Biomedical subjects
Publications and source records attributed to M Papamichail.
Fluorescamine has been used for labelling proteins present on the surface of normal human peripheral blood lymphocytes. Under the conditions of study, 12 labelled proteins could be detected by SDS gel electrophoresis. This method may be of value in biochemical studies of lymphocyte membranes.
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Isolated fluid phase mouse C3b adhered to human and to mouse lymphocytes. On human, but not on mouse cells it could be stained by fluoresceinated F(ab')2 anti-mouse C3. Binding to mouse cells was, however, shown by inhibition of C3-dependent rosette formation and by direct staining with fluorescein-conjugated C3b (FITC-mouse C3b). Optimal staining of lymphocytes by FITC-mouse C3b depended on a sufficient intensity of fluorochrome conjugation and on a degree of aggregation of the C3b. FITC-mouse C3b preparations which initially stained weakly, stained strongly after being aggregated with glutaraldehyde. The failure of immunofluorescent techniques to demonstrate binding by mouse lymphocytes of mouse C3b which had not been so aggregated was apparently due to inaccessibility to anti-C3 of the receptor-bound C3b. Aggregated mouse C3b-FITC induced sequential patching and capping of lymphocyte complement receptors followed by endocytosis, all of which was inhibited completely by cold (4 degrees), sodium azide (2 x 10(-3) m), cytochalasin B (28 microgram/ml) and chlorpromazine (10(-4) m), and partially by lignocaine (2 x 10(-3) m), and colchicine (10(-4) m). That mouse C3 which has interacted with mouse complement receptors may not be demonstrable by anti-C3 antibody may have important implications for immunohistochemical localization of C3 bound in vivo to lymphoid cells.
Overnight incubation of lymphocytes with certain fatty acids bound to albumin has previously been shown to modify the fatty acid composition of cellular phosphoglycerides without impairing viability, and also to inhibit mitogen and antigen-induced lymphocyte transformation. In the present study, incubation of human lymphocytes with palmitic acid or with linoleic acid impaired their capacity to form complement-dependent rosettes but had no effect on either spontaneous rosette formation with sheep erythrocytes or Fc(gamma)-dependent rosette formation. With mouse lymphocytes, complement-dependent rosette formation was suppressed only following incubation with linoleic acid and there was no effect on Fc(gamma)-dependent rosette formation. Although the mechanism of these effects remains to be elucidated the results suggest that control of membrane lipid composition may modify the important in vivo immunological functions which involve complement receptors.
The capacity of non-heat-aggregated monoclonal human immunoglobulins of different classes, to localize in murine splenic germinal centres within 24 h of intravenous injection has been investigated. It has been shown that at least trimerization of polyclonal IgG must occur before any germinal centre trapping is manifest. Studies of complement fixation by these IgG preparations in vivo, together with studies of the germinal centre trapping of various monoclonal immunoglobulins, have indicated that the sole structural requirement for germinal centre localization of immunoglobulin aggregates is the ability to fix complement. Results suggest that immunoglobulin aggregates are transported to germinal centres via membrane C3 receptors of mobile cells, and then are released with loss of complement to become fixed to dendritic macrophages by a separate mechanism.
Treatment of mouse spleen lymphocytes with a high concentration of trypsin (1 mg/ml) increases the number of EA positive cells to include nearly all the B lymphocytes. It seems that all B lymphocytes express Fc receptors but in some cells these are hidden by other proteins present on the cell surfaces.
The lymph node cell populations from 24 patients with non-Hodgkin's lymphoma were investigated for some of the surface characteristics which are used as T- and B-lymphocyte markers. Nineteen of the patients had had no treatment of any kind while the other 5 had had either recent radiotherapy or chemotherapy. Seven lymph nodes from patients without malignant disease and 7 others with reactive changes were also studied. It was found that lymphocytic lymphomas, nodular or diffuse, well or poorly differentiated, had a high proportion of cells which were positive for B-cell markers. Other non-lymphocytic lymphomas had a higher proportion of cells positive for T-cell markers, and were similar to the cells from non-malignant lymph nodes.
Lymphocytes from tumour-bearing lymph nodes in 15 patients with Hodgkin's disease, and lymphocytes from 6 spleens infiltrated by Hodgkin's tissue and 6 tumour-free spleens were studied using T and B lymphocyte markers. For comparison, lymphocytes from 16 tumour-free lymph nodes were similarly assessed. It was found that the tumour-bearing nodes showed a predominance of T lymphocytes compared with the control nodes, whereas in involved splenic tissue the proportion of T lymphocytes was slightly reduced compared with uninvolved spleens.
