bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of T cells on the expression of lymphokine-activated killer cell activity and in vivo tissue distribution.
Authors: Maghazachi A A , Goldfarb R H , Herberman R B
Published: December 2022

Lymphokine-activated killer (LAK) cells are currently being evaluated in several cancer centers for the immunotherapy of patients with a variety of cancers. Understanding the in vivo distribution of LAK cells should help to optimize their antitumor efficacy. As a model system to examine this issue, nylon wool column-passed rat lymphocytes were cultured in the presence of rIL-2 for 1 and 2 days. The resulting cells were divided into two populations; one that adhered to the plastic flasks and the second which did not adhere. The adherent cells were found to be highly cytotoxic against NK-sensitive and NK-resistant targets, whereas the nonadherent cells were unable to kill NK-resistant targets unless T cells were removed from this population. These results indicate that T cells present in IL-2 activated bulk splenocytes may interfere with the activity of LAK cells. Adherent or nonadherent LAK cells were evaluated for their pattern of in vivo distribution after i.v. inoculation. These cells were found to display a restricted pattern of distribution, localizing mainly in the lungs at 2 h after i.v. injection but redistributing into the liver and the spleen by 24 h. LAK cells were rarely recovered from the lymphoid tissues, including the peripheral lymph nodes and the mesenteric lymph nodes. However, if T cells were not removed from the LAK cell population, some radioactivity was recovered from the peripheral and mesenteric lymph nodes. Fractionation of 2 day-activated, nonadherent population on discontinuous Percoll resulted in the enrichment of large granular lymphocyte (LGL)/LAK activity in low density fractions (42% and 45% Percoll), whereas high density fraction (70% Percoll) contained T cells which showed no cytolytic activity. Upon transfer into syngeneic rats, the 42% fraction showed typical LAK migration. In contrast, the 70% fraction showed typical T cell migration. What is more important, removal of the granulated cells resulted in a population which have no granules and resemble large agranular lymphocytes known to be pre-LGL/LAK cells. Large agranular lymphocytes showed a pattern of distribution different from both T and LGL/LAK cells.
Tumor necrosis factor induction by Candida albicans from human natural killer cells and monocytes.
Authors: Djeu J Y , Blanchard D K , Richards A L , Friedman H
Published: December 2022

Other investigators have previously reported that TNF has been induced from macrophages by bacteria and, more recently, from NK cells by certain tumor cells. Sendai virus has also been reported to induce TNF from macrophages. We report here that an opportunistic fungi, Candida albicans, can also induce TNF, not only from human monocytes, but also from Percoll-fractionated large granular lymphocytes (LGL) which mediate NK function. Incubation of monocytes of LGL with C. albicans for 8 h was sufficient for detection of TNF release and peak induction was observed at 24 h. Induction of TNF from LGL did not require the participation of monocytes or T cells because treatment of the LGL with CD14 or CD15 to eliminate contaminating monocytes and CD3, CD4, or CD8 to eliminate contaminating T cells did not decrease the level of TNF produced from the treated LGL. Small T cells recovered from the denser fractions of the Percoll gradient had no ability to produce TNF, even when 10% monocytes were added to the T cells to provide accessory function. The phenotype of the TNF-producing LGL was CD2+, CD11+, CD16+, NKH1+, LEU7-. The TNF produced by both monocytes and LGL was neutralized by specific monoclonal and polyclonal anti-TNF but not by monoclonal antilymphotoxin. These results indicate that TNF production is a normal response of monocytes and LGL to stimulation by fungi such as C. albicans and that the release of TNF may be related to its ability to activate effector function to control Candida growth, which we have shown earlier for neutrophils with TNF.
Clonal analysis of autoantibody-producing cell precursors in the preimmune B cell repertoire.
Authors: McHeyzer-Williams M G , Nossal G J
Published: December 2022

