Biomedical subjects

N Romani

Publications and source records attributed to N Romani.

At least 73 records · Page 4Linked to original sources

PubMed1992

Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellular antigen of interdigitating dendritic cells and some B lymphocytes.

A monoclonal has been isolated that labels an intracellular antigen in dendritic cells and some B cells. The M342 hamster immunoglobulin was selected because it stained cells in the periarterial sheaths of spleen, the deep cortex of lymph node, and the thymic medulla--the same regions in which one finds interdigitating cells, the presumptive in situ counterparts of isolated lymphoid dendritic cells. M342 labeled an antigen within granules of isolated dendritic cells, but only in cells that had been cultured for a day and not in fresh isolates. This extends recent findings that most freshly isolated spleen dendritic cells are located in the periphery of the white pulp nodule and may serve as precursors for the periarterial pool of interdigitating cells, the site for M342 staining in situ. By electron microscopic immunolabeling, the M342 antigen was found exclusively in a type of multivesicular body. M342 staining was not found in mononuclear phagocytes from blood and peritoneal cavity. Peritoneal B cells expressed M342+ granules, and upon appropriate stimulation splenic B cells developed reactive granules as well. We conclude that M342 is a strong marker for interdigitating cells. Its existence reveals intracellular specializations in the vacuolar system of antigen-presenting cells including subsets of dendritic cells.

Animals↗

PubMed1992

The immunologic properties of epidermal Langerhans cells as a part of the dendritic cell system.

Dendritic cells form a system of antigen-presenting cells that is widely distributed in the body. They constitute trace populations in lymphoid and non-lymphoid tissues and in the circulation. They are characterized by their typical dendritic and "veiled" morphology, by their constitutive expression of high levels of major histocompatibility complex class II molecules on their surface, and by their outstanding capacity to initiate primary immune responses. Dendritic cells occur in two states of differentiation. In the immature state they are highly specialized for processing foreign protein antigens; in the mature state they efficiently stimulate resting antigen-specific T cells. Dendritic cells can migrate from the non-lymphoid tissues, where they reside in the immature state, via the afferent lymphatics or the blood to the T cell-dependent areas of the lymphoid organs (lymph nodes, spleen). There, they appear as mature dendritic cells. Therefore, dendritic cells are ideally suited to mediate important aspects of immunogenicity: they can acquire antigens in the tissues and process them in an immunogenic form; they can carry the immunogen to the lymphoid organs; and they can find and efficiently activate antigen-specific T cell clones and thus generate an immune response. Studies of epidermal Langerhans cells have greatly helped in establishing this concept. They can be investigated freshly isolated from the epidermis where they represent immature (tissue) dendritic cells. After 2-3 days in culture they develop into mature dendritic cells. The mechanisms of dendritic cell maturation, which can be studied best using epidermal Langerhans cells, and the specific functions of Langerhans cells in immunogenicity are discussed.

Animals↗

PubMed1992

Global degranulation of rat mast cells stimulated with DNP-polystyrene.

Mediator release was studied in rat peritoneal mast cells sensitized with a mouse monoclonal anti-DNP IgE antibody, and stimulated with DNP-ornithine covalently attached to radio-derivatized polystyrene petri dishes. Cells releasing serotonin at maximal rates were investigated by transmission electron microscopy. Generalized exocytosis of granules could be observed, suggesting non-directional release of mediators, and non-compartmentalized action of second messengers in mast cells stimulated with polystyrene-bound DNP. Stimulation of sensitized mast cells by DNP covalently bound to the rigid polystyrene surface is consistent with extrinsic mechanisms proposed for Fc(epsilon)RI receptor action, and suggests that internalization of Fc(epsilon)RI is not needed for triggering cell degranulation.

Animals↗

PubMed1992

Effective enrichment of murine epidermal Langerhans cells by a modified(mismatched) panning technique.

