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M H Kosco

Publications and source records attributed to M H Kosco.

28 records · Page 2Linked to original sources
PubMed1988

Thy-1 positive tingible body macrophages (TBM) in mouse lymph nodes.

This study was prompted by the observation that cells in mouse lymph nodes (LN) with cytological characteristics of tingible body macrophages (TBM) appeared to be Thy-1 positive. The objective of this study was to determine if these large cells were TBM and to conclusively demonstrate their reactivity for Thy-1. The cells were studied using monoclonal antibodies (MoAb) against Thy-1 and macrophage markers including F4/80 and Ia antigens at both light microscopic (LM) and electron microscopic (EM) levels. Immunocytochemical reactivity by TBM for Thy-1 antigen specific MoAb was demonstrated by LM in both in situ and in vitro LN preparations. Furthermore, ultrastructural examination of these germinal center cells in situ demonstrated that the Thy-1 reactivity visualized at the LM level was associated with ribosomes and endoplasmic reticulum as well as their plasma membrane. Similarly, these cells expressed intracytoplasmic and membrane reactivity for Ia antigens and also for the macrophage specific antigen F4/80. This indicates that the reactivity is due to active synthesis of the Thy-1 antigen and not attributable to reactivity of any phagocytosed Thy-1 positive cells. As defined by their germinal center location and morphological characteristics, these Thy-1 reactive macrophages were identified as TBM. Germinal center TBM thus represent a unique, vigorously phagocytic subset of mature macrophages which express both macrophage and thymocyte markers.

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PubMed1988

Germinal center B cells present antigen obtained in vivo to T cells in vitro and stimulate mixed lymphocyte reactions.

In the present study we sought to test the ability of GC B cells to activate T cells by antigen presentation and mixed lymphocyte reactions. The first set of experiments demonstrated that GC B cells obtained three or more weeks after immunization could induce IL 2 production by T cells only by adding antigen to the cell cultures. In the next (table; see text) series, it was demonstrated that GC B cells isolated as early as 1 day and for over a week following an antigenic challenge with OVA (i.e. where they obtained antigen from the FDC-derived iccosomes in vivo), were able to stimulate high levels of IL 2 production in the absence of exogenous OVA. This response was maximal on Day 5 which corresponded precisely with the keinetics of the ultrastructural studies which document the uptake of antigen by GC B cells in vivo (see Szakal et. al, this volume). Furthermore, it was shown that the FDC-derived antigen was remarkably immunogenic when compared with exogenous antigen. The data from the MLRs demonstrated that GC B cells, which are low density cells, are potent stimulators of T cells compared to the resting, low density B cells. In addition, GC B cells even stimulated a significant syngeneic response. These results indicate that GC B cells can present antigen and interact with T cells efficiently. This is believed to be important in the germinal center reaction and in the induction and maintenance of the immune responses.

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PubMed1988

A subpopulation of germinal center B cells differentiate directly into antibody forming cells upon secondary immunization.

In the present study we sought to test the hypothesis that GC B cells may be stimulated to differentiate into AFCs during the induction of the secondary antibody response. To test this we gave HRP immune mice a booster immunization and looked for AFCs in the GCs making anti-HRP specific antibody. Within 3 days cells in the GCs were making anti-HRP. Most of the cells were just starting to make immunoglobulin in the rough endoplasmic reticulum but some were nearly mature plasma cells like those in the medullary cords. Furthermore, we isolated GC B cells after a boost with OVA and tested their ability to differentiate directly into AFCs. Data obtained using an OVA specific RIA indicated that purified GC B cells would differentiate and produce specific antibody in the absence of added T cells or antigen. Examination of the cells after 5 days in culture indicated that many of them had differentiated into large plasma cells. These results obtained both in vivo and in vitro demonstrate that shortly after booster immunization GC B cells receive the appropriate antigen and T cell signals in vivo to differentiate and become AFCs.

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PubMed1986

Antigenic phenotyping of isolated and in situ rodent follicular dendritic cells (FDC) with emphasis on the ultrastructural demonstration of Ia antigens.

The antigenic phenotype of mouse lymph node follicular dendritic cells (FDCs) was studied by immunocytochemical techniques. Indirect fluorescence was used in conjunction with monoclonal antibodies to localize FDC surface antigens on FDC-enriched cell preparations and in cryostat sections. Lymph nodes from rats and mice were also labeled directly for Ia antigens with fluorescein- or peroxidase-conjugated Ia-specific monoclonal antibodies (i.e., MRC Ox4 and 10-2.16, respectively). Lymphoid tissue was also prepared for electron microscopy to allow clear distinction between Ia antigens of B lymphocytes and FDCs in situ. In these experiments, gold-labeled antigen was used to clearly identify FDCs and their processes among the Ia-positive cells of lymph node follicles. The labeling observed by light and electron microscopy showed that FDCs expressed Ia in situ and in vitro. Additional surface determinants shown to be expressed by FDCs included H2-K, common leukocyte antigen, and the receptor for the Fc portion of IgG1 and IgG2b. Neither macrophage antigens, such as Mac-1, Mac-2, Mac-3, and F4/80, nor the lymphocyte markers Ly-1, Ly-2, and Thy-1 were expressed by FDCs. Thus, the antigenic phenotype of FDCs, along with their distinctive dendritic morphology, their nonphagocytic and nonadherent nature, and their ability to trap and retain immune complexes on their plasma membrane, identifies them as a unique cell population.

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PubMed1986

Localization of antibody-forming cells in draining lymphoid organs during long-term maintenance of the antibody response.