Evidence for anti-self-reactivity in the preimmune B cell repertoire has been well documented. The present study aimed to determine the frequency of antibody-forming cell precursors in this repertoire whose Ig V regions impart reactivity to "self" or autologous Ag. Clones were activated in vitro with LPS and their secreted IgM antibody was assayed for reactivity by direct binding to cell surface or intracellular Ag. An IL-4-containing lymphokine mixture was added to the clonal cultures to induce the secretion of IgG1. The reactivity of secreted IgG1 with Ag would more closely resemble the binding required to activate B cells through their monomeric surface IgM and/or IgD. The results indicate a high frequency of precursors secreting IgM with reactivity to intracellular Ag, namely 1 in 37 +/- 6 B cells, with a marked paucity of response to cell surface molecules. The repertoire was markedly deficient in precursors secreting IgG1 able to bind to intracellular Ag, with only one clone detected by the screening of 3.0 x 10(6) spleen cells. No positives were detected for cell surface Ag. This suggested that the frequency of clones in the preimmune repertoire that express IgR with sufficient affinity to bind "self" molecules must be very low.
Helper T lymphocytes which recognize the MHC class I alloantigens in vivo are CD4+CD8-1.
Authors: Piguet P F
Published: December 2022

To determine the CD4 or CD8 phenotype of the Th lymphocyte which recognizes in vivo the MHC class I alloantigens, B10 recombinant mice were treated with anti-CD8 or anti-CD4 mAb and immunized with lymphoid cells from donors differing in the K or D region of the MHC. Alloantibodies were evaluated by a 51Cr-release assay or by indirect immunofluorescence. The production of IgG anti-Dd and anti-Kk alloantibodies was increased by the deletion of the CD8+ and absent in mice depleted of the CD4+ subset. These experiments indicate that the helper influence elicited by the recognition of a MHC class I alloantigen in vivo is due to cells of the CD4+CD8- phenotype.
IFN-gamma, tumor necrosis factor-alpha, and urokinase regulate the expression of urokinase receptors on human monocytes.
Authors: Kirchheimer J C , Nong Y H , Remold H G
Published: December 2022

The ability of macrophages to reach inflammatory loci is crucial in the function of cellular immunity. Invasive properties of macrophages may be due to the proteinase urokinase which binds to cell surface receptors, and thereby confers on macrophages the capacity for localized proteolysis of the interstitium. Here, we investigated the role of the macrophage-activating factors IFN-gamma, TNF-alpha, and granulocyte-macrophage-CSF and of urokinase on the expression of urokinase receptors by human cultured monocytes. IFN-gamma and TNF-alpha induced increased urokinase binding to human cultured monocytes in a time- and dose-dependent fashion. At optimal concentrations, IFN-gamma (200 U/ml) increased the number of receptors/cell from 14,000 to 64,000, TNF-alpha (50 U/ml) to 30,000, and combinations of IFN-gamma and TNF-alpha to 90,000. Granulocyte-macrophage-CSF had no effect. The enhanced urokinase binding is due to increased numbers of urokinase receptors and not an increased affinity of the receptor for urokinase. In the presence of urokinase during monocyte activation, IFN-gamma induced only 25,000 receptors/cell. However, urokinase does not inhibit increased receptor expression when the cells are activated with TNF-alpha. The effect of urokinase on induction of urokinase receptors by combinations of IFN-gamma and TNF-alpha varied with the dosage of TNF-alpha: A combination of IFN-gamma (200 U/ml) and TNF-alpha (15 U/ml) induced 38,000 receptors/cell in the presence and 90,000 receptors/cells in the absence of urokinase, whereas IFN-gamma (200 U/ml) and TNF-alpha (20 U/ml) induced 90,000 receptors/cell in the absence and presence of urokinase. These studies demonstrate that IFN-gamma, TNF-alpha, and urokinase collectively regulate the number of urokinase receptors on human monocytes. The induction of urokinase receptors may be responsible for increased invasiveness of the activated macrophage.
Functional analysis of mononuclear cells infiltrating into tumors. IV. Purification and functional characterization of cytotoxic cell-generating factor.
Authors: Murakami T , Uede T , Shijubo N , Torimoto Y , Ide T , Sugimura K , Azuma I , Takai Y , Kikuchi K
Published: December 2022

