bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemical characterization and biosynthesis of the Ki-1 antigen in Hodgkin-derived and virus-transformed human B and T lymphoid cell lines.
Authors: Froese P , Lemke H , Gerdes J , Havsteen B , Schwarting R , Hansen H , Stein H
Published: December 2022

The Hodgkin-associated Ki-1 antigen was analyzed in different cell lines. In Hodgkin analogous L428 cells, biosynthetically labeled with radioactive amino acids, the Ki-1 antibody precipitated three glycoproteins with 90, 105, and 120 kDa, respectively. Surface-labeling revealed that the two larger components were membrane-associated forms of the Ki-1 antigen, although the 90-kDa molecule was shown in pulse-chase experiments to be the precursor of the 105- and 120-kDa forms. All three forms of the Ki-1 antigen possess a tunicamycin-sensitive 6-kDa N-linked carbohydrate moiety. O-Linked oligosaccharides could not be detected. Thus, the differences in m.w. are probably not due to glycosylation. The ionophore monensin prevented the appearance of the membrane-associated molecules, which demonstrated that they are assembled between the transcompartment of the Golgi complex and their insertion into the cell membrane. The 90-kDa precursor molecule cannot be generated by disulfide reduction from the two larger forms. After internal labeling with P-32, only the 105- and 120-kDa bands became visible, indicating that the Ki-1 molecule is phosphorylated after its processing into the two larger membrane-associated forms. Analysis of the Ki-1 antigens from other cell lines demonstrated that after external labeling of two other Hodgkin-derived cell lines, six Epstein-Barr virus lymphoblastoid cell lines and one human T leukemia virus I-positive T cell line, both the 105- and the 120-kDa membrane molecules could be detected, regardless of the presence or type of virus integrated.
Biologic-response-modifier-induced indoleamine 2,3-dioxygenase activity in human peripheral blood mononuclear cell cultures.
Authors: Carlin J M , Borden E C , Sondel P M , Byrne G I
Published: December 2022

Degradation of tryptophan to kynurenine, catalyzed by indoleamine 2,3-dioxygenase (IDO), has been augmented in human epithelial cell lines treated with human interferon-gamma (HuIFN-gamma). Several human biologic response modifiers, including HuIFN-gamma, HuIFN-beta, HuIFN-alpha, interleukin 2 (HuIL-2), and tumor necrosis factor alpha, have now been assessed for their ability to enhance tryptophan degradation in human peripheral blood mononuclear cell (PMC) cultures. PMC were isolated from normal donors, cultivated in RPMI 1640 medium containing [3H]tryptophan, and treated with individual biologic response modifiers. At various intervals, culture supernatants were removed, fractionated by reversed-phase high performance liquid chromatography, and radioactivity in resultant fractions was determined. Significantly increased amounts of tryptophan catabolites were observed after treatment with HuIFN-gamma, HuIFN-beta, HuIFN-alpha, and HuIL-2, but not human tumor necrosis factor alpha. Often, greater than 30% of available tryptophan was degraded by treated PMC cultures. Although antibodies to HuIFN-alpha, HuIFN-beta, and HuIFN-gamma specifically neutralized the induction of IDO activity in PMC by their respective HuIFN, only anti-HuIFN-gamma antibody also neutralized HuIL-2-induced IDO activity. Furthermore, T24 bladder carcinoma cells, in which IDO was induced by HuIFN-gamma but not by the other biologic response modifiers, were induced to degrade tryptophan by supernatants of HuIL-2-stimulated PMC cultures, but not by HuIFN-beta-stimulated PMC culture supernatants. Thus, whereas HuIL-2 indirectly induced IDO in PMC cultures by stimulating production of HuIFN-gamma, all cases of interferons appeared to induce IDO directly in PMC cultures.
Inhibition of human T lymphocyte E rosette formation by neutrophils and hydrogen peroxide. Differential sensitivity between helper and suppressor T lymphocytes.
Authors: Staite N D , Messner R P , Zoschke D C
Published: December 2022

Co-incubation of human mononuclear cells with small numbers of autologous polymorphonuclear leukocytes resulted in a ratio-dependent inhibition of T lymphocyte rosette formation with sheep erythrocytes (SRBC). Inhibition by polymorphonuclear leukocytes was reversed with catalase, implicating H2O2 or associated products as the suppressive agent(s). Exposing T lymphocytes directly to micromolar concentrations of H2O2 also inhibited subsequent rosette formation. Inhibition was dose-dependent between 40 and 320 microM H2O2 and was not due to cytotoxicity at those H2O2 concentrations. Kinetic studies demonstrated that after exposing T lymphocytes to H2O2 a proportion of cells appeared to be relatively resistant in that they retained their ability to form E rosettes. T cell phenotype analysis of these cells showed that, in contrast to untreated T cells, H2O2-treated T rosette-forming cells were almost exclusively of the helper/inducer phenotype. Analysis with the monoclonal antibody 4B4 revealed that H2O2-resistant T rosette-forming cells contained significantly fewer 4B4-positive cells than predicted for the T helper population, indicating that H2O2-resistant T cells might be a subset of helper/inducer T lymphocytes. The interaction between H2O2 and T cells was rapid but did not lead to loss of Leu-5b binding sites. Preliminary functional analysis indicates that interleukin 2 production and phytohemagglutinin-induced proliferation by the relatively H2O2-resistant T cells is similar to untreated T cells. In contrast, H2O2-treated T cells which lost their ability to form E rosettes produced undetectable levels of interleukin 2 and proliferated poorly in response to phytohemagglutinin. Finally, mononuclear cells pretreated with H2O2 and subsequently stimulated with mitogens demonstrated a preferential expansion of the T helper population with concomitant loss of T suppressors. Our results show that, although neutrophil-released H2O2 inhibits effective interactions between SRBC and T lymphocyte sheep erythrocyte receptors, there appears to be a population of T helper cells which is relatively resistant to H2O2 both in terms of SRBC rosette formation and response to mitogens. These data suggest that at sites of inflammation phagocyte-released H2O2 could exert immunoregulatory effects on T cells by altering T cell subset survival and allowing the function of a particular lymphocyte population to predominate.
Lymphocyte subsets differentially induce class II human leukocyte antigens on allogeneic microvascular endothelial cells.
Authors: Pardi R , Bender J R , Engleman E G
Published: December 2022

