The Journal of Experimental Medicine

Piguet, P F; Dewey, H K; Vassalli, P

doi: N/Apmid: 1092790

Caryotypic analysis of the cells dividing in mouse parent-hybrid MLC showed an F1 hybrid cell proliferation, which varied depending upon the source of lymphoid cells used: strong in spleen MLC (sometimes equal to that of the parental cells), less marked in lymph node cell MLC, and most often absent in MLC between cortisone-resistant (CR) thymocytes. MLC between parental spleen cells and F1 CR thymocytes showed, however, that in certain conditions of culture F thymocytes can also proliferate. Using parental or F1 spleen cells lacking T lymphocytes, it was found that F1 cell proliferation is entirely dependent upon the presence of parental T cells, but does not require the presence of T lymphocytes among the F1 cells. Immunofluorescence analysis of the blasts observed in one-way MLC showed that about 70% of the parental blasts were T blasts, and 25%B blasts (containing a high proportion of plasmablasts); among the F1 blasts, there was also the same percentage of B blasts and plasmablasts, but many of the T blasts bore only small amounts of T-cell antigen (MTLA), and there was also about 20%of unstained blasts, possibly T blasts bearing MTLA in amounts undetectable by immunofluorescence. The possibility is discussed that the F1 responding T cells belong to a subpopulation performing a suppressive function; MLC lacking F1 T cells showed increased 3H thymidine incorporation. The proliferation and differentiation of parental and F1 B cells may result mainly from an unspecific, "polyclonal" triggering.

Nossal, G J; Pike, B L

doi: N/Apmid: 47889

This paper deals with the behavior of adult mouse bone marrow cells placed in tissue culture with or without antigen, and subsequently assessed for immune competence after adoptive transfer into lethally X-irradiated, syngeneic hosts. Attention was focussed on B lymphocytes through using hapten human gamma globulin (HGG) preparations as putative tolerogens in tissue culture, the T-cell-independent antigens DNP-POL and NIP-POL as challenge injections in adoptive hosts, and numbers of hapten-specific PFC in host spleens for the quantitation of immune competence. It was found that the capacity of bone marrow cells to mount an adoptive immune response rose by a factor of about fivefold over 3 days in tissue culture. This rise was completely abolished by the presence in the culture of hapten-HGG conjugates with about one mole of hapten per carrier molecule. The prevention of the emergence of immune competence amongst maturing B cells was termed clonal abortion tolerogenesis. Dose-response studies showed the lowest effective antigen concentration to be between 2.5 times 10- minus 10 and 2.5 times 10- minus 9 M, and a standard concentration of 2.5 times 10- minus 8 M was chosen as producing near maximal effects. The tolerance was antigen-specific and time-dependent, being maximal only when antigen was present continuously as the cultured cells was maturing. It did not depend on the presence of T lymphocytes in marrow, and was not of an "infectious" type. In contrast to tolerogenesis of mature B lymphocytes by high antigen concentrations, it could not be abolished by lipopolysaccharide. We speculate that clonal abortion may be a tolerance mechanism of great physiological significance for self-recognition, and discuss the results in the framework of other recent tolerance models, including those involving receptor blockade and suppressor T cells.

Dewald, B; Rindler-Ludwig, R; Bretz, U; Baggiolini, M

doi: N/Apmid: 236354

The subcellular localization of elastase and of neutral proteases hydrolyzing histone and casein was determined in human and rabbit polymorphonuclear leukocytes using fractionation by isopycnic centrifugation. Granule-rich fractions obtained by this technique were extracted and analyzed by acrylamide gel electrophoresis, and proteolytic activity on the gels was demonstrated by staining with either N-acetyl-D,L-alanine alpha-naphthyl ester or naphthol AS-D acetate as substrate. In both species, all neutral proteases assayed were found to be localized exclusively in the azurophil granules. Specific activities were about 10-30 times higher in human than in rabbit preparations. In extracts of human azurophil granules up to 10 proteins exhibiting esterolytic activity could be demonstrated after electrophoretic separation. Three major and two or three minor components of these esterases were shown to possess elastase activity. Similar zymograms prepared with extracts from rabbit azurophil granules revealed only one major elastase band. The electrophoretic analysis further showed that the most strongly cationic proteins of both human and rabbit PMNs were also confined to the azurophil granules.

Lilly, F; Duran-Reynals, M L; Rowe, W P

doi: N/Apmid: 165256

Tissue extracts from 6-wk old mice of the AKR strain (H-K) show high levels of infectious murine leukemia virus, and these mice show a near 100% incidence of spontaneous leukemia. In F1 mice of the cross, BALB/c times AKR (H-2K/H-2K), both the occurrence of virus and the incidence of spontaneous leukemia are suppressed to very low values, due largely to the presence of the FV-1b allele inherited from the BALB/c parent. Mice of the (BALB/c times AKR) F-1 times AKR backcross generation were observed for possible correlations between virus expressions at 6 wk of age, H-2 type and leukemia incidence. H-2 type showed at most a weak influence on the occurrence of infectious virus, but there was a very strong correlation between the level of virus expression and the occurrence of leukemia and a strong correlation between H-2 type and leukemia. In addition, there was a highly significant nonrandom distribution of virus-negative mice among the backcross litters, suggesting a maternal effect on virus expression.