Rat cytotoxic cell-generating factor (CGF) was purified from cell-free supernatants of a T cell hybridoma (6B2-B8) that constitutively produces CGF. CGF activity was assessed by its ability to generate cytotoxic cells against 51Cr-labeled T-9 cells from spleen cells of T-9-immunized rats. The purification scheme consisted of ammonium sulfate precipitation, AcA 54 gel permeation, Mono Q anion exchange chromatography, Superose 12HR 10/30 gel permeation, SDS-PAGE with subsequent electroelution, and ProRPC HR5/10 reverse phase column chromatography. Overall, CGF was purified approximately 13,000-fold, with a maximum 2.5% recovery of activity, and the sp. act. of the purified CGF was approximately 19,000 U/mg. The purified CGF is distinct from the other lymphokines such as IL-1, IL-2, IL-3, IL-4, T cell-replacing factor/IL-5, IL-6, and IFN-gamma. It is capable of promoting the generation of cytotoxic T cells from R1-10B5 (+) spleen cells of T-9-immunized rats and also stimulates a W3/25 (+) T cell hybridoma to express the IL-2R. The CGF has an apparent m.w. of 28,000 under non-reducing and 14,000 and 16,000 under reducing conditions. 125I-labeled CGF binds to normal thymocytes as well as splenic T cells. The highest level of binding of CGF was detected on splenic T cells derived from T-9-immunized rats that were previously shown to contain CTL precursors. The binding analysis with 125I-labeled CGF demonstrated that CGF binds to a specific cell surface molecule with an approximate m.w. of 60,000 to 70,000.
Expression of the beta-subunit of the human leukocyte adherence receptor depends upon cell type and stage of differentiation.
Authors: Hickstein D D , Howard M , Meuller L , Hickey M J , Collins S J
Published: December 2022

Using a cDNA clone for the beta-subunit of the receptor on human leukocytes that mediates cellular adherence (CD18), we investigated the lineage specificity of beta-subunit mRNA expression in human hematopoietic cells. Relatively high levels of the beta-subunit mRNA transcript were detected in mature peripheral blood leukocytes, including granulocytes and both resting and PHA-activated T lymphocytes. In contrast, relatively low levels of this transcript were observed in EBV-transformed B cells, in the immature Jurkat T cell line, and in chronic myelogenous leukemia myeloblasts and lymphoblasts. The beta-transcript was undetectable in the K562 chronic myelogenous leukemia blast crisis cell line with erythroblastic characteristics and in cultured skin fibroblasts. Two acute myeloid leukemia samples displayed unusually high levels of this transcript, comparable to levels observed in mature PBL. beta-subunit mRNA expression appeared to be primarily confined to leukocytes. In all cells examined the levels of surface beta-Ag expression paralleled levels of beta-mRNA.
CD15 monoclonal antibodies react with a phosphotyrosine-containing protein on the surface of human neutrophils.
Authors: Skubitz K M , Mendiola J R , Collett M S
Published: December 2022

mAb are useful as probes in the study of the roles of cell-surface components in neutrophil function. Many mAb that bind to human neutrophils react with the oligosaccharide lacto-N-fucopentaose III (CD15 antibodies). These antibodies, as well as several other widely used mAb reactive with human neutrophils, were employed to detect phosphoproteins present on these cells. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [gamma-32P]ATP revealed a 170 to 190-kDa phosphoprotein specifically reactive with CD15 antibodies. No phosphoproteins were immunoprecipitated by CD11 or CD18 mAb. Phosphoamino acid analysis of the 170- to 190-kDa protein showed that it contained predominantly phosphotyrosine and a low level of phosphoserine. Recently, it was shown that this phosphoprotein is one of the major substrates of ecto-protein kinase activity on human neutrophils. The roles for the 170- to 190-kDa phosphoprotein and the ecto-protein kinase in neutrophil function remain to be determined.
Murine immune response to the Neisseria meningitidis group C capsular polysaccharide. II. Specificity.
Authors: Rubinstein L J , Stein K E
Published: December 2022

As a means of further understanding the regulation of diversity and the development of protective immunity to the Neisseria meningitidis group C capsular polysaccharide (MCPS), we have generated and characterized, in detail, a panel of mAb against MCPS, a homopolymer of alpha(2----9)-sialic acid. Whereas the serum response to MCPS is restricted to the IgM and IgG3 isotypes, the panel of mAb includes, in addition, both IgG1 and IgG2b. Among 15 mAb of four isotypes, seven different specificities were observed based on direct binding in a fluorescence ELISA and precipitation in gel. Although all the mAb were derived from mice immunized with bacteria encapsulated with the native MCPS (strain C11), only 7 of 15 reacted with MCPS alone. Seven of 15 reacted with a natural O-acetyl-negative variant (OAc-, strain MC19) polysaccharide as well as with MCPS. Five of these reacted as much as 3 logs better with OAc- than MCPS and the other two reacted better with MCPS than OAc-. One mAb appeared to be alpha(2----9)-linkage specific as it reacted not only with MCPS and OAc-, but also with the capsular polysaccharide of Escherichia coli K92, a polymer of sialic acid linked alternately alpha(2----8) and alpha(2----9). None of the mAb reacted with the capsular polysaccharide of E. coli K1, a homopolymer of alpha(2----8)-sialic acid. In general, there was a good correlation between the ability to precipitate Ag in gel and to agglutinate bacteria; however, 3 of 15 mAb, all IgG3, did not conform to this rule in that they precipitated Ag but did not agglutinate bacteria of the relevant capsular specificity. Antibodies of both IgM and IgG isotypes and of both major specificities, MCPS-specific and those binding MCPS and OAc-, were bactericidal for strain C11, whereas only those reactive with OAc- were able to kill strain MC19.
Blocking of lymphokine activated killer (LAK) cell mediated cytotoxicity by cell-sized beads bearing tumor cell proteins.
Authors: Chong A S , Hersh E M , Grimes W J
Published: December 2022

