The Journal of Experimental Medicine

de Landazuri, Manuel Ortiz; Herberman, Ronald B.

doi: 10.1084/jem.136.5.969pmid: 5082673

Spleen cells from W/Fu rats 40 days or more after immunization with a syngeneic Gross virus-induced leukemia were unreactive in direct cytotoxic assays. Incubation of these immune cells at 37°C for 12 hr or longer, in the absence of antigen, resulted in the appearance of specific cytotoxic reactivity. Other lymphoid cells from the immune rats also were activated upon in vitro incubation, but to a lesser extent. Experiments were performed to define the necessary conditions and the mechanism for the in vitro incubation. Activation was temperature dependent, occurring at 37°C but not at 4°C. Immune serum suppressed the activation, but normal rat serum also had some inhibitory activity. Passage of immune cells through a nylon column, before preincubation, prevented activation. In contrast, exposure to nylon after preincubation did not remove cytotoxic reactivity. These findings demonstrate the reversal of a central inhibition of immune cell activity. The explanations offered for this phenomenon included change in surface characteristics of the immune cells during in vitro incubation, and the possible need for an adherent helper cell. Footnotes Submitted: 1 May 1972

Lamelin, J.-P.; Lisowska-Bernstein, B.; Matter, A.; Ryser, J. E.; Vassalli, P.

doi: 10.1084/jem.136.5.984pmid: 4117194

The simultaneous use on mouse lymphoid suspensions of heterologous antisera directed against thymus-derived (T) cell mouse-specific lymphocyte antigen and brain-associated theta antigen (MSLA and BAθ) or thymus-independent (B) cell mouse-specific bone marrow-derived lymphocyte antigen (MBLA) surface antigens allowed direct proof of the different specificity of these antisera by double immunofluorescence (IF) staining with selective visualization of fluorochromes. These antisera and antisera against mouse Ig and its different types of chains were then used with technique of either double IF staining or IF combined with radioautography, allowing the following conclusions: ( a ) Surface Ig (sIg) was found exclusively on B cells and never on T cells, but not all B cells had sIg. Cells containing detectable amounts of Ig were MBLA+, but had less sIg than other B cells or none at all. There was evidence for the existence of a significant number of MBLA+ lymphocytes, neither bearing nor containing detectable Ig. ( b ) µ-Chains were the most frequent but not the only heavy chains found on spleen cells; however, it could not be decided with the technique used, if a single cell can bear more than one type of heavy chain. No cell containing γ-chains was found to bear surface µ-chains, although a very few cells containing both µ- and γ-chains were observed. ( c ) The antigen-binding cells detected after immunization with bacteriophage T4, bovine serum albumin, Maia squinado hemocyanin, and sheep erythrocytes were analyzed for MSLA, MBLA or sIg using double IF, a combination of IF and radioautography, or inhibition of "rosette" formation. Practically all the antigen-binding cells detected were MSLA-, MBLA+, sIg+. ( d ) More B cells than T cells were found among short-lived lymphoid cells labeled by repeated in vivo injections of tritiated thymidine, but the results did not support a simplified concept equating T cells to long-lived and B cells to short-lived lymphocytes. ( e ) Cells dividing rapidly in the lymph nodes draining the sites of immunization with various antigens were predominantly T cells 2 days after immunization and in majority B cells a few days later. ( f ) Incubation of lymphoid cells at 37°C with rabbit anti-mouse Ig or anti-κ chains led to complete disappearance of sIg and to decrease of MBLA ("antigenic modulation"). In the same conditions, anti-MBLA gave partial modulation of MBLA and of sIg; MBLA, however, reappeared much faster than sIg. No modulation of T cell surface antigens by the appropriate antisera was observed. Cell treatment with Pronase could remove MBLA, sIg, MSLA, and BAθ, which reappeared within a few hours. Neuraminidase treatment was without detectable effect on these antigens. Footnotes Submitted: 4 May 1972

Matter, A.; Lisowska-Bernstein, B.; Ryser, J. E.; Lamelin, J.-P.; Vassalli, P.