Increased expression of major histocompatibility complex class II (Ia) antigens on vascular endothelium is a common observation in allografts undergoing acute rejection. This phenomenon is generally ascribed to the host immune response directed against graft alloantigens, but its cellular and molecular basis are incompletely understood. In the present study we show that constitutively Ia-negative human microvascular endothelial cells (EC) can be induced to express surface class II human leukocyte antigens shortly after exposure to allogeneic lymphocytes in vitro. CD16+ (natural killer) and CD8+ (cytotoxic/suppressor) lymphocytes were efficient in triggering Ia antigen expression by EC, whereas CD4+ (helper/inducer) lymphocytes induced EC Ia expression only if cultured in the presence of autologous monocytes. Binding of lymphocytes to EC was shown to be essential for the subsequent induction of EC Ia, and anti-CD18 (LFA-1) antibody, which blocks lymphocyte-EC adhesion, was the only antibody of a panel of antilymphocyte antibodies that completely blocked the induction of EC Ia. Antibodies to interferon-gamma, which is a potent inducer of EC Ia, and to the CD3 T cell-surface antigen partly inhibited the induction of EC Ia by T cells, but neither antibody had any effect on Ia induction mediated by CD16+ cells, suggesting that T cells and natural killer cells utilize different mechanisms to induce Ia on EC. When combined with data from other laboratories indicating that Ia+ but not Ia- EC stimulate allogeneic T cell proliferation and cytotoxicity, our results suggest that the binding of EC by lymphocyte subpopulations followed by the induction of Ia antigen may represent the initial stage of incompatible allograft rejection.
Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells.
Authors: Ransom J , Fischer M , Mercer L , Zlotnik A
Published: December 2022

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.
Detection and partial characterization of a human mast cell carboxypeptidase.
Authors: Goldstein S M , Kaempfer C E , Proud D , Schwartz L B , Irani A M , Wintroub B U
Published: December 2022

Using a high performance liquid chromatography assay that detects the cleavage of the C-terminal leucine from angiotensin I, we have identified a carboxypeptidase activity in mast cells from human lung and in dispersed mast cell preparations from human skin. The enzyme activity was detected in a preparation of dispersed human mast cells from lung of greater than 99% purity and was released with histamine after stimulation with goat anti-human IgE. In nine preparations of dispersed human mast cells from lung of 10 to 99% purity, net percentage of release of carboxypeptidase correlated with the release of histamine, localizing carboxypeptidase to mast cell secretory granules. The enzyme activity was also detected in preparations of dispersed human mast cells from skin and in extracts of whole skin. The inhibitor profile and m.w. of carboxypeptidase activity from preparations of dispersed mast cells from skin was similar to that from dispersed mast cells from lung. Mast cell carboxypeptidase had a m.w. on gel filtration of 30,000 to 35,000. The enzyme in crude lysates of dispersed mast cell preparations had optimal activity between pH 8.5 and 9.5 and was inhibited by potato inhibitor, which distinguished it from carboxypeptidase in cultured human foreskin keratinocytes and adult fibroblasts, and from other proteolytic mast cell enzymes. The enzyme activity was also inhibited by EDTA, o-phenanthroline, and, to a small extent, by 8-OH quinoline, but not by Captopril, soybean trypsin inhibitor, or pepstatin. These findings demonstrate that human mast cell secretory granules contain carboxypeptidase in addition to tryptase and chymase. It appears that mast cells from skin may have a higher content of carboxypeptidase than do mast cells from lung.
Thymus-dependent in vivo suppression of IgE synthesis in a murine IgE-secreting hybridoma.
Authors: Mathur A , Kamat D M , Van Ness B G , Lynch R G
Published: December 2022