Lymphokine activated killer cells (LAK) have been demonstrated to be cytotoxic for a variety of tumor-derived cells. Little is known of the nature of the cell surface molecules that mediate LAK cell-target cell interactions. Reported here are studies designed to develop the methodology that can lead to the identification and characterization of tumor cell surface molecules recognized by LAK cells. Results from experiments involving the pre-treatment of LAK cells and target cells (51Cr-labeled target cells or cold-blocking cells) with trypsin, neuraminidase, or sodium periodate suggest that proteins on the surface of LAK cells specifically recognized trypsin-sensitive molecules on the tumor cell surface. We extracted tumor cell membranes with detergents, and incorporated membrane proteins together with phospholipids and cholesterol onto the surfaces of cell-sized hydrophobic beads. The resulting "pseudocytes" block LAK mediated killing of 51Cr-labeled targets. Trypsin pretreatment of these pseudocytes significantly reduced their blocking activity. These observations suggested that we have incorporated onto the surface of pseudocytes tumor-membrane derived molecules that are specifically recognized by LAK cells. When membrane proteins from LAK resistant PBMC were incorporated onto beads, the resulting pseudocytes did not block LAK mediated cytotoxicity. It is of interest that beads coated with membrane proteins from one tumor were able to reduce LAK cell lysis of a different tumor target. Our results are consistent with the possibility that each LAK cell is polyspecific or that the LAK cell recognizes a common marker on many tumors. The methodology using pseudocytes should allow the purification and characterization of target acceptor molecule(s) and permit us to distinguish between these possibilities.
Differentiation potential of 14-day fetal mouse thymocytes in organ culture. Analysis of CD4/CD8-defined single-positive and double-negative cells.
Authors: Ceredig R
Published: December 2022

A two-color flow cytometric analysis was carried out on 14-day fetal mouse thymus lobes maintained for various lengths of time in organ culture. Kinetic analysis showed that the proportions of CD4- and CD8-defined subpopulations changed with time. In particular, there appeared to be accumulation of J11d-, CD4/CD8 double-negative and J11d-, Ly-6A+ single-positive cells, a phenotype associated with "mature" cells. Gated two-color analysis of CD4/CD8 single-positive cells showed that they contained distinct subpopulations as defined by mAb to CD3, TCR (F23.1), Ly-6A, Ly-6C, LFA-1, and J11d. Thus, the precursor cells found in the 14-day fetal mouse thymus were capable of generating all the subpopulations of CD4/CD8-defined single-positive and double-negative cells seen in the normal adult mouse thymus.
Isotype, VH genes, and antigen-binding analysis of hybridomas from newborn normal BALB/c mice.
Authors: Glotz D , Sollazzo M , Riley S , Zanetti M
Published: December 2022

