STIMULATED LYMPHOCYTE CULTURESLohrmann, Hans-Peter; Graw, Carole M.; Graw, Robert G.
doi: 10.1084/jem.139.5.1037pmid: 4825240
Lymphocytes, stimulated with different doses of plant mitogens or allogeneic cells, incorporate varying amounts of 3 Hthymidine. Theoretically, this may be due to different numbers of responding cells, to earlier proliferative response of these cells, and/or to their more or less rapid transit through the cell cycle. Dog peripheral blood lymphocytes were stimulated in vitro with different doses of phytohemagglutinin (PHA), or with allogeneic lymphocytes. After their synchronization by incubation with hydroxyurea (4 mM), the mean durations of the cell cycle, and of the different cell cycle phases were constant and unrelated to strength or type of stimulation. PHA-stimulated lymphocyte cultures were maintained in the presence of colchicine, to prevent clonal proliferation of responding lymphocytes. DNA uptake in this setting, attributed to first generation responders, was related to the strength of proliferative lymphocyte response in control cultures without colchicine. Furthermore, cell proliferation occurred earlier with greater stimulation. It is concluded that higher 3 Hthymidine uptake in vitro by stimulated lymphocytes is due to greater numbers of responding cells, which are triggered into proliferative response earlier, and not to a more rapid transit of the responding cells through the cell cycle. Footnotes Submitted: 12 December 1973
CYTOTOXIC ACTIVITY OF MOUSE MACROPHAGES STUDIED BY VARIOUS INHIBITORSMelsom, Haakon
doi: 10.1084/jem.139.5.1049pmid: 4825241
The influence of various inhibitors on the cytolytic potential of mouse macrophages against syngeneic erythrocytes has been investigated in vitro by isotope techniques. Intact macrophage membrane and cell metabolism was essential for full cytotoxic activity. The process was completely blocked by anaerobiosis and cold. ATP from both mitochondrial respiration and glycolysis seems to be the high energy intermediate which is utilized during the cytotoxic activity of macrophages leading to target cell lysis. The process did not depend on concomitant DNA transcription, translation, or protein synthesis. Footnotes Submitted: 7 January 1974
ANTIBODY-INDUCED MOVEMENT OF MEMBRANE COMPONENTS OF LEISHMANIA ENRIETTIIDoyle, John J.; Behin, Reza; Mauel, Jacques; Rowe, David S.
doi: 10.1084/jem.139.5.1061pmid: 4596509
Incubation in vitro of viable Leishmania enriettii with antibodies from infected or immune guinea pigs and a fluorescein-labeled antiguinea pig Ig conjugate induced aggregation of surface antigens to form a "cap" over the anterior pole of the amastigote and over both the anterior and posterior poles of the promastigote form of the parasite. Cap formation occurred only with optimum quantities of guinea pig antibodies and was inhibited by low temperature and the metabolic inhibitors, sodium azide and iodoacetamide. The aggregated antigens were rapidly lost from the surface of the parasite but reappeared after 3 h of incubation at 23°C. This phenomenon of ligand-induced membrane antigen movement is apparently similar to that described in mammalian cells, and may represent the first stage of the interaction between host antibodies and the surface membrane of protozoal parasites. Footnotes Submitted: 9 December 1974
UPTAKE AND REDUCTION OF OXIDIZED AND REDUCED ASCORBATE BY HUMAN LEUKOCYTESBigley, Robert H.; Stankova, Libuse
doi: 10.1084/jem.139.5.1084pmid: 4825242
Incubation of human leukocytes with dehydroascorbate (DHA) results in an increase in their reduced ascorbate (AA) content and hexose monophosphate shunt (HMS) activity, independent of oxygen supply. Incubation with AA induces these changes only in the presence of oxygen. The increase in HMS activity observed as cell AA increases by 1 µmol is the same during incubation with either DHA or AA. We propose that human leukocytes take up ascorbate as DHA (AA after oxidation to DHA) and reduce it promptly to AA, and that HMS stimulation upon incubation with either AA or DHA is a result of DHA reduction. Footnotes Submitted: 4 February 1974
MECHANISMS OF GENETIC RESISTANCE TO FRIEND VIRUS LEUKEMIA IN MICEKumar, Vinay; Bennett, Michael; Eckner, Robert J.
