Steven D. Douglas

Human lymphocyte-sheep erythrocyte rosette formation: Some characteristics of the interation☆

Abstract Sheep red blood cell rosette (SRBC-R) formation was evaluated and utilized as a human T cell marker for lymphocytes from normals and selected patients; 69–82% of the blood lymphocytes of normal individuals and 90–100% of human thymocytes form SRBC-R. In double-labeling experiments in which the B lymphocytes are first labeled with either aggregated human immunoglobulin or anti-immunoglobulin and then SRBC added, the vast majority of B cells do not form SRBC-R. However, rare cells are noted that show B cell markers and are also SRBC-R. Lymphocytes from most patients with chronic lymphocytic leukemia (CLL) showed predominence of anti-lg and aggregate staining and low percentages of SRBC-R. Therefore, these appeared to represent B cell leukemias. One case, however, showed no anti-lg or aggregate staining but 88% SRBC-R and thus appeared to be a T cell leukemia. These relationships were also found in hypogammaglobulinemia patients with relative B cell deficiency. Rosette formation was not inhibited by anti-immunoglobulin sera including specific anti-light chain sera. Electron microscopic studies of the SRBC-R suggest that there are relatively few sites of attachment between the reactive lymphocyte and SRBC. This binding is distinguished from binding of complement-coated SRBC to the C3 receptor-bearing lymphocyte, in which broad zones of attachment between lymphocyte and erythrocyte are observed.

Electron Microscopic and Functional Aspects of Human Lymphocyte Response to Mitogens

There have been numerous reports on human lymphocyte response to phytomitogens in vitro and this subject has recently been reviewed by the author (Douglas 1971) and by others (Sell & Asofsky 1968, Meuwissen et al. 1969, Cooper 1972). In this report I shall consider the evidence for the selectivity of different mitogens for lymphocyte populations, with emphasis on the electron microscopic findings and the response of lymphocytes from patients with immunologic diseases. Studies of the various phytomitogens have shown that these substances differ significantly in their physicochemical and biological properties. Concanavaiin A (Con A) derived from the jackbean has a molecular weight of 71,000, lacks carbohydrate constituents, and reacts with sugars which have the D-arabinopyranoside configuration at carbons 3, 4, and 6 (Goldstein et al 1965). Phytohemagglutinin (PHA) has a molecular weight of about 128,000 and its reactive group appears to involve an oligosaccharide binding site (Kornfeld & Kornfeld 1970). The pokeweed mitogen (PWM) is unique in that it has 33 intrachain disulfide bonds; its molecular weight is approximately 32,000 (Borjeson et al. 1966a, Reisfeld et al. 1967) and its specificity is unknown. Our early studies (Chessin et al. 1966, Douglas et al. 1967a) as well as those in other laboratories (Barker et al. 1969) suggest significant differences in the effects of PHA and PWM on human lymphocyte cultures in vitro. Our

Ultrastructural comparison between phytomitogen transformed normal and chronic lymphocytic leukemia lymphocytes

The ultrastructural features of lymphocytes stimulated in vitro with phytohemagglutinin (PHA), pokeweed mitogen (PWM), and concanavalin A (Con A) from normal individuals and patients with chronic lymphocytic leukemia (CLL) were studied. With PHA and Con A the transformed cells had characteristics of blasts, and with PWM both blast cells and plasmacytoid cells occurred. The mean total cell area, the nuclear and cytoplasmic size, and the number of lysosomes were diminished in mitogen- transformed CLL cells as compared to transformed normal lymphocytes. Some stimulated CLL lymphocytes were indistinguishable from normal cells. The results suggest that the CLL lymphocytes which are transformed by phytomitogens could be derived from defective leukemic cells and/or a residual population of normal T and B cells.

T AND B LYMPHOCYTES AND CUTANEOUS ANERGY IN INFLAMMATORY BOWEL DISEASE

The cellular immune status of patients with inflammatory bowel disease (IBD) has been a focus of attention and controversy for over 35 yr. Some studies of in vitro and in vivo indicators of cellular immunity have shown impairments in the immune mechanisms of patients with Crohn’s disease (CD),’-7 whereas other investigators have considered these mechanisms to be normaL8-” Meanwhile, most published reports seem to agree that cellular immunity is not defective in patients with ulcerative colitis (UC).4*677,’o*12-’4 Our own previous studies, on the other hand, led us to the conclusion that there was indeed a defect in cellular immunity in a substantial proportion of patients with IBD, both CD and UC alike. This conclusion was based principally upon our finding of impaired peak lymphocyte responsiveness to graded doses of the nonspecific plant mitogen phytohemagglutinin (PHA) among approximately one-third of our IBD patients with either CD or UC.” In the present study we have strengthened our previous conclusion by demonstrating depressions in circulating T-lymphocyte populations among 32 1BD patients with either CD or UC. We have also found elevations in B lymphocytes among 77 IBD patients of both categories as well as a high incidence of anergy upon skin testing with 2,4-dinitrochlorobenzene (DNCB) among 29 IBD patients of both types.