In this study we report on the characterization of a panel of 62 hybridomas generated by fusing unstimulated spleen cells from neonatal (less than 24 hr old) normal BALB/c mice with the non-secreting Sp 2/0 cell line. The vast majority (98%) of these hybridomas secreted Ig but only 20% produced IgM. The isotype of the remaining hybridomas was determined as being IgG2b. Interestingly, when splenocytes from 1-day-old mice were stimulated with LPS for 48 h prior to the fusion event, 84% of the hybridomas were secreting IgM. The hybridoma supernatants were screened either by ELISA or RIA for binding reactivity using a panel of 17 Ag, proportionally divided between self and non-self. A binding reactivity could be assigned in 44% of cases. Of these, 29% were monoreactive, i.e., reactivity occurred with one Ag only, while the remaining 15% were multireactive. The majority (21 of 27) of hybridomas with a defined reactivity were directed against self-Ag. These included autologous red blood cells, DNA, histone H1, thyroglobulin, and Ag of the cell surface of T cells. The frequency of utilization of VH genes was determined using DNA probes for eight VH gene families. While all VH gene families appeared to have been used, one, VH 7183, had a slight but significant (p less than 0.02) higher utilization than expected by random expression. The frequency of all the other VH gene families was not significantly different from random utilization. No correlation was found between Ag reactivity in the supernatants and the utilization of a particular VH gene family. These findings indicate that early in the ontogeny the predominant reactivity of B cells is for self-Ag and, unlike what it is commonly believed, the IgM isotype is not dominant within these endogenously activated B cells at this time of ontogeny when genes from all VH families are utilized.
T cell stimulation via CD2 molecules is regularly accompanied by an increase in cytoplasmic pH. Different effects of lectins and CD3 antibodies.
Authors: Fischer G F , Holter W , Majdic O , Cragoe E J , Knapp W
Published: December 2022

The stimulation of different cell types with growth factors is often accompanied by a rapid intracellular alkalinization. By using mitogenic lectins, cluster of differentiation (CD)2 and CD3 mAb, as stimuli, we studied early changes of the intracellular pH in the activation process of resting human PBL. We found increases in free cytoplasmic Ca2+ levels and DNA synthesis but no intracellular alkalinization in the early activation phase upon stimulation with the mitogenic lectins, Con A, and PHA. Similarly stimulation with CD3 mAb led in most instances to no detectable pH shifts. Only in 7 out of 30 experiments was CD3 mAb-induced alkalinization observed. In contrast, stimulation with mitogenic combinations of anti-CD2 mAb led in all instances to rapid and clear-cut intracellular pH shifts very similar to those observed upon stimulation with PMA. In medium lacking sodium bicarbonate the intracellular alkalinization via the CD2 structure could be blocked by the amiloride analogue 5-(N-methyl-N-isobutyl)amiloride (MIA), which indicates that this increase in pH is mediated by the amiloride-sensitive Na+/H+ antiporter. Blockade of this antiporter had no negative effect, however, on T cell proliferation as measured by thymidine incorporation. In contrast, significantly enhanced proliferation rates were observed after stimulation with mitogenic combinations of anti-CD2 antibodies in the presence of MIA. No such effect of MIA could be observed in lectin induced T cell stimulation. These findings indicate that stimulation of the Na+/H+ antiporter via the CD2 structure is neither a prerequisite for T cell proliferation nor does it promote T cell growth. It rather seems to function in a regulatory role. In its absence, superinduction of proliferation can be achieved.
Suppression of B cell function by methotrexate and trimetrexate. Evidence for inhibition of purine biosynthesis as a major mechanism of action.
Authors: Rosenthal G J , Weigand G W , Germolec D R , Blank J A , Luster M I
Published: December 2022

Methotrexate (MTX) is a widely used drug in the treatment of a variety of human neoplasms. Trimetrexate (TMQ) is a lipid-soluble quinazoline derivate of MTX that, unlike MTX, is not dependent upon membrane folate transport for cellular entry. A number of studies have demonstrated that MTX and, more recently, TMQ possess potent immunosuppressive properties. To examine the cellular events associated with the immunomodulatory effects of anti-folates on humoral immunity, a murine B cell maturation model was used. In vitro, MTX and TMQ reduced the number of antibody-forming cells to SRBC, as well as IgM production. B cells stimulated with anti-Ig demonstrated a dose-related suppression in [3H]UdR incorporation after addition of either drug, suggestive of a decrease in de novo DNA synthesis. B cell activation events preceding S phase were also suppressed by both anti-folates, as evidenced by inhibition of RNA synthesis. However, neither drug affected surface expression of Ia Ag nor inositol phosphate accumulation. Addition of TdR caused a slight non-significant increase in the antibody-forming cell response in the presence of 10(-7) M MTX. However, addition of hypoxanthine or adenine, but not guanine, resulted in complete restoration. Timed addition revealed that the ability of MTX to suppress antibody responses was diminished if added after 48 h of culture, similar to the reversal of this suppression mediated by hypoxanthine. Cell cycle analysis of LPS-stimulated B lymphocytes demonstrated that both drugs modulated events preceding, as well as during, the S phase. The present studies suggest that although drug-induced impairments in dTMP biosynthesis may be responsible for deficient lymphoid proliferation, anti-folate-induced impairment in purine biosynthesis is a major mechanism in anti-folate-induced suppression of humoral immunity.
Possible role of neuraminidase in activated T cells in the recognition of allogeneic Ia.
Authors: Taira S , Nariuchi H
Published: December 2022