In previous studies we demonstrated that BALB/c mice bearing ascitic tumors of the IgE-secreting hybridoma B53 (epsilon, kappa, anti-dinitrophenyl) developed large numbers of Lyt-1-2+ Fc epsilon R(+) T lymphocytes (T cells with membrane Fc receptors) in response to the elevated serum IgE concentration. The development of Fc epsilon R(+) T lymphocytes was followed by a progressive decrease in the levels of serum IgE in spite of continued proliferation of the hybridoma cells. This sequence of events suggested that the IgE-secreting hybridoma triggered a suppressive immunoregulatory circuit of the host that inhibited IgE expression by the hybridoma cells. The present studies were undertaken to investigate the basis for the subsequent decline in serum IgE levels in mice with B53 tumors and to identify host factors that might be involved in this process. We observed that ascitic B53 cells recovered at increasing time points from BALB/c mice exhibited a selective decline in steady state levels and rates of synthesis of epsilon-heavy chain protein and mRNA. The expression of kappa-light chain protein and mRNA appeared relatively unchanged. The decrease in epsilon-heavy chain gene expression did not occur when B53 tumors were passaged in nu+/nu+ mice or in BALB/c mice depleted of Lyt-2+ cells (suppressor/cytotoxic cell lineage), but did occur in nu+/nu+ mice reconstituted with neonatal BALB/c thymus and in BALB/c mice depleted of L3T4+ cells (helper/inducer cell lineage). That Fc epsilon R(+) T lymphocytes were directly involved in the inhibition of IgE expression was supported by the earlier and more pronounced inhibition of B53 IgE in mice infused with Fc epsilon R(+) T lymphocytes. We conclude from these findings that: 1) the decline in serum IgE levels that occurs toward the end of each generation of in vivo passage of the B53 hybridoma is due to decreased production of IgE by the hybridoma cells, 2) the decreased production of IgE is due to a selective loss of epsilon mRNA expression, 3) the decrease production of IgE by B53 cells is dependent on the presence of Lyt-2+ cells, and 4) Fc epsilon R(+) T lymphocytes participate in the mechanism by which IgE production is suppressed.
Formyl peptide leukocyte chemoattractant uptake and release by cultured human umbilical vein endothelial cells.
Authors: Rotrosen D , Malech H L , Gallin J I
Published: December 2022

Recent observations support an active role for the vascular endothelial cell in the induction and evolution of the inflammatory response. Since prior studies suggested that cultured bovine endothelial cells express high affinity binding sites for the neutrophil chemotactic oligopeptide formyl methionyl-leucyl-phenylalanine (f-Met-Leu-Phe), we sought to further characterize the interaction between formyl peptide chemoattractants and human vascular endothelial cells. Cultured human umbilical vein endothelial cells and peripheral blood neutrophils specifically bound f-Met-Leu-[3H]Phe, whereas specific binding to cultured fibroblasts, smooth muscle, and epithelial cells was negligible. Endothelial cells expressed 3.6 +/- 0.7 X 10(5) binding sites/cell with a Kd of 210 +/- 31 nM. Although the hexapeptide formyl norleucyl-leucyl-phenylalanyl-norleucyl-tyrosyl-lysine (f-Nle-Leu-Phe-Nle-Tyr-Lys) and the tetrapeptide f-Met-Leu-Phe-Lys completed with f-Met-Leu-[3H]Phe for binding to endothelial cells, specific binding of 125I-f-Nl-Leu-Phe-Tyr-Lys or f-Met-Leu-Phe-Lys-fluorescein to endothelial cells was not observed, suggesting that steric constraints on formyl peptide binding differ between endothelial cells and leukocytes. At 37 degrees C, cell-associated f-Met-Leu-[3H]Phe greatly exceeded that bound at 0 degrees C and was incorporated predominantly into a nondisplaceable compartment. Release of f-Met-Leu-[3H]Phe or radioactive breakdown products from this compartment was time- and temperature-dependent with a t1/2 of approximately equal to 20 min at 37 degrees C. Resolution of the radioactive products released from f-Met-Leu-[3H]Phe-loaded endothelial cells by thin layer chromatography indicated that greater than or equal to 57% of the released material co-migrated with intact f-Met-Leu-[3H]Phe. Degradative release was blocked by agents that interfere with lysosomal acidification. The radioactive material released from f-Met-Leu-[3H]Phe-loaded endothelial cells bound specifically to neutrophils. This binding was inhibited 50.2 +/- 6.4% by a greater than or equal to 10(3)-fold excess of nonradioactive f-Met-Leu-Phe whereas binding of authentic f-Met-Leu-[3H]Phe was inhibited 89.4 +/- 3.0%. Supernatant obtained from f-Met-Leu-[3H]Phe-loaded endothelial cells elicited a rise in neutrophil cytosolic free calcium ([Ca2+]i) measured by quin2 fluorescence. The change in neutrophil [Ca2+]i depended on ligand binding to the neutrophil formyl peptide receptor since endothelial supernatants were devoid of activity in the presence of the f-Met-Leu-Phe antagonist, tert-butoxycarbonyl-Phe-Leu-Phe-Leu-Phe.(ABSTRACT TRUNCATED AT 400 WORDS)
Cationization of protein antigens. IV. Increased antigen uptake by antigen-presenting cells.
Authors: Apple R J , Domen P L , Muckerheide A , Michael J G
Published: December 2022