A method for the enrichment of murine epidermal Langerhans cells (LC) is described in detail. It is based on positive selection of LC from pre-enriched fresh or cultured epidermal cell suspensions derived from ear skin by a modified panning technique. The method uses the interspecies cross-reactivities of anti-immunoglobulin antibodies: when LC in an epidermal cell suspension are labeled with mouse anti-major histocompatibility complex (MHC) class II antibodies they bind to petri dishes coated with anti-rat immunoglobulin antibodies. We therefore call this method "mismatched panning." After rinsing off non-adherent cells, the adherent LC can easily be dislodged by adding excess amounts of rat immunoglobulins, which effectively compete with the LC-bound mouse anti-MHC class II antibodies for binding to the petri dish. Using this modified panning technique, both fresh and cultured LC could be enriched up to more than 90% purity. From one ear, 2.0-3.0 x 10(4) fresh LC and 3.0-4.5 x 10(4) cultured LC could be obtained. Of all LC present in a primary, unenriched epidermal cell suspension, 40-60% were recovered when panned immediately after isolation of the epidermal cells and 50-75% when panned after 3 d of epidermal cell culture. Viability of panned LC was consistently more than 90%. Antigen presenting and T-cell-stimulating capacity of LC and responses to the cytokines granulocyte/macrophage colony-stimulating factor and tumor necrosis factor-alpha were not impaired by this panning procedure. The major advantage of this method compared to pre-existing panning techniques is the ease with which adherent LC can be dislodged from the panning dishes. Because the elution procedure is very gentle, virtually all panned LC are viable. As a consequence, good yields of highly enriched LC can be obtained in a reasonable time.

Animals↗

PubMed1991

Class II major histocompatibility complex molecules of murine dendritic cells: synthesis, sialylation of invariant chain, and antigen processing capacity are down-regulated upon culture.

Dendritic cells (DCs), such as Langerhans cells (LCs) of the epidermis and the DCs of lymphoid organs such as spleen, are potent antigen presenting cells. DCs express high levels of major histocompatibility complex (MHC) class II molecules, but, partly because of the low numbers of primary DCs in any tissue, there has been no detailed study of the biochemistry of their class II molecules. This information may be needed to help explain recent findings that DCs process native protein antigens when freshly isolated from epidermis and spleen. Processing ceases during culture, yet a strong accessory function for activating resting T cells develops. We studied immunoprecipitates of DC class II and invariant chain (Ii) molecules by two-dimensional gel electrophoresis. We found that (i) freshly isolated LCs synthesize large amounts of class II and Ii polypeptides; (ii) Ii molecules that are known to be involved in antigen processing display an unusually large number of sialic acids in fresh LCs; (iii) with culture, class II and Ii synthesis decreases dramatically and has virtually ceased at 3 days; and (iv) the turnover of class II in pulse/chase experiments is slow, being undetectable over a 12- to 32-hr culture period, whereas the turnover of Ii is rapid. We conclude that MHC class II molecules of DCs do not seem to be qualitatively unique. However, the regulation of class II and Ii expression is distinctive in that biosynthesis proceeds vigorously for a short period of time and the newly synthesized class II remains stably on the cell surface, whereas Ii turns over rapidly. This may enable DCs to process and retain antigens in the peripheral tissues such as skin and migrate to the lymphoid organs to activate T cells there.

Animals↗

PubMed1990

Disappearance of certain acidic organelles (endosomes and Langerhans cell granules) accompanies loss of antigen processing capacity upon culture of epidermal Langerhans cells.

Freshly isolated epidermal Langerhans cells (LC) can actively process native protein antigens, but are weak in sensitizing helper T cells. During culture, when LC mature into potent immunostimulatory dendritic cells, T cell sensitizing capacity develops but antigen processing capacity is downregulated. Processing of exogenous antigens for class II-restricted antigen presentation involves acidic organelles. We used the DAMP-technique to monitor acidic organelles at the ultrastructural level in fresh, as well as cultured, mouse and human LC. We observed that the loss of antigen processing capacity with culture of LC was reflected by the disappearance of certain acidic organelles, namely endosomes (particularly early ones), and the hitherto enigmatic LC granules ("Birbeck Granules"). Our findings support the notion that endosomes are critical for antigen processing and suggest that LC granules might be involved as well.