doi: 10.1084/jem.139.5.1093pmid: 4596510
Resistance to malignant erythropoiesis induced by Friend spleen focus-forming virus and resistance to marrow stem cell allografts are under genetic control. Strains of mice, e.g., C57BL/6 and B10.D2, which are homozygous for resistance at the Fv-2 locus, are also good rejectors of most bone marrow allografts. 89 Sr, a bone-seeking isotope, irradiates marrow but not other lymphoid organs and abrogates resistance to marrow allografts without suppressing T- or B-cell functions. Thus, marrow-dependent effector cells (M cells) seem to resist allogeneic stem cells. To test if the genetic resistance to Friend virus (FV) is also mediated by M cells, B6 mice were treated with 89 Sr using a dosage schedule known to abrogate resistance to allogeneic marrow cells. 9 days after FV infection of such mice, the spleens showed malignant erythroblastosis which could not be suppressed by prior hypertransfusion, a procedure which suppresses physiologic erythropoiesis. Such 89 Sr-treated B6 mice also supported extensive virus replication, while control mice did not. FV markedly suppressed the ability of 89 Sr-treated B6 mice to produce antisheep red blood cell (SRBC) antibodies, a feature seen normally only in genetically susceptible mice. Thus, 89 Sr-treated B6 mice behaved in these respects as if they were susceptible to FV. When increasing doses of 89 Sr were administered to B6 mice, a dose-related loss of resistance to FV was seen. Therefore, it appears that 89 Sr-sensitive M cells mediate the genetic resistance to FV. The results of experiments with 89 Sr indicated that genetically resistant mice would be expected to possess target cells which are susceptible to transformation by FV. To verify this corollary, bone marrow cells from B10.D2 ( Fv-2 rr ) mice were transplanted into previously infected and lethally irradiated DBA/2 ( Fv-2 ss ) recipients which share the same H-2 d alleles. 5–15 days later, the spleens of DBA/2 primary recipients yielded transformed cells which were capable of producing splenic tumor colonies upon transplantation into adult, unirradiated B10.D2 secondary recipients. Various control experiments clearly indicated that the tumor colonies so induced were of B10.D2 marrow origin. This indicated that B10.D2 stem cells could be transformed when allowed to interact with FV in the spleens of susceptible DBA/2 mice. However, 30 days after transplantation of B10.D2 bone marrow cells into DBA/2 recipients, no transformed cells were detected. Apparently, in the 30-day interval precursors in the B10.D2 marrow gave rise to mature M cells which resisted the leukemic process. Since M cells recognize hybrid or hemopoietic histocompatability antigens expressed on primitive normal and transformed hematopoietic cells, we suggest that M cells may exert surveillance by rejecting leukemic cells. Thus, marrow transplantation from genetically resistant donors may provide a new mode of treatment for leukemia, by providing precursors of M cells and other immunocompetent cell types. Footnotes Submitted: 16 December 1973
EVENTS AFTER THE BINDING OF ANTIGEN TO LYMPHOCYTESAult, Kenneth A.; Unanue, Emil R.