Cationization of BSA generates a molecule that mounts antibody responses of increased magnitude and duration and induces T cell proliferation at concentrations 500 times less than native BSA (nBSA). To explain the alteration in immunogenic properties of this Ag, the uptake of nBSA and cationized BSA (cBSA) by splenic APC has been investigated. T cell proliferation assays were conducted with nBSA and cBSA preparations with varying degrees of substitution. An inverse correlation between the degree of cationization and the amounts of Ag needed for optimal T cell reactivity was observed. To determine whether affinity for APC resulted in an increased uptake of cBSA, splenic APC were incubated with nBSA or cBSA for varying amounts of time. Comparisons were made at each time point between untreated Ag-pulsed APC (Ag uptake) and paraformaldehyde-fixed Ag-pulsed APC (processed Ag). Proliferation of T cells primed with nBSA or cBSA increased in proportion to the amount of time of APC exposure to high concentrations of nBSA, first appearing after a 2-h pulse and peaking at 8 h. Conversely, untreated APC needed only a 30-min cBSA exposure to induce either nBSA- or cBSA-primed T cell proliferation, indicating a rapid uptake of cBSA. Comparisons with proliferation induced by paraformaldehyde-fixed cBSA APC indicate that nBSA T cells recognize a lag phase-processed form of cBSA, whereas a majority of cBSA T cells recognize either a rapidly processed form of cBSA, or a membrane-processed cBSA molecule without a classical lag phase processing event. When monensin was used as an inhibitor of fluid phase pinocytosis in splenic APC, the presentation of nBSA was inhibited by 85%, but the presentation of cBSA was inhibited by only 20%. These results imply that nBSA enters the cell by fluid phase pinocytosis, whereas cBSA enters by a nonspecific adsorptive mechanism. The different modes of cellular entry for the two molecules, nBSA and cBSA, resulting in a rapid uptake of cBSA, may have important ramifications on T cell activation and immunoregulation.
In vitro analysis of the interactions of recombinant IL-2 with regenerating lymphoid and myeloid cells after allogeneic marrow transplantation.
Authors: Heslop H E , Price G M , Prentice H G , Cordingley F T , Webster A D , Hoffbrand A V , Brenner M K
Published: December 2022

Although administration of rIL-2 post-T depleted allogeneic bone marrow transplantation (TD-BMT) offers the prospect of augmenting immune reconstitution and thereby reducing the risks of infection and relapse, it has been unclear what direct or indirect effects this agent would have on the regenerating myeloid system. We find that addition of 200 IU or rIL-2 to patient lymphocytes obtained within 6 wk of TD-BMT results in a substantial (2 to 3 log) increase in INF-gamma secretion and the production of TNF. Cytokines present in supernatants obtained from IL-2-stimulated patient lymphocytes have two contrasting effects on myeloid cells from normal donors and from marrow recipients. They prime granulocytes for enhanced oxidative metabolism as measured by ability to generate chemiluminescence in response to FMLP, whereas IL-2 added directly to neutrophils has no effect. However, these IL-2-induced cytokines also act to inhibit myeloid progenitor growth and reduce granulocyte macrophage (GM) colony formation by a mean of 53%. Preincubation of supernatants with anti-IFN-gamma antibody partially abrogates both enhancement of granulocyte chemiluminescence and suppression of marrow CFU-GM. Addition of IL-2 directly to recipient marrow also produces inhibition, leading to a 25% reduction of GM-colony growth. This effect is not due to direct interaction between myeloid progenitor cells and IL-2, because it is completely abrogated by removal of CD8 and Leu-7+ lymphocytes from the marrow. Although the suppressive effects on marrow growth in vitro are of particular concern after BMT, the potential of IL-2 to promote granulocyte function, immune reconstitution, and anti-leukemic activity after TD-BMT justify further consideration of IL-2 therapy in this setting.
Molecular linkage of the Ly-3 and Ly-2 genes. Requirement of Ly-2 for Ly-3 surface expression.
Authors: Gorman S D , Sun Y H , Zamoyska R , Parnes J R
Published: December 2022

We have isolated the Ly-3 gene by chromosomal walking from the previously cloned Ly-2 gene. These genes are shown to be 36 kb apart and in the same transcriptional orientation. Transfection of the Ly-3 gene into mouse L cells results in cell surface expression of Ly-3 protein only in the presence of Ly-2 (or its human homolog, CD8), although Ly-2 surface expression is not similarly dependent on Ly-3. cDNA clones encoding Ly-3 have been isolated and sequenced and show little sequence similarity to Ly-2, whereas both Ly-2 and Ly-3 are homologous to Ig variable regions. One cDNA clone encodes a form of Ly-3 lacking a transmembrane region. Although two alleles of Ly-3 have been previously defined serologically, Northern blot analyses of Ly-3 mRNA from a series of inbred mouse strains show unusual polymorphisms in the lengths of Ly-3 mRNA species defining at least three allelic variants of this gene.
Prerequisite for the induction of lymphokine-activated killer cells from T lymphocytes.
Authors: Sawada H , Abo T , Sugawara S , Kumagai K
Published: December 2022

Human blood mononuclear cells were separated into Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells by means of a combination of the Percoll gradient method and C-mediated cytolysis using mAb. When purified Leu-11+7-NK, Leu-11-7+, and Leu-11-7-T cells were cultured with rIL 2 (500 U/ml) for 6 days in a medium supplemented with 10% FCS, Leu-11+7-NK cells responded at the maximum level and Leu-11-7+ cells responded moderately as shown by both cell-proliferation response and cytotoxic activity generated. On the other hand, Leu-11-7-T cells did not respond at all to rIL-2. However, when Leu-11-7-T cells were cultured with rIL-2 in a medium supplemented with 10% autologous serum, they showed considerable responsiveness to rIL-2. In addition, much greater response to Leu-11-7-T cells were produced by the addition of monocytes. Monocyte cytokines, neither IL 1, IFN-gamma, TNF, nor their combination were able to substitute for monocytes in the induction culture. In contrast, the response level of Leu-11+7- NK cells remained unchanged irrespective of supplementation with autologous serum to medium or the addition of monocytes to the culture. These results indicated that culture conditions in the experiments significantly affected the results as to determination of lymphokine-activated killer cell precursors, especially the result pertaining to the conversion of T lymphocytes to lymphokine-activated killer cells. Under appropriate conditions, not only NK cells but also T cells are important precursors of lymphokine-activated killer cells.
Identification of a murine pan-T cell antigen which is also expressed during the terminal phases of B cell differentiation.
Authors: Gulley M L , Ogata L C , Thorson J A , Dailey M O , Kemp J D
Published: December 2022