Animals↗

PubMed1990

"Intravascular lymphomatosis" (angioendotheliomatosis): evidence for a T-cell origin in two cases.

Intravascular lymphomatosis (IL) is a rare and potentially fatal multifocal intravascular proliferative disorder, most often involving the skin and the central nervous system. Originally considered an endothelial disorder, IL has recently been reclassified as an angiotropic lymphoma, most often of B-cell origin. We report immunocytochemical and ultrastructural findings in two patients with IL, both representing angiotropic T-cell lymphomas. In one patient, lesional tissue was examined by Southern blot analysis and monoclonal T-cell receptor rearrangement was found. As an additional feature in one patient, a myelosuppressive serum factor was demonstrated in peripheral blood progenitor cell cultures as the cause of underlying chronic anemia and leukopenia; this factor is thought to be a cytokine product of the lymphoma cells.

Aged↗

PubMed1990

Dendritic cell production of cytokines and responses to cytokines.

Dendritic cells (DC) are a family of bone marrow-derived MHC class II expressing cells which occur in small numbers in most lymphoid and nonlymphoid tissues. They represent a distinct lineage of leukocytes which can be found in two distinct maturational stages: immature dendritic cells are exemplified by the Langerhans cells in the epidermis, and are considered to be precursors to the mature dendritic cells in the lymphoid organs. These maturational stages can be distinguished by phenotypic and functional characteristics. Immature dendritic cells are weak stimulators of resting T lymphocytes but are excellent in processing soluble protein antigens for presentation to T cell clones. Mature dendritic cells show exactly reciprocal features. In this review the relatively few available data on cytokine production by DC and responses of DC to cytokines are collected. Our goal is to consider the role of cytokines in DC function including the transition from immature to mature stages.

Animals↗

PubMed1990

Expression of the C3d/EBV receptor and of other cell membrane surface markers is altered upon HIV-1 infection of myeloid, T, and B cells.

Human cell lines (the T-cell lines H9, Jurkat, and HUT102, the myeloid lines U937 and HL60, and the Raji B cell line) were infected with HIV-1. HIV-1 antigen could be detected by immunofluorescence analysis in more than 50% of T cells and myeloid cells 15 days after infection. Infection of Raji cells took more than 2-3 months. Studies of cell surface marker expression revealed remarkable changes after HIV-1 infection of Raji cells: expression of CR2 (C3d/EBV receptor, CD19, CD20, CD22, CD23, CD10, and surface IgM) were highly reduced, in the case of CR2 and membrane-IgM from 100 to 0%, whereas levels of CD37 and CD38 remained unaltered by HIV-1 infection. U937 cells showed a reduction of CD4 expression from 14 to 5% after HIV-1 infection; the CR3 expression slightly increased from 25 to 30%. In contrast, HLA-DR was only expressed (21%) after HIV-1 infection but not in uninfected U937 cells. Expression of HLA-DR could be detected also in HL60 cells (33%) after HIV-1 infection. In H9 cells, CD4 was reduced from 60 to 30% after HIV-1 infection, whereas HLA-DR and CD25/IL-2 receptor expression increased from 16 to 90% and from 0 to 50%, respectively. CD4 was reduced from 70 to 0% from Jurkat cells after HIV-1 infection, whereas expression of CR2 was only slightly diminished from 8 to 4%. Expression of CR1 and HLA-DR was slightly increased in these cells (1 to 3%).(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, Differentiation, B-Lymphocyte↗

PubMed1989

An antigen-independent contact mechanism as an early step in T cell-proliferative responses to dendritic cells.

