 to 31 weeks estimated gestational age. Monoclonal antibodies to EGFR and TFR were applied on frozen skin sections using an amplification biotin-streptavidin-fluorescein technique. TFR was faintly expressed on epidermal basal cells throughout embryonic and fetal development, as it is in adult epidermis. Up to week 12, EGFR was uniformly expressed on cells of the basal, intermediate and periderm cell layers. From the midfetal period onwards, the suprabasal cell layers showed a decreased staining compared with the basal layer. During the third trimester the cornified cell layer was completely negative. The hair germ and heir peg cells were positive. Later, the outer root sheath and hair bulb remained labelled, with less staining of the hair cone. The sebaceous and eccrine sweat glands were also labelled. These results suggest that in embryonic and fetal epidermis, TFR expression is not correlated with cellular proliferation, whereas EGFR appear to be associated with proliferating and undifferentiated cells.
CD4+ T lymphocytes recognize multi-molecular complexes, formed by major histocompatibility complex class II molecules and exogenous antigens, on the surface of antigen-presenting cells (APC). For most protein antigens, processing is required to produce immunogenic peptide fragments that can then form stable associations with class II molecules. These two processes, the modification of antigen and its coupling to class II molecules, are thought to occur in acidic endosomal compartments. Furthermore, membrane class II molecules are endocytosed in APC and may provide ligands for the immunogenic peptides. To gain insight into these processes, we examined the internalization and acidification of membrane HLA-DR molecules by three APC populations: 1) freshly isolated Langerhans cells (LC), 2) LC after 48-72 h of bulk epidermal cell culture, and 3) peripheral blood monocytes (PBM). Using FITC-conjugated anti-HLA-DR monoclonal antibodies (MoAb), endocytosis was studied by fluorescence microscopy and by flow cytometry (pulse width analysis), while acidification was assessed by exploiting the pH sensitivity of fluorescein fluorescence. We observed both freshly isolated LC and PBM to internalize surface HLA-DR molecules into acidic compartments with great efficiency. Endocytosis was inhibited by the addition of azide and 2-deoxy-D-glucose, whereas acidification was partially blocked by treatment with ammonium chloride or chloroquine. The degree of internalization and acidification of HLA-DR molecules was greatly influenced by the degree of Ab cross-linking. On the other hand, cultured LC were capable of internalizing HLA-DR molecules, but were not able to acidify the environments to which these molecules were delivered; this loss of acidification capacity was partially restored by treatment with phorbol 12-myristate 13-acetate.(ABSTRACT TRUNCATED AT 250 WORDS)
The distribution and localization of several neuropeptides were investigated in the lichenified lesions of 11 patients with atopic dermatitis using indirect immunofluorescence. Substance P-positive nerve fibres were observed in most of the cases of atopic dermatitis, but not in normal controls. Somatostatin immunoreactive nerves were not found in the skin of atopic dermatitis, whereas a normal pattern of immunoreactivity could be detected in most of the healthy subjects. Neuropeptide Y-positive dendritic epidermal cells were observed in lesional skin from patients with atopic dermatitis, but not in controls. Calcitonin gene-related peptide and vasoactive intestinal polypeptide immunoreactivity in patients with atopic dermatitis did not differ from that in healthy subjects. With galanin antiserum a diffuse intracellular staining was observed in the epidermis of both atopic patients and controls, while no positive staining was found with either neurotensin or neurokinin A antibodies in either group. These findings suggest a possible involvement of some neuropeptides in the pathomechanisms of atopic dermatitis.
Adults with atopic dermatitis (AD), with respiratory atopy only and healthy non-atopic controls were given intradermal injections of substance P (SP), neurokinin A (NKA), neurotensin (NT), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP) and histamine into the normal-appearing skin on the back. The weal and flare responses were evaluated after 3, 5 and 15 min and the areas calculated using an automatic image analyser. With the three different concentrations used (1, 3 and 30 pmols) a statistically significant (P less than 0.05) reduction in both the weal and flare response to SP, NKA, NT and histamine and a reduced flare to CGRP was observed only in AD patients. Among those with AD there was no uniformity of response to the individual neuropeptide and in general the more severely affected showed a lower reactivity. Dose-response relationships were evaluated for SP and NT (10-320 pmols) in AD and healthy controls. In AD dose-response curves and time-course relationships were similar to controls, but at significantly reduced levels. The itch response to the neuropeptides and histamine was not different in atopics and controls. We suggest that this hyporesponsiveness in AD is the result of natural tachyphylaxis of the target structures (mast cells and blood vessels) and possibly due to a higher availability of neuropeptides in the skin or to a primary abnormal sensitivity of the blood vessels and mast cells to these peptides.
A 52-year-old male patient with bronchial carcinoma developed pemphigus vulgaris 3 weeks after the end of cobalt therapy. The possible role of gamma-radiation in inducing the skin disease is discussed.