doi: 10.1084/jem.136.5.1008pmid: 4563148

The ultrastructural features of B-, T-, and surface Ig(sIg)-bearing cells have been studied on cell suspensions from lymphoid organs of mice at different stages of immunization. The cells were identified by exposure to rabbit antibodies against mouse-specific lymphocyte antigens (MSLA) or brain-associated θ antigen (BAθ) for T cells, mouse-specific bone marrow-derived lymphocyte antigens (MBLA) for B cells, and mouse Ig for sIg-bearing cells. The rabbit antibodies fixed on the cell surfaces were detected by peroxidase-labeled sheep anti-rabbit Ig antibodies or by a "bridge" technique using southern bean mosaic virus or bacteriophage T4 as the final markers. In some experiments, short-lived lymphoid cells were labeled in vivo with repeated tritiated thymidine and the ultrastructural detection of their surface antigens was combined with radioautography. MBLA+ lymphoid cells showed a whole range of ultrastructural patterns. Most were small and medium-sized lymphocytes with a clear cytoplasm containing mono- and polyribosomes, but they comprised also blasts and large cells with various amounts of endoplasmic reticulum, as well as plasma cells at different stages of maturation. sIg-bearing cells appeared to be identical with MBLA+ cells, except that sIg was less easily detectable on large blasts, and only very rarely observed on plasma cells. MSLA+ and BAθ+ cells fell into three categories. One of them (T 1 cells) consisted of small to medium-sized lymphocytes with a clear cytoplasm and few organelles, mostly monoribosomes. A second consisted of large cells (T 2 cells) characterized by numerous polyribosomes often in a "rosette"-like pattern, occasional dark, membrane-bound granules, and a developing "filamentous network." The third, very characteristic type, (T 3 cells) was represented by dark small to medium-sized lymphocytes, usually containing large amounts of closely packed ribosomes and showing a striking accumulation of filamentous network, often condensed in large areas devoid of cell organelles. This filamentous network appeared to correspond to the cytochalasin B-sensitive system of microfilaments found in other cells and considered to be one of the contractile elements of the cell. The T 3 lymphocytes showed frequently vesicles suggestive of a strong pinocytic activity, and assumed a variety of shapes, including uropods. Evidence is presented that T 1 lymphocytes represent "virgin" T cells, T 2 "activated," and T 3 "differentiated" lymphocytes. Footnotes Submitted: 4 May 1972

Lee, Chi-Jen; Dubos, Rene

doi: 10.1084/jem.136.5.1031pmid: 5082669

The effects of neonatal infection, perinatal malnutrition, and crowding on the metabolism of brain catecholamine were studied in specific pathogen-free mice. Metabolic turnover of catecholamine was determined by measuring the incorporation of precursor tyrosine- 14 C into brain tissue, catabolic activity of norepinephrine- 3 H at various times after intracisternal injection, and tissue levels of dopamine and norepinephrine. The rate of tyrosine incorporation was decreased by neonatal infection but was increased by perinatal malnutrition and crowding. There was no difference in catabolic activity of norepinephrine between infected, crowded, and control groups. In the malnourished group, however, the total radioactivity from norepinephrine was significantly higher than in the control group ½ and 2 hr after injection. The brain contents of dopamine and norepinephrine were depressed in the malnourished group. There was no significant difference in catecholamine levels between infected, crowded, and control groups. In the malnourished group, treatment of the mothers with growth hormone prevented almost completely weight loss during lactation, and also resulted in higher fetal weight. Hormone treatment restored to normal the levels of brain catecholamine and the enzymatic activity of brain tyrosine hydroxylase in progeny of malnourished mothers. Footnotes Submitted: 14 June 1972

Kappas, Attallah; Bradlow, H. Leon; Gillette, Peter N.; Gallagher, T. F.

doi: 10.1084/jem.136.5.1043pmid: 4263649

A variety of 5ß steroid metabolites derived from hormones natural to man are potent inducers experimentally of δ-aminolevulinate synthetase, the rate-limiting enzyme in porphyrin-heme formation. This mitochondrial enzyme is found at high levels of activity in the livers of patients with the genetic disease, acute intermittent porphyria (AIP). In this study the metabolism of 14 C-labeled testosterone was examined in AIP patients to determine whether there was a disproportionate conversion of the hormone to its 5ß, compared to its 5α metabolite. The results indicate that AIP subjects do generate a substantially greater than normal fraction of 5ß metabolite from this steroid; the excessive degree of ring A reduction of testosterone taking place via the 5ß pathway in the porphyric patients averages 350% greater than in the nonporphyric subjects. In one asymptomatic AIP patient the disproportionate generation of 5ß metabolite from the hormone reached a level 10 times the normal mean. Studies with a second 14 C-labeled hormone, dehydroisoandrosterone, whose metabolism in man resembles that of testosterone, confirmed the derangement in reductive transformation of steroids found in the individuals carrying the genetic lesion of AIP. These findings define a new endocrine abnormality in AIP patients and raise the possibility that endogenously derived 5ß steroids may contribute by an induction mechanism to the increased levels of hepatic δ-aminolevulinate synthetase activity found in AIP patients. Footnotes Submitted: 7 June 1972

Douglas, Tommy C.

doi: 10.1084/jem.136.5.1054pmid: 4117187

A rat antigen system parallel to the mouse theta system has been described • All rat strains tested expressed an antigen cross-reactive with the θ-AKR specificity of mice while none cross-reacted strongly with θ-C3H. The rat antigen may be demonstrated by either absorption or direct complement-mediated killing of rat thymocytes. Patterns of organ distribution and developmental appearance in the nervous system of rats also parallel those previously reported for theta in mice. Footnotes Submitted: 26 May 1972