Dendritic cells bearing antigen efficiently aggregate and stimulate antigen-specific T cells. We describe an experimental model in which an initial, apparently antigen-independent binding step is followed by ligation of the TCR. The model is the polyclonal response to mAb to the CD3 portion of the TCR complex. Epidermal and thymic dendritic cells utilize low levels of Fc receptors to present the anti-CD3 mAb and induce mitogenesis. Within 3 h of coculture, most of the dendritic cells have formed clusters with the resting T lymphocytes, and these clusters are the site for subsequent DNA synthesis and cell growth. However, the binding of dendritic cells to T cells proceeds as efficiently in the absence of anti-CD3 as in its presence, and anti-FcR mAb does not block. CD3 and Fc receptors are essential for the subsequent mitogenesis response in dendritic-T cell clusters. Because an exogenous ligand for the TCR does not seem to be required for the extensive polyclonal clustering of resting lymphocytes to dendritic cells, we suggest that an antigen-independent mechanism mediates the initial interaction. This clustering seems essential for T cell growth since we do not detect, in two-chamber experiments, soluble lymphocyte-activating factors that originate from dendritic-T cell aggregates and that activate anti-CD3-coated T cells.

Animals↗

PubMed1989

A small number of anti-CD3 molecules on dendritic cells stimulate DNA synthesis in mouse T lymphocytes.

Resting T cells enter cell cycle when challenged with anti-CD3 mAb and accessory cells that bear required Fc receptors (FcR). Presentation of anti-CD3 is thought to be a model for antigens presented by accessory cells to the TCR complex. We have obtained evidence that the number of anti-CD3 molecules that are associated with the accessory cell can be very small. We first noticed that thymic dendritic cells and cultured, but not freshly isolated, epidermal Langerhans cells (LC) were active accessory cells for responses to anti-CD3 mAb. DNA synthesis was abrogated by a mAb to the FcR but not by mAb to other molecules used in clonally specific antigen recognition, i.e., class I and II MHC products or CD4 and CD8. The requisite FcR could be identified on the LC but in small numbers. Freshly isolated LC had 20,000 FcR per cell, while the more active cultured LC had only 2,000 sites, using 125I-anti-FcR mAb in quantitative binding studies. Individual LC had similar levels of FcR, as evidenced with a sensitive FACS. FcR could not be detected on T cells or within the dendritic cell cytoplasm, at the start of or during the mitogenesis response. When the response was assessed at 30 h with single cell assays, at least 20 T cells became lymphoblasts per added LC, and at least 8 T cells were synthesizing DNA while in contact with the LC in discrete cell clusters. To the extent that anti-CD3 represents a polyclonal model for antigen presentation to specific T cell clones, these results suggest two conclusions. First, only 200-300 molecules of ligand on dendritic cells may be required to trigger a T cell. Second, the maturation of LC in culture entails "sensitizing" functions other than ligand presentation (anti-CD3 on FcR) to clonotypic T cell receptors.

Animals↗

PubMed1989

Presentation of exogenous protein antigens by dendritic cells to T cell clones. Intact protein is presented best by immature, epidermal Langerhans cells.

The capacity of dendritic cells to present protein antigens has been studied with two MHC class II-restricted, myoglobin-specific, T cell clones. Spleen dendritic cells and cultured epidermal Langerhans cells (LC) presented native myoglobin weakly and often not at all. These same populations were powerful stimulators of allogeneic T cells in the primary MLR. Freshly isolated LC were in contrast very active in presenting proteins to T cell clones but were weak stimulators of the MLR. Both fresh and cultured LC could present specific peptide fragments of myoglobin to the clones. These results suggest that dendritic cells in nonlymphoid tissues like skin can act as sentinels for presenting antigens in situ, their accessory function developing in two phases. First antigens are captured and appropriately presented. Further handling of antigen then is downregulated while the cells acquire strong sensitizing activity for the growth and function of resting T lymphocytes. The potent MLR stimulating activity of cultured epidermal LC and lymphoid dendritic cells probably reflects prior handling of antigens leading to the formation of allogeneic MHC-peptide complexes.

Animals↗

PubMed1989

Murine epidermal Langerhans cells express significant amounts of class I major histocompatibility complex antigens.

