The effect of PHA, PPD, allogeneic cells, and sheep erythrocytes on albumin gradient-fractionated mouse spleen cell populations

Adler, William H.; Peavy, Duane L.; Smith, Richard T.

doi:10.1016/0008-8749(70)90062-6

Cited by 32 publications

(14 citation statements)

References 9 publications

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“…The cells lost in the pellet (40% of the original population) consisted of red cells, dead and aggregated lymphocytes, and a number of viable lymphocytes. These observations are consistent with previous reports (35,39).…”

Section: Cell Fractionation and Recovery Of Radioactive Protein Andsupporting

confidence: 94%

Cell Surface Immunoglobulin

Vitetta

Baur

Uhr

1971

The Journal of Experimental Medicine

353

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There is accumulating evidence that the capacity of lymphocytes to bind antigen specifically (1-6) is due to Ig on the cell surface: anti-Ig causes the transformation of lymphocytes (7-12) and other changes in membrane properties (13,14); anti-L chain and anti-Ig inhibit both binding of specific antigen and stimulation of cellmediated immune reactions by antigen (15-18); anti-Ig of allotype or class specificity binds to lymphocytes (19-23); "rosette" formation around lymphocytes occurs when hybrid antibody to both Ig and specific antigen is added along with red cells coated with antigen (24, 25); lymphocytes treated with anti-Ig form rosettes with Ig-coated red cells (26,27); and specific adherence of lymphocytes to antigen-coated columns is blocked by pretreatment of cells with anti-Ig serum (28). There is conflicting evidence, however, as to whether the major class of cell surface immunoglobulin on normal splenic lymphocytes is IgM (12,20) or IgG (7, 21), as well as the distribution of Ig on cells (20,21).In evaluating the above results, I t should be emphasized that the methodologies used rely on the capacity of anti-Ig antibody to make effective contact with antigenic determinants of the receptor. Such contact depends upon the presence of determinants on portions of the receptor available to anti-Ig antibody in the medium and retention of antigenicity when the receptor is bound to the plasma membrane. These problems may account for the several contradictory reports mentioned above and the finding that individual antisera directed against the same immunoglobulin can give conflicting results, presumably depending on differences in the specificities of the antisera (20,29).For these reasons we developed an approach in which surface Ig could be studied by standard immuno-and biochemical techniques after its removal from the celt surface (30).

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Section: Cell Fractionation and Recovery Of Radioactive Protein Andsupporting

confidence: 94%

Cell Surface Immunoglobulin

Vitetta

Baur

Uhr

1971

The Journal of Experimental Medicine

353

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“…Ii Mean cpm in unfractionated cells. rated according to density has been previously described (13). In both groups the highest proportion of all spleen cells was recovered in the C layer, and the representation in each of the other three subpopulations was nearly identical.…”

Section: Effect Of Thymectomy On In Vitro Stimulation Of Subpopulatiomentioning

confidence: 98%

“…Tissue Culture Method.--The method of culture used in these studies has been described in detail in previous papers from this laboratory (11,13,14). The medium employed was RPMI 1640, containing 100 units of penicillin and 100/zg of streptomycin per ml, with 5% fresh, heated (56 ° for 30 rain) human serum.…”

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confidence: 99%

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Cellular Recognition in Vitro by Mouse Lymphocytes

Takiguchi

Adler

Smith

1971

The Journal of Experimental Medicine

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The thymus is influential in establishing and maintaining elements of immune function throughout life (1, 2) and appears to be required for elicitation of delayed hypersensitivity reactions, for effective rejection of allografts (3, 4), for resistance to tumor induction (5, 6), and for response to certain classes of antigens (7,8). The objective of the studies to be reported here was to examine further the role of the thymus in cellular recognition. The susceptibility of whole and albumin gradient-separated subpopulations of cells taken from mice thymectomized at birth to stimulation in vitro by alloantigens and P H A was assayed. The capacity of thymus grafts of differing origins to restore cellular reactivity was also explored. These studies confirm that the thymus is required for normal levels of responsiveness of lymphoid cells to primary stimulation in vitro by alloantigens and PHA. Materials and MethodsExperimental Animals.--Mice of the inbred strains, C57BL/6J, A/J, BALB/c, DBA/2, and CBA/J, and the F~ hybrids of these strains, were derived from inbred stocks maintained in this laboratory or purchased directly from the Jackson Laboratories, Bar Harbor, Maine.Neonatal Thymectomy and Thymus Grafting Proceclure.--Thymectomy was performed within 24 hr of birth by the method described by Sjodin et al. (9). Macroscopic examination when the animals were sacrificed for an experiment verified the success of thymectomy.The thymus graft procedure was a modification of that of Metcalf et al. (10) in which a polyethylene trocar containing one lobe of newborn thymus tissue was inserted under the left kidney capsule under direct vision. The trocar was approximately the size of an 18 gauge needle and was mounted on a syringe containing 0.5 ml of air.