A new rat anti-mouse mAb, designated S7, is described. The antibody, an IgG2a, reacts with all Thy-1.2-positive cells and all granulocytic cells in the marrow. Resting B cells are non-reactive but, during the terminal phases of LPS-induced B cell differentiation, the S7 determinant appears and reaches high levels of expression. S7 will probably be useful in studies aimed at subdividing the later stages of the B cell maturation process.
Effects of in vivo administration of anti-T3 monoclonal antibody on T cell function in mice. I. Immunosuppression of transplantation responses.
Authors: Hirsch R , Eckhaus M , Auchincloss H , Sachs D H , Bluestone J A
Published: December 2022

Anti-T3 mAb are being increasingly used clinically in the treatment of organ graft rejection. However, there has not previously been a murine model in which the effects of these mAb on the immune system could be studied in vivo. We have established such a model using the anti-murine-T3 mAb, 145-2C11. Administration of 145-2C11 led to rapid depletion of T cells from peripheral blood and suppression of skin graft rejection. However, depletion of T cells from spleen and lymph node was both delayed and incomplete. Full recovery of T cell number was dependent on the presence of a thymus, but treatment of thymectomized animals revealed that depletion was not the mechanism by which the mAb induced immunosuppression. Rather, alterations in TCR expression may play a more important role. TCR had modulated from T cells in spleen and lymph node early after treatment, and TCR expression remained subnormal for at least 51 days posttreatment. However, subnormal TCR expression alone could not fully explain the observed T cell dysfunction, inasmuch as a period of time existed after TCR re-expression during which T cells appeared to be anergic to CTL and MLR reactivity. These findings implicate T cell dysfunction as an important element in the induction of immunosuppression after anti-T3 administration.
Mechanism involved in the mobilization of neutrophil calcium by 5-hydroxyeicosatetraenoate.
Authors: O'Flaherty J T , Jacobson D , Redman J
Published: December 2022

5-Hydroxyeicosatetraenoate (5-HETE), like leukotriene B4 and platelet-activating factor, stimulated human polymorphonuclear neutrophils to mobilize intracellular calcium. The three compounds acted through mechanisms that were inhibited by pertussis toxin, cholera toxin, and PMA. Each agonist, furthermore, desensitized (or down-regulated) the neutrophil's calcium mobilization response to a second challenge with the same agonist. However, 5-HETE and leukotriene B4 had little or no activity in cross-desensitizing neutrophil responses to each other or to platelet-activating factor. Furthermore, 5-HETE interfered minimally or not at all with the binding of radiolabeled leukotriene B4 and platelet-activating factor to their respective receptors on neutrophils. Thus, 5-HETE mobilizes neutrophil calcium by a mechanism different from those used by leukotriene B4 and platelet-activating factor. This mechanism appears to involve specific 5-HETE receptors that couple to pertussis toxin-inhibitable, GTP-binding proteins.
Inhibition of macrophage tumoricidal activity by glucocorticoids.
Authors: Hogan M M , Vogel S N
Published: December 2022

In this study, the effect of corticosteroids on the activation of macrophages to a fully tumoricidal state was examined. Thioglycolate-elicited peritoneal exudate macrophages from C3H/HeJ mice were rendered cytolytic for P815 mastocytoma cells in a two-signal tumoricidal assay that used recombinant interferon-gamma (rIFN-gamma; 1 to 10 U/ml) as a "priming" signal and butanol-extracted lipopolysaccharide (But-LPS; 0.1 to 5 micrograms/ml) as a "trigger" signal. Treatment of macrophages with either rIFN-gamma alone or But-LPS alone failed to result in significant cytolytic ability. Tumoricidal activity was markedly inhibited in a dose-dependent fashion when glucocorticoids were added simultaneously to the cultures with rIFN-gamma and But-LPS at concentrations ranging from 1 X 10(-10) to 1 X 10(-5) M. Nonglucocorticoid sex hormones failed to inhibit tumoricidal activity in this system under identical culture conditions. Inhibition was most effective if the glucocorticoids were added simultaneously with the priming and triggering signals (rIFN-gamma and But-LPS); however, if the glucocorticoids were added 24 hr after the signals were provided to the cultures, suboptimal inhibition was observed. Experiments that dissociated the priming phase of activation from the triggering phase showed that glucocorticoids inhibited both the rIFN-gamma-induced priming stage as well as the But-LPS-induced triggering stage of activation. These observations provide evidence that glucocorticoids, but not other steroid hormones, inhibit the activation of macrophages to a fully tumoricidal state by interfering with either the priming or triggering signals in this two-signal model of macrophage activation.
IgA:IgM and IgA:IgA hybrid hybridomas secrete heteropolymeric immunoglobulins that are polyvalent and bispecific.
Authors: Urnovitz H B , Chang Y , Scott M , Fleischman J , Lynch R G
Published: December 2022