Silver, Donald M.; McKenzie, Ian F. C.; Winn, Henry J.

doi: 10.1084/jem.136.5.1063pmid: 5082670

In response to repeated injections of sheep red blood cells, C57BL/10J mice produce predominantly 19S antibody in increasingly higher amounts, while A/J mice initially produce 19S antibody and then switch to produce increasing 7S antibody titers. In an F 1 generation all mice responded like the C57BL/10J mice. Backcross data implied genetic control involving at least three loci. Footnotes Submitted: 6 June 1972

E. W. Lamon; H. M. Skurzak; E. Klein; H. Wigzell

doi: 10.1084/jem.136.5.1072pmid: 4117188

Rapaport, F. T.; Watanabe, K.; Cannon, F. D.; Mollen, N.; Blumenstock, D.; Ferrebee, J. W.

doi: 10.1084/jem.136.5.1080pmid: 4404277

17 Cooperstown beagles of known DL-A genotypes were exposed to supralethal total body irradiation and received a bone marrow allograft from a DL-A-identical donor; 11 littermate and 6 nonlittermate donor-recipient pairs were studied. The recipients are surviving uneventfully for 315, 364, 424, 440, 531, 531, 584, 605, 625, 635, and 649 days in the littermate group and for 211, 279, 280, 368, 479, and 480 days in the nonlittermate group. The radiation chimeras underwent bilateral nephrectomy and received a kidney allograft obtained from their respective marrow donor within 43–120 days after bone marrow transplantation. The renal allografts are surviving for 191, 200, 221, 234, 313, 349, 361, 377, 378, 405, 441, 444, 482, 557, 580, 581, and 586 days, respectively. 12 of 13 skin allografts obtained from the marrow donor are at present surviving in the recipients for 107, 110, 110, 110, 116, 122, 128, 143, 143, 162, 178, and 199 days, respectively; one graft was rejected at 84 days. In contrast, the radiation chimeras rejected 25 skin allografts obtained from DL-A-incompatible donors within 10.5–21 days (MST = 15.2 days). Skin transplants obtained from DL-A-identical siblings of the bone marrow donors were rejected within 20–36 days (MST = 25.8 days) in recipients of bone marrow cells obtained from littermate donors. Recipients of nonlittermate bone marrow transplants accorded such allografts a prolonged survival time of 27–76 days (MST = 56.2 days). Prospective selection of genotypically DL-A-identical donor-recipient pairs results in the regularly reproducible long-term survival of bone marrow allografts. The radiation chimeras produced in this manner have developed a donor-specific state of unresponsiveness to kidney and skin allografts. The results are consistent with the existence in the canine species of at least three closely linked genetic systems relevant to transplantation, including DL-A, MLC, and a possible bone marrow transplantation locus. Footnotes Submitted: 22 May 1972

Eidinger, David; Garrett, Thomas J.

doi: 10.1084/jem.136.5.1098pmid: 4538840

The primary and secondary immune responses to thymus-dependent and -independent antigens were evaluated in normal male and female mice and in castrated male mice. Both IgM antibody production in the primary response and IgG antibody production in the secondary response were enhanced in females vs. males of equivalent age. Castration of the male converted this animal to a female in terms of responsiveness to the thymus-dependent group of antigens, while inducing equivalent or even greater enhanced responsiveness over the female to the thymus-independent antigen, polyvinylpyrrolidone. Further characteristics of the changes in lymphoid organs were determined in the castrated animal vs. normal males and females. It was shown that the spleen and thymus became markedly hyperplastic, the organ weights exceeding the female, which in turn were greater than in the male. The enhanced weight of the thymus was shown to be due to increased numbers of cortisone-sensitive cells, the absolute number of cortisone-resistant cells remaining equivalent to normal males and females. Thus, the increased thymic weight of the female also resided in the cortisone-sensitive population. Peripheral lymphocyte counts in castrated animals exceeded both normal males and females. Further experiments in gonadectomized males provided evidence that increased thymic cell activity per se played a role in enhanced response to thymus-dependent antigens, but that a thymic-derived hormone mediated the enhanced effect to the thymus-independent antigen in the castrated animal. The capacity for loss of androgenic hormone-producing tissue to generate enhanced differentiation of stem cells was denoted by experiments in which numbers of spleen colonies and uptake of 59 Fe, employed as an index of hematopoiesis 1 wk after reconstitution of lethally irradiated castrated and normal recipients, were enhanced in gonadectomized male animals. Thus, in summary, changes in sex hormone levels exerted a marked influence on immune responsiveness and stem cell differentiation, by increasing numbers of functioning cells, by promoting cellular differentiation, as well as by promoting cellular function via hormonal effects. Footnotes Submitted: 1 June 1972