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“…We harvested these cells 9 days after challenge, when they had just completed a cycle of blast transformation and several cell divisions in the draining lymph nodes (17)(18)(19); they might be expected to differ physiologically from their unstimulated precursors. Sensitized cells have been described as intensely pyroninophilic (20)(21)(22), medium-sized lymphocytes (3,5,18,23) of low to moderate density (24), possessing a uropod unlike their precursors (25,26) and ultrastructural features resembling those of a cell activated by mitogen (26). Thus, they may be regarded as already partially activated, and this would account for their decreased sensitivity to PHA or cytochalasin B.…”

Section: 5ogmentioning

confidence: 99%

Regulation of Lymphocyte Responses In Vitro: Potentiation and Inhibition of Rat Lymphocyte Responses to Antigen and Mitogens by Cytochalasin B

Yoshinaga

Waksman

1972

Proc. Natl. Acad. Sci. U.S.A.

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Cytochalasin B, at concentrations between 0.02 and 0.2 ;Lg/ml, was slightly stimulatory to lymph-node cells from normal rats and greatly potentiated their response to phytohemagglutinin and low concentrations of concanavalin A (mitogens for thymus-derived lymphocytes); it also potentiated the response of thymocytes to phytohemagglutinin. The response of lymphnode cells to lipopolysaccharide endotoxin (a mitogen for thymus-independent lymphocytes) was also enhanced, but only at concentrations in the usual inhibitory range, possibly by inhibition of a "suppressor T-cell" response. Sensitized lymphocytes responding to antigen were not stimulated at all, except at a very high cell density, where inhibition of a "suppressor cell" response was also considered likely. At concentrations of 5-10 Mg/ml or higher, cytochalasin 1B inhibited all responses tested.Cytochalasin B is a fungal product (1) widely used as an inhibitor of cell functions that involve contraction of microfilaments (2). Its target within the cell was undetermined (3) until recently; new evidence establishes firmly its ability to interact directly with the actin moiety of actomyosin, producing a loss of viscosity and of ATPase activity (4). Cytochalasin B, in a restricted low-concentration range (<1 Mug/ml), stimulates chemotaxis (5) and phagocytosis ( §), but inhibits these functions at higher concentrations (>5 Mkg/ml). We report here its ability, in a concentration range of 0.02-0.5 ,g/ml, to stimulate lymphocyte DNA synthesis and to potentiate the responses of lymphocytes to mitogens. MATERIALS AND METHODSAnimals and Sensitization. Inbred DA rats of both sexes, 3-5 months of age, were sensitized by injection of ovalbumin (Nutritional Biochemicals Corp., Cleveland, Ohio), 100 ,g in Freund's complete adjuvant in both hind foot pads (6). Their inguinal and iliac nodes, 9 days after sensitization, were used as sources of sensitized lymph-node cells (LNC). As sources of normal LNC, cervical, axillary, inguinal, and popliteal lymph nodes were harvested from untreated rats. (Grand Island Biological Co., Grand Island, N.Y.), 100 units of penicillin per ml, and 100 ,lg of streptomycin per ml, was used throughout.Reagents. Ovalbumin, PHA (phytohemagglutinin P; Difco Laboratories, Detroit, Mich., ¶), Con A (concanavalin A, twice crystallized, Nutritional Biochemicals Corp., Cleveland, Ohio), and lipopolysaccharide B, Escherichia coli 0111:1B4 (Difco Laboratories, Detroit, Mich.) were dissolved in phosphate-buffered saline (pH 7.4) at various concentrations, and 0.1 ml of the solutions were added to appropriate cultures. Cytochalasin B (Imperial Chemical Industries Ltd., Macclesfield, Chesire, England) was dissolved at 3 mg/ml in (CH3)2SO. Just before use, the stock solution was diluted with phosphate-buffered saline to the desired concentrations, and corresponding dilutions of (CH3)2SO were prepared as controls.Estimation RESULTSEffect of Cytochalasin B on Nonstimulated Cells. Normal LNC (2 X 106/ml) were incubated with cytochalasin B at concentrati...

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The effect of PHA, PPD, allogeneic cells, and sheep erythrocytes on albumin gradient-fractionated mouse spleen cell populations

Cited by 32 publications

References 9 publications

Cell Surface Immunoglobulin

Cell Surface Immunoglobulin

Cellular Recognition in Vitro by Mouse Lymphocytes

Regulation of Lymphocyte Responses In Vitro: Potentiation and Inhibition of Rat Lymphocyte Responses to Antigen and Mitogens by Cytochalasin B

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