Polyvalent bispecific antibodies were secreted by hybrid hybridoma cells when both parental clones expressed a naturally polymerizing immunoglobulin. Hybrid hybridomas made from IgA lambda 2 anti-trinitrophenyl (TNP) and IgA kappa anti-phosphocholine (PC) parental cells secreted polymeric IgA antibodies that bound both TNP and PC. Some of the TNP binding was dissociated from the PC binding under conditions of mild reduction and alkylation suggesting that the bispecific polymeric IgA contained disulfide-linked parental monomers as well as bispecific hybrid monomers. Hybrid hybridomas constructed from IgA lambda 2 anti-TNP and IgM kappa anti-ox erythrocyte parental cells secreted bispecific, polymeric immunoglobulin that contained mu-, alpha-, kappa-, and lambda 2-chains. The mu and kappa-chains dissociated from the alpha- and lambda 2-chains under conditions of mild reduction and alkylation, indicating that both parental monomers had been incorporated into the same polymeric immunoglobulin to form a heteropolymeric antibody molecule. Heterologous pairing of alpha and mu heavy chains in monomers was not detected. Hybrid hybridomas constructed from IgA lambda 2 and IgG3 lambda 2 or IgA lambda 2 and IgG1 kappa parents co-secreted both parental immunoglobulins, but the antibodies secreted by these clones did not form heteropolymers or exhibit heterologous heavy chain pairing. These findings establish that polyvalent, bispecific, polymeric immunoglobulin molecules can be produced by hybrid hybridomas when both parents express a naturally polymerizing class of heavy chain but not when only one parent does. Hybrid hybridomas that produce heteropolymeric immunoglobulins are sources of high avidity bispecific antibodies that may find a number of basic and practical applications. The hybridoma cells that produce these antibodies may provide useful tools for investigating the in situ determinants of immunoglobulin chain association and the regulation of antibody assembly and secretion.
Motility of murine lymphocytes during transit through cell cycle. Analysis by a new in vitro assay.
Authors: Ratner S , Jasti R K , Heppner G H
Published: December 2022

The relationship between the basal (spontaneous) motility of murine lymphocytes and their position in the cell cycle was examined in a new collagen gel motility assay system. Concanavalin A-stimulated or control lymphocytes were allowed to locomote into slabs of type I collagen gel. The assay configuration permitted extraction of both total populations and locomotory subpopulations as viable, single-cell suspensions suitable for phenotypic and cell analysis. Concanavalin A stimulation resulted in a significant increase in the mean distance traveled by the leading cell front in 4 hr, from 23 microns (controls) to 67 microns. The estimated percentage of motile cells increased from 0.9 to 2.8%. Similar increases were observed after 18 hr of locomotion. The SIg+, Thy-1+, L3T4+, and Ly-2+ subsets exhibited equivalent increases in motility. Total populations and locomotory subpopulations were allowed to incorporate 5-bromo-2'-deoxyuridine, and their cell cycle profiles were compared by dual parameter anti-5-bromo-2'-deoxyuridine, propidium iodide fluorescence analysis. Total population and locomotory subpopulations did not differ significantly with respect to the ratio G0/G1:S, indicating that lymphocytes in these two phases exhibited approximately equal motility. Cells in late S and G2 + M were significantly less motile; locomotory subpopulations contained 60 to 75% fewer G2 + M cells than the total populations from which they were derived. Taken together, the results indicate that the concanavalin A-induced increase in motility commences before S phase and that motility diminishes shortly before or during G2 + M.
Methylation patterns of the T cell receptor beta-chain gene in T cells, large granular lymphocytes, B cells, and monocytes.
Authors: Sakamoto S , Ortaldo J R , Young H A
Published: December 2022

We evaluated the methylation state of the T cell receptor beta-chain gene (T beta) DNA of T cells, large granular lymphocytes (LGL), B cells, and monocytes to determine whether methylation can be correlated with the reported transcriptional activity of this gene and whether this methylation pattern can be used as a marker for different leukocyte populations. By using the restriction enzyme isoschizomers, MspI and HpaII, we found that T beta of T cells was highly unmethylated, the gene in B cells and monocytes is highly methylated, and the methylation state of T beta of LGL is intermediate between that of T cells and B cells or monocytes. These results indicate that the methylation pattern of T beta can be a marker for T cells and LGL. The difference in methylation pattern of T beta between LGL and T cells indicated that most LGL were different from T cell populations. The methylation pattern of T beta in LGL DNA was also heterogeneous, in contrast to that of other leukocytes, suggesting that human peripheral blood LGL include several cell subsets. Only T cells, which synthesize full length 1.3-kb transcripts of T beta, have almost completely unmethylated CCGG sequences, indicating a good correlation between the reported level of 1.3-kb mRNA transcription from T beta and hypomethylation of this gene.
Expression and the functional role of a p70/75 interleukin 2-binding molecule in human B cell.
Authors: Saiki O , Tanaka T , Doi S , Kishimoto S
Published: December 2022