doi: 10.1084/jem.139.5.1110pmid: 4596511
The behavior of the immunoglobulin antigen receptor on lymphocytes was studied using both fluorescent antiimmunoglobulin antibody to detect B cells and autoradiography with radiolabeled antigens to detect antigen-binding cells. It was shown that after binding of antiimmunoglobulin antibody to the lymphocyte there was a rapid loss of surface immunoglobulin and then a progressive reappearance over 18 h. This could be quantitated using an inhibition assay for surface immunoglobulin. Similarly, after binding various dinitrophenyl-conjugated proteins or keyhole limpet hemocyanin to their specific antigen-binding cells, there was a loss of the antigen receptor from the surface and then a progressive reappearance of the receptor. The reappearance of surface immunoglobulin and of the antigen receptor proceeded at about the same rate. Repeated exposure to antibody or prolonged exposure to antigen did not diminish the capacity of the lymphocyte to re-express its receptor. These events, which follow the interaction of antigen and its receptor, are of possible importance in understanding the mechanism of triggering of the immune response and of tolerance. Footnotes Submitted: 31 January 1974
ONTOGENY OF B LYMPHOCYTESGelfand, Michael C.; Elfenbein, Gerald J.; Frank, Michael M.; Paul, William E.
doi: 10.1084/jem.139.5.1125pmid: 4545162
Many bursa-equivalent (B) lymphocytes of adult mice bear surface Ig and receptors for C3. The frequency of Ig-bearing cells increases rapidly immediately after birth, but these cells lack complement (C) receptors. Lymphocytes bearing C receptors are not found in the spleens of BALB/c, DBA/2, and C57BL/6 mice until 2 wk of age and do not attain substantial numbers until 3–4 wk of age. In AKR mice, a lag between appearance of Ig-bearing and complement receptor lymphocytes (CRL) is also observed but it is of much shorter duration. AKR mice have a frequency of CRL at 2 wk of age of 28% in comparison to a frequency of 4.8% for DBA/2 mice. The difference in frequency between young and adult mice and between "low" and "high CRL" strains cannot be explained by a nonspecific inability to form rosettes as similar results are obtained with soluble antigen-antibody-complement complexes. Analysis of CRL frequency in (AKR x DBA/2)F 1 mice and F 1 x parental backcross progeny suggests that two independent genes control the rate of appearance of CRL. Furthermore, the genetic difference in the ontogeny of CRL is recapitulated in the repopulation of the B-lymphocyte line in adult-irradiated mice restored with syngeneic bone marrow. Thus, the "CRL genes" described here appear to control B-cell differentiation throughout life. Footnotes Submitted: 21 January 1974
ONTOGENY OF B LYMPHOCYTESGelfand, Michael C.; Sachs, David H.; Lieberman, Rose; Paul, William E.
doi: 10.1084/jem.139.5.1142pmid: 4545163
The frequency of lymphocytes bearing complement receptors in the spleens of 2-wk old mice appears to be controlled by two independent genes. The presence of a "high" allele at either locus leads to intermediate or high frequency of CRL at 2 wk of age. One of the genes controlling complement receptor lymphocyte (CRL) frequency ( CRL-1 ) is linked to the H-2 complex. Thus, in progeny of (AKR x DBA/2)F 1 x DBA/2, all mice with a low frequency of CRL at 2 wk of age are homozygous for the H-2 type of the low CRL parent (DBA/2). Furthermore, in the B10 series of congenic mice, CRL frequency at 2 wk of age is similar to the frequency in the donor of the H-2 region. Thus, C57BL/10, B10.BR, and B10-D2 mice are all of the low CRL type while B10.A mice are intermediate in CRL frequency at 2 wk. C57BR and DBA/2, the donors of the H-2 complex of the B10.BR and B10.D2, respectively, are of low CRL type while the A/WySn, the donor of the H-2 complex in the B10.A, is an intermediate CRL strain. Similarly in the A/WySn series of congenic mice, A.CA, A.SW, and A.BY are all low CRL strains while the A/WySn is intermediate. Studies of CRL frequency in mice with recombinant H-2 chromosomes (B10.A(2R), (4R), and (5R); B6/TL + ; and A/TL - ) indicate that CRL-1 is to the right of the Ss-Slp genes and to the left of Tla . Footnotes Submitted: 21 January 1974