In a primary MLR, predominant stimulators in spleen cells are adherent cells and not B cells, although B cells are one of the cell types expressing a large amount of Ia molecules. Our previous experiments showed that T cells treated with neuraminidase (Nase) responded to an allogeneic Ia on B cells. In our experiments, the relationship between the responsiveness to the allogeneic Ia molecules on B cells and Nase activity of T cells was examined. The results showed that T cells increased in Nase activity with the acquisition of the reactivity to Ia on B cells. T cells from normal mice increased in Nase activity after the incubation for 3 days or more in MLR, and these T cells responded to allogeneic Ia on B cells. However, T cells from mice genetically deficient in Nase responded poorly to the Ia on allogeneic B cells even after the incubation in MLR for 3 days. T cells incubated for 3 days in MLR decreased in electrophoretic mobility, indicating the decrease of net negative charge of the cells, and increased in their binding of peanut agglutinin which has been reported to bind to galactosyl residues exposed on T cell surface by removing sialic acids. These results suggest that Nase in T cells was activated by the cultivation in MLR for 3 days, and sialic acids of some molecules on T cell surface were removed by the enzyme and, in turn, T cells acquired the responsiveness to allogeneic B cells in a secondary MLR. Thus, Nase was suggested to play a regulatory role in the recognition of Ia molecules in T cells.
Direct identification of the murine IL-2 receptor p55-p75 heterodimer in the absence of IL-2.
Authors: Saragovi H , Malek T R
Published: December 2022

The proteins cross-linked to the IL-2R p55 subunit were biochemically compared in distinct cell populations that varied in their capacity to express high affinity IL-2R. We directly cross-linked p75 to p55 in the absence of IL-2 for the cell populations that bear only high affinity IL-2R. Furthermore, although no endogenous IL-2 production was detected, p75 was readily cross-linked to p55 for EL4J-3.4, a p55 transfectant of EL4 that bears high affinity IL-2R. These results strongly suggest that high affinity IL-2R exist as a preformed heterodimer of p55 and p75 which do not require IL-2 for their association. Furthermore, cross-linking of three other proteins of apparent Mr of 100,000, 135,000, and 180,000 to p55 was also seen, raising the possibility of a more complex subunit composition for the IL-2R.
Tumor necrosis factor and IL-1 associated with plasma membranes of activated human monocytes lyse monokine-sensitive but not monokine-resistant tumor cells whereas viable activated monocytes lyse both.
Authors: Ichinose Y , Bakouche O , Tsao J Y , Fidler I J
Published: December 2022

The purpose of our study was to determine some of the mechanisms involved in macrophage-mediated lysis of tumorigenic cells. A375 human melanoma cells (A375-R) resistant to lysis mediated by TNF and IL-1 were selected from the TNF- and IL-1-sensitive A375 parental melanoma cells subsequent to continuous (2 mo) exposure to rTNF. Peripheral blood monocytes isolated by centrifugal elutriation from healthy donors were incubated with rIFN-gamma and muramyl dipeptide, with a lipoprotein derived from Escherichia coli (CG-31362) or with LPS for 24 h. These activated monocytes lysed both the A375 (monokine-sensitive) and A375-R (monokine-resistant) melanoma cells. Activated tumoricidal macrophages fixed in 2% paraformaldehyde lysed only the TNF- and IL-1-sensitive A375 cells. These fixed monocytes contained both IL-1 and TNF activities as determined by D10 cell proliferation and L929 cytolysis assays, respectively. Nearly identical results were obtained with preparations of plasma membranes from activated human monocytes. Anti-IL-1 and/or anti-TNF sera neutralized the cytolysis of tumor cells mediated by free monokines, by fixed monocytes, or by plasma membrane preparations. In contrast, anti-TNF and/or anti-IL-1 sera did not inhibit tumor cell lysis by viable activated monocytes. We conclude that IL-1 and TNF molecules associated with the plasma membranes of activated monocytes mediate lysis of susceptible target cells. However, because activated monocytes lysed IL-1-and TNF-resistant target cells, molecules other than these monokines must also be involved in the antitumor activity of monocytes.
Synergistic and overlapping activities of tumor necrosis factor-alpha and IL-1.
Authors: Last-Barney K , Homon C A , Faanes R B , Merluzzi V J
Published: December 2022