Expression of p70/75 IL-2-binding molecules and their functional roles in induction of Ig secretion by IL-2 were examined in human B cells. IL-2, at high concentrations induced higher levels of Ig secretion in Staphylococcus aureus strain Cowan I (SAC)-activated B cells than at low concentrations. About 50% of SAC-activated B cells, lacking Tac antigen, were also responsive to Ig secretion by IL-2, although the required dose of IL-2 was higher than that for Tac-positive B cells. H-31 antibody which recognizes Tac antigen did not inhibit the induction of Ig secretion by high concentrations of IL-2 in both Tac-negative and Tac-positive B cells, suggesting that IL-2 might induce Ig secretion through a receptor distinct from Tac antigens. In contrast, IL-2 was ineffective in the absence of SAC stimulation even at high concentrations. Upon analysis by SDS-PAGE, p70/75 IL-2-binding molecules were detected on Tac-negative SAC-activated B cells. Similar IL-2-binding molecules distinct from Tac antigen (p55) were detected in both Tac-positive B and T cells. However, neither p55 nor p70/75 IL-2-binding molecules could be detected in the absence of SAC stimulation. These observations suggest that p70/75 IL-2 binding molecules are induced in human B-cells in the presence or absence of Tac antigen by SAC stimulation and these determinants play an important function in the transduction of IL-2 associated signal for B cell differentiation.
Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions.
Authors: Di Virgilio F , Steinberg T H , Swanson J A , Silverstein S C
Published: December 2022

Fura-2, loaded into J774.2 macrophages as the acetoxymethyl ester, is sequestered into intracellular vacuoles within 90 min after the beginning of the loading at 37 degrees C. The dye is also efficiently secreted from the cells. Sequestration and secretion of fura-2 reduce the accuracy of measurements of cytosolic free Ca2+ concentration in this cell line. Fura-2 is also sequestered and secreted by J774.2 when the dye is loaded into the cytoplasm as the pentapotassium salt by reversible permeabilization of the plasma membrane. Regardless of the mechanism by which fura-2 is loaded into the cytoplasm, both sequestration and secretion are prevented by 2.5 mM probenecid, a blocker of organic anion transport. Probenecid has no effect on resting or stimulated cytosolic free Ca2+ levels or on FcR-mediated phagocytosis. These findings suggest that macrophages express a transport mechanism for the anionic form of fura-2. This transport system is responsible for the clearance of fura-2 from the cytoplasm of this cell type. Furthermore we suggest that use of probenecid to block secretion and intracellular sequestration of fura-2 may overcome problems arising in the application of this Ca2+ indicator to macrophages and perhaps to other cell types.
Effects of murine recombinant interleukin 1 alpha on the host response to bacterial infection.
Authors: Czuprynski C J , Brown J F , Young K M , Cooley A J , Kurtz R S
Published: December 2022

The effects of exogenously administered rIL-1 alpha on elimination of viable listeriae from the liver and spleen during the course of a primary Listeria monocytogenes infection was studied. Similar numbers of L. monocytogenes were recovered from rIL-1 alpha-treated and control mice at up to 24 h after infection; however, by 48 h after infection more than 1 log10 fewer viable L. monocytogenes were recovered from the spleens of rIL-1 alpha-treated mice than from Listeria-infected controls. The difference in bacterial burden between IL-1 alpha-treated and control mice increased with time; by 7 days after infection viable L. monocytogenes had been eliminated from most rIL-1 alpha-treated mice, whereas control mice still harbored 10(4) to 10(5) L. monocytogenes per spleen and liver. Histopathologic examination confirmed that rIL-1 alpha-treated mice suffered considerably less damage to the spleen, liver, lung, and brain than did control mice. To determine whether rIL-1 alpha-mediated protection indirectly by augmenting the release of other cytokines, we determined serum levels of colony-stimulating activity and IFN activity in rIL-1 alpha-treated and control Listeria-infected mice. Treatment with rIL-alpha elicited an early burst of serum colony-stimulating activity as compared with sera from Listeria-infected control mice. These data suggest that exogenous administration of rIL-1 initiates release of colony-stimulating activity, and perhaps other cytokines, that accelerate the protective response of the infected host. Prophylactic augmentation of antimicrobial resistance by administration of rIL-1 alpha may be worthy of further evaluation.
Down-regulation of cytotoxic T lymphocyte development by a minor stimulating locus-induced suppressor cascade that involves Lyt-1+ suppressor T cells, IA- macrophages, and their factors.
Authors: Chow K P , Battisto J R
Published: December 2022

Down-regulation of the development of CTL has been studied in mice both in vivo and in vitro. To generate CTL to hapten-altered self Ag in vivo, an immunization protocol has been used in which the host's Th cells are stimulated by a minor locus histocompatibility Ag (Mlsd) and its precursor CTL are activated by trinitrophenylated syngeneic spleen cells. Injecting the H-2 compatible Mls-disparate spleen cells along with the TNP-coupled self cells into the hind paws causes TNP-self specific CTL to appear in popliteal lymph nodes within 5 days. We have previously reported that inducing Ts cells by i.v. injecting Mlsd-bearing cells prevents in vivo generation of TNP-self specific CTL after immunization in this way. Here the induced Ts cell as well as the mechanism by which it functions have been further examined. The suppression was seen to extend to allogeneic as well as TNP-self Ag, provided the Mlsd-tolerized animal was reexposed to Mlsd-bearing cells at the time of immunization for CTL. By transferring the Mlsd-induced suppression adoptively we have learned that the splenic suppressor cell bears Thy-1.2 as well as Lyt-1.1 Ag and inhibits the generation of CTL at the afferent limb. In addition, Mlsd-induced PEC of Mlsd-tolerized mice, but not of normal mice, mediated suppression of development of CTL in vivo. The active cells within the tolerized PEC have been identified as T cells and macrophages (M phi). Furthermore, PEC from mice tolerized to Mlsd suppressed generation of CTL directed toward TNP-self targets in vitro. T cells and M phi separated from PEC of Mlsd-tolerized mice achieved suppression best in culture when present together. In addition, Lyt-1+ splenic cells from tolerized but not normal mice cooperated to down-regulate CTL generation in vitro with peritoneal M phi from either tolerized or normal mice. Supernatants of 24- to 72-h cultures of PEC from tolerized mice were suppressive of CTL generation when incorporated at 40 to 50% of culture volume. Supernatants of T cells from tolerized PEC or spleen were suppressive in culture only when M phi from normal mice were also present. To achieve suppression dialyzed supernatants of M phi from tolerized mice could replace the M phi.(ABSTRACT TRUNCATED AT 400 WORDS)
IFN-gamma induces islet cell MHC antigens and enhances autoimmune, streptozotocin-induced diabetes in the mouse.
Authors: Campbell I L , Oxbrow L , Koulmanda M , Harrison L C
Published: December 2022