Peripheral blood lymphocyte subpopulations in sarcoidosis

Abstract We have determined the numbers of thymus-derived (T) and bone marrow-derived (B) lymphocytes in the peripheral blood of 20 patients with sarcoidosis and 15 healthy controls. T cells were estimated from the number of lymphocytes forming rosettes in vitro with unsensitized sheep red blood cells, and B cells were enumerated by immunofluorescent assesssment of membrane-bound immunoglobulins. The total lymphocyte count was lower in patients with sarcoidosis owing to a depletion of T lymphocytes from the blood. Nonetheless, the relative and absolute numbers of B lymphocytes were significantly increased. These alterations in lymphocyte subpopulations did not show any consistent correlation with the duration of the disease, clinical stage, activity, or treatment. Changes in the subpopulations may be related to both decreased cellular immunity and increased reactivity of the antibody-forming system as commonly seen in sarcoidosis.

Human monocyte activation by supernatants from concanavalin A (con A) stimulated lymphocytes.

Abstract Human peripheral lymphocytes were stimulated with Concanavalin A (Con A) in the absence of serum. Supernatants were collected from control and mitogen stimulated lymphocyte cultures and fractions pooled according to the elution before, together with or after human serum albumin which was added as a marker. Only one fraction derived from Con A stimulated lymphocyte culture Supernatants which eluted immediately after human serum albumin had a significant effect on the metabolism and structure of human monocytes in vitro . Monocytes separated by human serum albumin and incubated with this fraction for 20 hr had an increase in nuclear RNA synthesis. Monocytes attached to cover slips in Leighton tubes showed an increase in the percentage of phagocytizing cells and phagocytic activity. Electron microscopy demonstrated highly phagocytic cells containing numerous Golgi associated granules and strands of nondilated rough surfaced endoplasmic reticulum in presence of the active fraction.

Acid phosphatase cytochemistry of mitogen-transformed normal and chronic lymphocytic leukemia lymphocytes☆

Abstract Acid phosphatase cytochemistry was performed on lymphocytes stimulated in vitro with phytohemagglutinin, pokeweed mitogen, or concanavalin A. These electron microscopic studies demonstrated that activated lymphocytes from both normals and patients with chronic lymphocytic leukemia (CLL) had an increased number of lysosomes relative to resting cells. At the time of maximum thymidine incorporation, a reduced number of lysosomes was present in many transformed CLL lymphocytes, mainly medium-sized blast cells, in comparison to transformed normal cells. The findings demonstrate a lysosomal abnormality in phytomitogen transformed CLL lymphocytes which may be related to functional defects of these cells or to an incomplete transformation of a residual population of normal lymphocytes.

In vitro lymphocyte response to phytohemagglutinin and pokeweed mitogen in Hodgkin's disease

Lymphocytes from eight patients with Hodgkins disease were incubated with phytohemagglutinin (PHA) or pokeweed mitogen (PWM) over 7 days. Thymidine incorporation into DNA and ultrastructural features of transformed cells were studied. Response to these mitogens was either normal or diminished and/or delayed. In seven patients lymphocyte response to PHA was paralleled by a corresponding response to PWM. PHA‐transformed lymphocytes showed fine structural features similar to transformed normal cells. After PWM stimulation, blast cells and plasmacytoid cells in various stages of differentiation were observed. The number of transformed cells corresponded to the magnitude of thymidine incorporation and in cultures with normal PWM response, plasmacytoid cells occurred with almost normal frequency. If the specificity of the mitogens is as postulated, then patients with Hodgkins disease do not have a selective loss of T lymphocytes. Furthermore, the findings suggest that in some patients there may be a functional impairment of both T and B lymphocytes.

Monocyte receptors for immunoglobulin and complement in immunologic deficiency diseases.

Semiquantitative assessment of the human monocyte IgG receptor in normal subjects and patients with acquired agammaglobulinemia, chronic granulomatous disease chronic mucocutaneous candidiasis and the Wiskott‐Aldrich syndrome revealed no detectable differences. The monocyte C3 receptor was found to be normal in cells from patients with the Wiskott‐Aldrich syndrome. Monocyte receptors should be further investigated in patients with various forms of immunologic deficiency.

Hemolytic anemia with serum and erythrocyte-bound low-molecular-weight IgM

Abstract A 49-yr-old woman with severe hemolytic anemia had marked depression of serum IgG and IgA along with monoclonal serum IgM detectable in both high and low molecular-weight forms. Antiglobulin tests by instrumented and manual techniques showed only IgM coating of the erythrocytes and an eluate contained low-molecular-weight IgM. The IgM fraction of the patients serum contained anti-I agglutinins active at 37°C. Lymphocyte response to Concanavalin A was diminished, whereas response to phytohemagglutinin and pokeweed mitogen was normal. Blood monocyte Fc receptor activity was increased in comparison to controls. The finding of a low-molecular-weight IgM possibly related to hemolysis in addition to 19S IgM anti-I activity with high thermal optimum in a patient with hypogammaglobulinemia represents an unusual form of immune hemolytic anemia.