TNF-alpha and IL-1 induce the production of PGE2 from human chondrosarcoma, fibrosarcoma, and carcinoma cell lines. When combined at sub-optimal concentrations, TNF-alpha and IL-1 synergistically stimulate PGE2 production. The synergy of TNF-alpha and IL-1 on the induction of PGE2 is partially neutralized by specific antibodies. In vitro, human rTNF-alpha is directly cytotoxic to several human transplantable tumor cell lines. These include a human carcinoma, human chondrosarcoma, and a human transformed fibroblast cell line. The cytotoxic effect of TNF-alpha was abrogated by a specific, neutralizing, polyclonal antibody. IL-1 had no direct cytotoxic effect on these cell lines; however, IL-1 enhanced the cytotoxic effects of TNF-alpha. The synergy of these two cytokines in the cytotoxic assay was neutralized by the addition of specific neutralizing antibodies.
The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage.
Authors: Haziot A , Chen S , Ferrero E , Low M G , Silber R , Goyert S M
Published: December 2022

CD14 is a myeloid differentiation Ag expressed primarily on peripheral blood monocytes and macrophages. Although its function is unknown, the CD14 gene maps to a region encoding several myeloid growth factors and receptors. Analysis of the CD14 protein sequence deduced from the cDNA shows that although the CD14 protein contains a characteristic leader peptide, it lacks a characteristic transmembrane region, suggesting that CD14 may be anchored to the membrane via glycosylphosphatidylinositol (PI). Treatment of monocytes as well as a CD14-expressing neuroglioma cell line with PI-phospholipase C removed CD14 from the cell surface. Furthermore, monocytes from a patient with paroxysmal nocturnal hemoglobinuria, a disease characterized by lack of expression of other PI-linked proteins, failed to express CD14. Interestingly, the CD14-expressing neuroglioma cell line, which had been transfected with a single CD14 cDNA, released a soluble form of CD14 into the supernatant. Soluble forms of CD14 have previously been observed in serum of normal individuals and in culture supernatants of CD14+ cells. Biosynthetic experiments reveal that this soluble form of CD14 (48 kDa), which is smaller than the form released from the membrane by PI-phospholipase C (53 kDa), does not contain ethanolamine, the first constitutent of the PI-anchoring system. These studies demonstrate that CD14 is a member of the family of PI-anchored proteins and suggest that soluble forms of CD14 represent molecules that completely lack the PI-anchoring system.
The C terminus of the anaphylatoxin C3a generated upon complement activation represents a neoantigenic determinant with diagnostic potential.
Authors: Burger R , Zilow G , Bader A , Friedlein A , Naser W
Published: December 2022

Activation of the C component C3 results in generation of the anaphylatoxin C3a. The C3a polypeptide chain consists of 77 amino acids. The active site of this potent mediator, which also has immunoregulatory function resides in its C terminus. This report demonstrates that the C terminus of C3a (C3a-desArg) exposed by proteolytic cleavage from C3 represents a neoantigenic determinant. Two mAb specific for this epitope were obtained after immunization with the synthetic octapeptide (OP) Arg-Ala-Ser-His-Leu-Gly-Leu-Ala [C3a(69-76)] coupled to the carrier keyhole limpet hemocyanin (KLH). These anti-C3a(69-76) antibodies (H453 and H454) reacted in an ELISA system with C3a and KLH-OP but not with C3 or with KLH alone. Free OP efficiently blocked binding of the antibodies to C3a, whereas binding of another anti-C3a mAb (H13) remained unaffected. In immunoblotting analysis, the anti-C3a(69-76) mAb reacted with purified C3a but failed to react with the denatured, noncleaved C3. A novel quantitative C3a-ELISA was established with the anti-C3a(69-76) mAb. It had a sensitivity in the nanogram range (1 to 5 ng/ml). The C3a determination was not impaired by the presence of high concentrations of C3. Therefore, C3 removal was not required in contrast to the previously described C3a assays. This C3a ELISA might facilitate clinical C3a quantitation, e.g., in samples from patients with adult respiratory distress syndrome. In these patients, C3a determination in the early phase of the disease is of diagnostic relevance and has prognostic value.
Heterogeneous nature of the acute phase response. Differential regulation of human serum amyloid A, C-reactive protein, and other acute phase proteins by cytokines in Hep 3B cells.
Authors: Ganapathi M K , Schultz D , Mackiewicz A , Samols D , Hu S I , Brabenec A , Macintyre S S , Kushner I
Published: December 2022