Epidermal Langerhans cells (LC) are leukocytes that express major histocompatibility complex (MHC) class II antigens and function as antigen-presenting and accessory cells. Caughman et al. [Caughman, S. W., Sharrow, S. O., Shimada, S., Stephany, D., Mizuochi, T., Rosenberg, A. S., Katz, S. I. & Singer, A. (1986) Proc. Natl. Acad. Sci. USA 83, 7438-7442] reported that LC are deficient in surface expression of MHC class I antigens, implying a specialization of these cells to class II-restricted antigen presentation. To readdress this obviously important issue, we have studied murine epidermal sheets prepared from B6 X BALB/c----B6 bone marrow chimeras 5 months after irradiation and bone marrow reconstitution. This enabled us to distinguish class I of LC from that of surrounding keratinocytes. When sheets were analyzed by immunofluorescence microscopy with monoclonal antibodies specific for donor class I antigens, donor-derived LC but not LC of recipient origin were stained. Appropriate controls for antibody isotype and MHC haplotype were negative. LC in epidermal cell suspensions, prepared from normal BALB/c and BALB/cBy mice (MHC haplotype d), were analyzed by flow cytometry as well as immunofluorescence microscopy. LC were stained by monoclonal antibodies to class I antigens of haplotype d, but not by isotype-matched control antibodies to class I antigens of haplotype k. We also found that LC were virtually depleted from epidermal cell suspensions by treatment with monoclonal antibodies to class I antigens of haplotype d and complement but not by treatment with control monoclonal antibodies and complement. Our data, therefore, show that LC express MHC class I molecules on their surface.

Animals↗

PubMed1989

Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function.

Freshly isolated murine epidermal Langerhans cells (LC) are weak stimulators of resting T cells. Upon culture their phenotype changes, their stimulatory activity increases significantly, and they come to resemble lymphoid dendritic cells. Resident murine LC, therefore, might represent a reservoir of immature dendritic cells. We have now used enzyme cytochemistry, a panel of some 80 monoclonal antibodies, and immunofluorescence microscopy or two-color flow cytometry, as well as transmission electron microscopy, to analyse the phenotype and morphology of human LC before and after 2-4 d of bulk epidermal cell culture. In addition, LC were enriched from bulk epidermal cell cultures, and their stimulatory capacity was tested in the allogeneic mixed leukocyte reaction and the oxidative mitogenesis assay. Cultured human LC resembled human lymphoid dendritic cells in morphology, phenotype, and function. Specifically, LC became non-adherent upon culture and developed sheet-like processes (so-called "veils"), decreased their surface ATP/ADP'ase activity, and lost nonspecific esterase activity. As in the mouse, surface expression of MHC class I and II antigens increased significantly, and FcII receptors were significantly reduced. Markers that are expressed by dendritic cells (like CD40) appeared on LC following culture. Cultured human LC were potent T-cell stimulators. Our findings support the view that resident human LC, like murine LC, represent immature precursors of lymphoid dendritic cells in skin-draining lymph nodes.

Antibodies, Monoclonal↗

PubMed1988

Macrophages phagocytose thymic lymphocytes with productively rearranged T cell receptor alpha and beta genes.

The thymus gland is important for the formation of competent T lymphocytes. However, there is long-standing evidence that greater than 95% of newly formed thymocytes do not emigrate to peripheral lymphoid tissues but instead die locally. We have identified a rapid and selective pathway for thymocyte turnover in vitro. The mechanism entails binding, uptake, and digestion by macrophages. The susceptible cells are a subpopulation of double-positive thymocytes. These thymocytes can be enriched by virtue of their high buoyant density in Percoll and prove to have low levels of surface CD3 and little or no surface TCR. However TCR-alpha and -beta genes have undergone rearrangement, and full length alpha and beta transcripts are abundant. Therefore many double-positive cells rearrange and express TCR genes but do not have normal levels of TCR on the cell surface. We propose that thymocytes that undergo high turnover in situ are unable to form receptors that can be selected by MHC molecules in the thymus, and that these cells are recognized and cleared by the macrophage.

Gene Rearrangement, T-Lymphocyte↗

PubMed1988

Rejection, after a slightly prolonged survival time, of Langerhans cell-free allogeneic cultured epidermis used for wound coverage in humans.