Kolb, W P; Muller-Eberhard, H J

doi: N/Apmid: 47885

Isolation of the C5b-9 complex from inulin-activated whole human serum was effected by molecular sieve column chromatography employing Biogel A-15 M, preparative Pevikon block electrophoresis, and removal of low density beta-lipoproteins by flotation in CsCl. The final product was homogeneous upon cellulose acetate strip electrophoresis and analytical ultracentrifugation. Ouchterlony analyses indicated that the complex reacted with antisera to C5, C6, C7, C8, and C9 to form a continuous, circular precipitin line without spurs. The C5b-9 complex was dissociated by sodium dodecyl sulfate (SDS) in the absence of reducing agents, and analytical SDS-polyacrylamide gel electrophoresis revealed seven protein bands after straining with Coomassie Blue. Bands 1, 2, 3, and 6 were identified as C5b, C7, C6, and C9, respectively. Bands 4 and 7 were identified as two noncovalently bound subunits of C8. Molar ratios among C5b, C6, C7, C8, and C9 dissociated from the complex by SDS were estimated to be 1:1:1:1:3. Band 5 protein, which had an estimated mol wt of 88,000 and was found to occur with a molar ratio of 3, has not yet been identified. Its nature and possible biological functions are discussed.

Coutinho, A; Gronowicz, E

doi: N/Apmid: 1092788

Spleen cells from C3H/HeJ mice fail to respond with polyclonal antibody synthesis to mitogenic concentrations of lipopolysaccharide (LPS) which are optimal for activating spleen cells from a high-responder strain (B10.5M). This unresponsiveness is selective for LPS, since C3H/HeJ cells respond as normals to another B-cell mitogen, purified protein derivative of tuberculin. Spleen cells from low-responder mice also fail to mount a specific anti-NNP plaque-forming cell (PFC) response, when challenged in vitro by NNP-LPS. However, C3H/HeJ cells develop normal responses to another thymus-independent hapten conjugate, DNP-AECM-Ficoll. C3H/HeJ mice fail to mount a specific anti-LPS antibody response, when challenged in vivo with doses of soluble LPS which are fully immunogenic for the high-responder strain. However, C3H/HeJ mice develop normal direct and indirect PFC responses to LPS, when challenged with a thymus-dependent form of the immunogen. These results are interpreted as indicating as absolute requirement for functional mitogenicity of the antigen, in the induction of specific thymus-independent antibody responses.

Drake, W P; Pokorney, D R; Chipman, S; Levy, C C; Mardiney, M R

doi: N/Apmid: 1092792

Ribonuclease activity in cell-free thymus homogenates was elevated for five strains of mice genetically predisposed toward leukemia or reticulum cell neoplasms (AKR, C58, PL, RF, and SJL). Such increased activity was directed against polyuridylic acid and was observed in 8-wk old mice, well before the onset of neoplastic transformation. Similarly, white blood cell ribonuclease activity was elevated in mice of the strains AKR, C2H/He, PL and RF. Statistical analysis indicated that such elevated activity in these strains related to their high incidence of spontaneous neoplastic disease. Elevated ribonuclease activity thus represents a new biochemical marker relating to the genetic propensity of some strains of mice to die prematurely of spontaneous neoplasia.

Joseph, B S; Cooper, N R; Oldstone, M B

doi: N/Apmid: 1092789

In these studies, a number of human cell lines including epithelial, neural, glial, and lymphoid cells infected with several strains of measles virus were found to be lysed upon incubation with fresh sera from humans containing antibody measles virus. In all instances, the cytolytic event was mediated by alternative complement (C) pathway without a significant contribution from classical pathway. In contrast, isolated measles virus in conjunction with antibody was found to selectively activate the classical C pathway. Measles antibodies of the IgG class, but not the IgA class, possessed cytolytic potential against cells infected with measles virus. Human IgG antibodies could directly activate the alternative C pathway. No defect was found in cytolytic measles antibody in sera or cerebrospinal fluid from patients with subacute sclerosing panencephalitis, nor was the alternative C pathway impaired in sera from these patients. Sera from newborn humans exhibited a functional alternative C pathway.

Steinman, R M; Adams, J C; Cohn, Z A

doi: N/Apmid: 1127378

White pulp nodules of mouse spleen contain a minor population of cells with morphologic features that are identical to those of dendritic cells, a cell type recently described in vitro. They have characteristic large, irregularly shaped nuclei with distinctive chromatin patterns and small nucleoli. The cytoplasm is extended in processes that contain relatively few organelles. These presumptive dendritic cells can be distinguished from other cell types that are known to exist in spleen including those that have irregular or branching cell shapes. In particular, dendritic cells do not contain the large number of lysosomes seen in phagocytes, and do not actively interiorize intravenously administered colloidal thorium dioxide particles. They also lack the well developed secretory apparatus (rough endoplasmic reticulum and Golgi zone) and microfilament bundles that are noted in connective tissue cells. These morphologic observations, combined with previous in vitro work, substantiate the existence of a novel class of cells in mouse lymphoid organs.

Kung, P C; Siverstone, A E; McCaffrey, R P; Baltimore, D

doi: N/Apmid: 1127379

The mouse thymus contains two forms of terminal deoxynucleotidyl transferase (TdT) which are distinguishable by the salt concentration necessary to elute them from a phosphocellulose column, by their distrubtion among the thymocyte subpopulations, and by their sensitivity to cortisone treatment. In the whole thymus the later eluting peak (peak II) is the predominant one with about 3-10% of the total activity appearing in peak I. Both peak I and peak II activities are most sensitively assayed by the polymerization of dGMP onto an oligo(dA) primer. The minor population of thymocytes which is less dense and cortisone-resistant contains a higher specific activity of peak I TdT. The majority of TdT activity is, however, found in the major population of thymocytes which occurs in the center region of a bovine serum albumin gradient and is cortisone-sensitive. A very low level of an activity indistinguishable from peak II TdT activity is also detected in the mouse bone marrow. Other tissues, such as spleen, liver, heart, and brain lack detectable amounts of TdT activity.