The effect of nonspecific mitogens on the trapping of 125I-labeled aggregated human IgG (125I-AHGG) in germinal centers (GC) of mouse spleens has been investigated by both radioactivity uptake and immunofluorescence. Phytohemagglutinin and concanavalin A (Con A) significantly decreased trapping. Lipopolysaccharide produced less inhibition, and pokeweed mitogen had no significant effect. The maximum inhibition occurred with 250--500 mug Con A. This had no effect on 125I-AHGG uptake in liver kidney and blood. No differences were found between i.p. and i.v. routes of Con A injection. The effect of mitogens on the 125I-AHGG trapping in GC is due more likely to modification of the migratory properties of lymphocytes brought about by surface binding, than to their mitogenic properties, since Con A decreased 125I-AHGG localization in thymectomized, x-irradiated and bone marrow-reconstituted animals.
When the mononuclear cell population separated from normal human blood is passed through a nylon fibre column, monocytes and surface Ig bearing cells are retained; the eluted cells are a mixed properties. It appears that a proportion of the T cells eluted form EA-rosettes, and the eluted cell subpopulation includes non-T cells which share with B cells the ability to bind both EA and aggregated human IgG.
Mouse fibroblasts form spontaneous rosettes with sheep erythrocytes and with red cells from several other species. They do not form C3 or Fc rosettes, and they do not bind small amounts of aggregated gammaglobulin. Spontaneous rosette formation is maximum at 37 degress and minimum at 4 degrees. The reaction is complete in 30 min. Antisera to fibroblast membrane components do not inhibit rosette formation. Rosetting is inhibited by fixation of the cells with formaldehyde or glutaraldehyde as well as by killing with phenol or by prolonged incubation with sodium azide. The rosette reaction is inhibited by vinblastine but not by cytochalasin B. From these data on the kinetics, temperature and drug sensitivities it is suggested that T cells and fibroblasts form spontaneous rosettes by different mechanisms.
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Rosette formation with mouse red cells was observed in the "hairy" cells of four cases of leukaemic reticuloendotheliosis. A similar observation was made on B-lymphocytes from chronic lymphoid leukaemia but the monocytes in monocytoid leukaemia did not form rosettes. This finding provides new evidence for the B-cell nature of the "hairy" cell.
Lymphocytes and red cells from various mammalian species have been mixed in vitro in conditions which favor their aggregation in the form of rosettes. The frequencies of rosette formation taken in conjunction with observations on surface bound immunoglobulin on the lymphocytes favor the interpretation that, in many species of animal, rosette formation can be used as an indicator of the thymic (T) or bursal equivalent (B) origin of lymphocytes.
In vivo decomplementation of mice with cobra factor completely prevented trapping of aggregated human IgG within splenic germinal centres. Observations of the intrasplenic position of the aggregated material within 8 hr of its injection into normal and decomplemented mice suggest that the early phase of localisation in germinal centres depends on binding to lymphocyte C3 receptors.
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1. A microscopy system giving good fluorescence at 400 times magnification or greater is essential, and in effect this means a vertical illumination incident light system of the Ploem type, with optical quality lenses and objectives. 2. Enumeration of positive cells must allow for the probability of contamination of separated mononuclear cell preparations by monocytic cells. 3. The specificity of the immunofluorescent reagents used must be rigorously checked, and the presence of aggregated labelled IgG excluded. 4. The concurrent use of an independent method of identifying T-cells provides an important check, especially when there is a possibility of anti-lymphocytic antibodies being present. 5. In chronic lymphocytic leukemia, tests for surface markers for T and for B cells may permit detection of the less common T-cell leukaemia, which may have a graver prognosis. 6. Immunofluorescence tests a morphological lymphocytic marker. In the more subtle forms of lymphocytic disturbance, there is a need for tests of lymphocyte function.
Evidence is presented which indicates the presence on murine lymphocytes of a membrane receptor for determinants on the hinge region of human IgG. These determinants are exposed following pepsin scission of IgG molecule, i.e. on the F(ab')2 fragment. The evidence for a hinge receptor derives, in vivo, from splenic localization of F(ab')2 in germinal centres and, in vitro, from immunofluorescent binding studies. The sequential immunofluorescent pattern for the uptake of human F(ab)2 fragments into murine spleen germinal centres was identical with that previously observed for heat-aggregated human IgG, but F(ab')2 fragments appeared to be retained in the germinal centres for a shorter time than aggregated IgG. Experiments with nude mice and T cell-deprived mice showed that the localization of F(ab')2 fragments does not require T cells. Competition experiments suggest that the receptor for F(ab')2 may bear little relation to the receptor for aggregated IgG. The relevance of such a lymphocyte membrane receptor to the immunopathology of rheumatoid arthritis is discussed in the light of previous findings that a proportion of the serum IgG of patients with rheumatoid arthritis has a structural anomaly compared with control IgG, characterized exposure of new determinants at the hinge region.