Autologous cultured epidermis (CE) grown from small skin biopsies in vitro has been successfully applied for wound grafting in humans. Since it has been reported recently that allogeneic CE might be tolerated as permanent wound cover, we investigated the properties of CE and its use as autologous and allogeneic grafts. Except for some differences, such as the absence of Langerhans cells and the lack of a stratum corneum, CE resembled its natural analogue. Autologous CE applied for grafting of leg ulcers and various surgical skin defects adhered firmly and permanently to the wound bed within 2 weeks, became regularly stratified, and formed a stratum corneum. Langerhans cells gradually entered the grafts; the dermis contained no inflammatory infiltrate. Allogeneic CE unmatched for MHC and blood group antigens used to partially cover tangentially excised third-degree burns, donor sites of split-thickness skin, and a defect after tumor excision initially survived well like the autografts. However, they were completely rejected after 10-22 (mean, 14.5) days, which is 4-5 days later than reported for split-thickness skin allografts. Clinically, rejection presented as "melting" of the graft. (Immuno)histologically, we found a dense mononuclear dermal infiltrate consisting predominantly of activated T cells, vacuolization, and single-cell necrosis of keratinocytes, as well as HLA-DR expression on keratinocytes, and finally separation and lysis of the epidermis. Limiting dilution analysis in 2 out of 4 allograft recipients revealed a considerable increase of circulating donor-specific cytotoxic T cell precursors during graft rejection. We conclude that grafting of allogeneic CE does not lead to permanent but to slightly prolonged graft survival.

Adolescent↗

PubMed1988

Hemangiosarcoma in chronic leg ulcer.

We recently evaluated a patient with an angiosarcoma arising in a long-standing ulceration of his leg, which appeared representative of a characteristic, though fairly uncommon, subgroup of angiosarcomas. The clinical and immunohistochemical features of the tumor enabled us to define its origin from vascular endothelial cells.

Aged↗

PubMed1987

Phagocytosis of keratin filament aggregates following opsonization with IgG-anti-keratin filament autoantibodies.

IgG-anti-keratin intermediate filament autoantibodies occur in low titers in all normal human sera. These same antibodies are present in high titers in the sera of patients who have diseases in which cells containing keratin intermediate filaments (KIF) have been damaged, such as systemic lupus erythematosus, graft-versus-host disease, and cutaneous tumors. Since some human autoantibodies are thought to function in part as opsonins promoting the removal of insoluble cellular proteins after tissue injury, we investigated the influence of IgG-anti-KIF autoantibodies on the phagocytosis of insoluble KIF aggregates by human monocytes and polymorphonuclear neutrophils (PMN). Keratin intermediate filaments assembled in vitro were reconstituted into dense spherical KIF aggregates 0.3-2.5 microns in diameter by dialysis against phosphate-buffered saline. Immunoelectron microscopy revealed that, as expected, human IgG-anti-KIF autoantibodies bound to the KIF aggregates. Human monocytes or PMN were incubated either with nonopsonized KIF aggregates or with KIF aggregates that had been reacted with IgG-anti-KIF autoantibodies. The uptake of KIF aggregates was visualized by indirect immunofluorescence, immunoperoxidase staining, and immunoelectron microscopy. Monocytes rapidly and efficiently bound and phagocytosed KIF aggregates that had been coated with IgG-anti-KIF autoantibodies. Nonopsonized KIF aggregates, in contrast, were taken up much less efficiently. Differences were most marked at 4 degrees C for 60 min with phagocytosis of opsonized KIF aggregates by 23 +/- 8% of monocytes in contrast to phagocytosis by only 0.2 +/- 3% monocytes when nonopsonized KIF aggregates were used. Similar results occurred at 37 degrees C for 5 min with phagocytosis by 38 +/- 28% vs 1.8 +/- 0.4% of monocytes of opsonized and nonopsonized KIF aggregates, respectively. A high percentage of PMN also phagocytosed opsonized KIF aggregates, whereas nonopsonized KIF aggregates were ingested less avidly. These data indicate that the opsonization of extracellular KIF aggregates by IgG-anti-KIF autoantibodies plays an important role in promoting the phagocytosis of KIF aggregates. The subsequent phagocytosis represents a rapid and very effective mechanism for the removal of insoluble KIF following keratinocyte cell death.

Autoantibodies↗