Previous studies on the "spontaneous antibody response" have included in vitro steps and it is possible that the response is an in vitro artifact. The objective of the present study was to induce a spontaneous antibody response entirely in vivo and determine if the response is localized and if the magnitude of the response is related to the location of persisting antigen. Antigen was injected into the right hind footpads of mice, and lymph nodes on the right side were draining and lymph nodes on the left side were controls. Antibody-forming cells (AFCs) were enumerated in both draining and nondraining nodes 2 weeks, 2 months, and 1 year after secondary immunization. Four days prior to determining AFC number, the mice were severely bled to stimulate AFC production. Thousands of AFCs were found in the draining lymph nodes and the numbers were dramatic in nodes closest to the injection site that retain the most antigen. In contrast, the vast majority of nondraining nodes lacked any AFCs. One year after immunization, the response was almost exclusively in the popliteal node, draining the foot where antigen was administered a year earlier. These results are consistent with previous data on the spontaneous response and support the hypothesis that antigen retained on FDCs is essential in the maintenance of serum antibody levels.

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PubMed1985

Isolated follicular dendritic cells: cytochemical antigen localization, Nomarski, SEM, and TEM morphology.

The objectives of the present study were to determine the cytological features of isolated follicular dendritic cells (FDC), which distinguish them from other leukocytes or dendritic cell types. Consequently, we have developed methods for the fixation, peroxidase cytochemistry, and visualization of FDC, which are applicable to cytological evaluations by Nomarski optics, scanning, and transmission electron microscopy. A functionally supported identification of FDC in vitro was made possible by utilizing, in conjunction with the dendritic morphology, the cytochemically identifiable antigen, horseradish peroxidase (HRP), and the known capacity of FDC to sequester immune complexes (i.e. HRP-anti-HRP) on their plasma membranes. The observations showed that FDC constitute a relatively pleomorphic, nonphagocytic group, distinct from other dendritic type cells such as lymphoid dendritic cells, Langerhans cells, and interdigitating cells (LDC, LC, and IDC), as well as typical leukocytes. Morphologically distinct FDC were identified as cells either with filiform dendrites or with "beaded" dendrites. FDC possessed a single or sometimes a double, lymphocyte-size cell body, which contained an irregular, lobated nucleus, Golgi apparatus, numerous small vesicles, and some mitochondria. Mitochondria were not abundant in the dendritic processes. Filiform dendrites tended to branch and anastomose near the cell body and form a radiating "sunburst"-like pattern. On the average, dendrites measured 15-20 microns in length and 0.1-0.3 micron in diameter. Occasional dendrites were extremely elongated, reached several hundred microns in length, and terminated in an enlargement measuring nearly a micron in diameter. Other filiform dendrites usually had a club-shaped terminal enlargement. The microspheres of "beaded" dendrites ranged between 0.3 and 0.6 micron in diameter. The dendritic processes were also shown to have a highly ordered pattern of immune complex attachment on their surface, suggestive of a periodic arrangement of receptor sites.

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PubMed1985

Follicular dendritic cells in suspension: identification, enrichment, and initial characterization indicating immune complex trapping and lack of adherence and phagocytic activity.

Antigen-retaining follicular dendritic cells (FDC) have been identified and studied in sections of lymph nodes and spleen, but studies of these cells in culture have been extremely limited. The purpose of this study was to establish techniques to release these fragile cells from mouse lymph nodes in a viable state and to identify these cells routinely in lymph node cell suspensions. FDC were obtained from passively or actively immunized popliteal lymph nodes of mice injected in the footpads with 125I-labeled human serum albumin (HSA) or horseradish peroxidase (HRP). Lymph nodes were removed 1 hr after the footpads had been injected with collagenase. After another hour of incubation in vitro with collagenase, protease, and deoxyribonuclease, FDC were released by gentle teasing and enriched by centrifugation on a low density bovine serum albumin (BSA) or Percoll gradient. Most FDC with the associated radiolabel floated at densities greater than 1.06 g/ml on BSA or Percoll gradients. Slides of the FDC-enriched fraction were prepared, using a cytobucket which allowed the cells to be affixed to glass slides by centrifugation in a less disruptive manner than by cytocentrifugation. FDC that were air-dried and fixed with 3% glutaraldehyde had a characteristic pink acidophilic cytoplasm after Wright's staining, and had a faintly basophilic euchromatic nucleus frequently with peripherally-clumped chromatin. In addition, these cells were large and irregularly shaped (up to 60 micron long). Fixation of FDC with 0.6% paraformaldehyde/ 0.9% glutaraldehyde on poly-L-lysine-coated slides resulted in a preservation of FDC which made possible visualization of long dendritic processes by Nomarski optics. Antigen presence on the cell surface was confirmed by autoradiography and, in the case of HRP, was also visualized enzymatically using diaminobenzidine. In contrast to resident peritoneal macrophages or some contaminating lymph node macrophages present on the same slides, FDC did not phagocytize opsonized sheep red blood cells (SRBC) or adhere to plastic surfaces although they did form rosettes with opsonized SRBC. Cell marker studies indicated FDC have a distinctive phenotype. They were positive for Ia, Fc receptor, and leukocyte common antigen, but negative for Thy-1, Ly-1, Ly-2, endogenous Ig, Mac-1, Mac-2, Mac-3, and F4/80, and negative to weakly positive for nonspecific esterase. Cultured FDC remained viable and retained radiolabeled antigen-antibody complexes on their surfaces and were significantly enriched for FDC.(ABSTRACT TRUNCATED AT 400 WORDS)

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