To explore the role of the lymphokine, IFN-gamma, in the development of autoimmune-mediated insulin-dependent diabetes, we examined the effects of systemically administered IFN-gamma on the clinical features and pancreatic immunohistology of CBA mice made diabetic with multiple low doses of the pancreatic islet beta-cell toxin, streptozotocin. Mice given streptozotocin and IFN-gamma were significantly more hyperglycemic than those given streptozotocin alone and had significantly decreased body weight. Mice given IFN-gamma alone did not differ in glycemia or weight from vehicle-injected mice. On day 11, Ia proteins were detected on islet cells from mice given streptozotocin and their expression was potentiated by IFN-gamma; they could not be detected on islet cells from mice given IFN-gamma alone or vehicle. H-2K protein expression was increased on islet cells from mice given streptozotocin and was potentiated by IFN-gamma. IFN-gamma alone also increased H-2K protein expression on islet cells compared with vehicle-treated mice. These findings show that IFN-gamma enhances the severity of diabetes in mice given multiple-low doses of streptozotocin, in association with enhanced expression of Ia and H-2K proteins on islet cells. They indicate an important role for IFN-gamma in amplifying the autoimmune process leading to beta-cell destruction in diabetes. The ability of IFN-gamma to worsen autoimmune disease has implications for its use in man.
Toxic shock syndrome toxin-1 induces inositol phospholipid turnover, protein kinase C translocation, and calcium mobilization in human T cells.
Authors: Chatila T , Wood N , Parsonnet J , Geha R S
Published: December 2022

Toxic shock syndrome toxin-1 (TSST-1) is a 22-kDa exotoxin produced by most Staphylococcus aureus strains responsible for toxic shock syndrome. TSST-1 is a mitogen for human T cells. The mechanism of T cell activation by TSST-1 was investigated. TSST-1 induced IL-2R expression, IL-2 synthesis, and proliferation in T cells in a monocyte-dependent fashion. Neither IL-1 nor IL-2, alone or in combination, substituted for monocytes in supporting TSST-1-induced mitogenesis. We investigated the mechanism by which TSST-1 induces initogenesis. TSST-1 failed to induce ADP-ribosylation of T cell membrane proteins. However, the toxin induced transient translocation of protein kinase C from cytosol to plasma membranes and also induced the mobilization of cellular Ca2+ stores in both PBMC and the Jurkat human tumor T cell line, suggesting that TSST-1 triggered inositol phospholipid turnover. This was directly demonstrated to be the case in both cellular preparations studied. TSST-1 induced the increased synthesis of the inositol phospholipid phosphatidyl inositol, phosphatidyl inositol-4 phosphate, and phosphoinositol inositol-4,5-bisphosphate, and induced the breakdown of inositol phospholipid as evidence by the accumulation of phosphatidic acid and inositol phosphates. We conclude that the action of TSST-1 involves the induction of inositol phospholipid turnover, protein kinase C activation, and mobilization of cellular Ca2+ stores. This effect is similar to that of mitogenic lectins and of anti-CD3 antibodies.
Role of T cells in regulating expression of the B cell repertoire. Anti-ssDNA precursor frequency of DBA/2 B cells is increased in the presence of T cells from NZB mice.
Authors: Cowdery J S , Pitts A K
Published: December 2022

Splenic B cells from DBA/2 and NZB mice were compared with regard to precursor frequency of anti-ssDNA-producing cells. Using a modification of the splenic fragment assay, we show that NZB T cells are capable of increasing the frequency of expression of anti-ssDNA precursors in DBA/2 splenic B cells. When limiting numbers of splenic B cells of DBA/2 origin were adoptively transferred into an irradiated (1200 rad) recipient, the co-transfer of NZB T cells markedly increased the frequency of anti-ssDNA precursors in cultured splenic fragments. The anti-ssDNA produced under these conditions was exclusively IgM and exhibited a high degree of cross-reactivity with TNP and fluorescein. Thus, the increase in anti-ssDNA precursor frequency reflected an expansion of the B cell repertoire to include precursors of polyspecific antibody-producing cells that under normal circumstances are not expressed. The ability of NZB T cells to increase the anti-ssDNA precursor frequency was further defined by the CBA/N immunodeficiency gene xid, in that B cells from DBA/2.xid donors did not exhibit increased anti-ssDNA precursor frequency in the presence of NZB T cells. When NZB splenic B cells were co-transferred with DBA/2 T cells, the anti-DNA precursor frequency of the NZB B cells was not reduced. This study demonstrates that T cells can influence the emergency of B cell clones in an Ag-nonspecific manner. The well documented in vivo spontaneous polyclonal activation of NZB B cells may be secondary to T cell-mediated expansion of the B cell repertoire.