Because a number of different cytokines have been reported to regulate the synthesis of human, murine, and rat acute phase proteins (APP), we studied the effect of cytokines on production of several major human APP in a single system, the human hepatoma cell line Hep 3B. Conditioned medium (CM) prepared from human blood monocytes activated with LPS in the presence of dexamethasone led to substantial induction of serum amyloid A (SAA) and C-reactive protein (CRP) synthesis whereas the defined cytokines IL-1 beta, TNF alpha, and medium from a human keratinocyte cell line (COLO-16), containing hepatocyte-stimulating factor activity, failed to induce these two major APP. Induction of SAA and CRP was accompanied by an increase in concentration of their specific mRNA. Size fractionation of CM from activated monocytes by fast protein liquid chromatography indicated that SAA- and CRP-inducing activity eluted as a single peak with a Mr of approximately 18 kDa. alpha 1-Antitrypsin, which also failed to respond to IL-1 beta or TNF alpha, was induced by both CM and medium from COLO-16 cells. The induction of AT by CM was accompanied by an increase in specific mRNA. Induction of ceruloplasmin and alpha 1-antichymotrypsin and decrease in the synthesis of albumin was achieved by both CM and IL-1 beta. Ceruloplasmin and albumin responded in a comparable fashion to both TNF alpha and medium from COLO-16 cells; the response of ACT to these cytokines was not evaluated. These results indicate that human SAA and CRP are induced in Hep 3B cells by products of activated monocytes but not by IL-1 beta, TNF-alpha, or some hepatocyte-stimulating factor preparations and that a group of heterogeneous mechanisms are involved in the induction of the various human APP.
Exact definition of species-specific and cross-reactive epitopes of the 65-kilodalton protein of Mycobacterium leprae using synthetic peptides.
Authors: Anderson D C , Barry M E , Buchanan T M
Published: December 2022

With the use of solid phase synthesis of peptides corresponding to major and minor peaks in a Hopp-Woods hydrophilicity plot, the epitopes for 10 of 14 known different mAb to the Mycobacterium leprae 65-kDa protein, a prominent T and B cell Ag of this bacillus, have been located in the primary structure. Five epitopes have been precisely mapped by using the synthetic peptides in inhibition ELISA experiments, and five others have been located on peptides of 22 amino acids or less in length. The epitope of an important species-specific antibody, IIIE9, which may be useful for seriodiagnosis of leprosy, appears to be distinguished from the epitope of the antibody IVD2, widely cross-reactive among mycobacteria, not by its sequence, but only by its critical residues. All epitopes studied appear continuous insofar as can be determined by this approach.
Molecular cloning of bovine class I MHC cDNA.
Authors: Ennis P D , Jackson A P , Parham P
Published: December 2022

Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.
A chronic inflammatory response. Its role in supporting the development of c-myb and c-myc related promonocytic and monocytic tumors in BALB/c mice.
Authors: Wolff L , Mushinski J F , Shen-Ong G L , Morse H C
Published: December 2022

This study demonstrates that an inflammatory response caused by the injection of pristane into the peritoneal cavity of mice provides a useful system for rapid induction of myeloid tumors by retroviruses. Two such tumors, which developed in the peritoneal cavity with average latencies of 68 to 71 d and incidences of greater than 50%, are 1) the McML, mature monocyte-macrophage tumors induced by retroviral constructs containing exons 2 and 3 of c-myc cDNA, and 2) the MML, promonocytic tumors induced by Moloney murine leukemia virus infection and its integration into the c-myb locus. Development of both neoplasms is clearly dependent on the intense i.p. inflammatory response, inasmuch as mice given the viruses and not pristane fail to develop these tumors. Although both types of tumors appear in the peritoneal cavity, the MML tumors that arise by i.v. injection of Moloney murine leukemia virus may actually originate via infection, and perhaps transformation, of precursor hemopoietic cells outside the peritoneal cavity, followed by migration of the cells to the peritoneal cavity. This is suggested by the fact that i.v.v but not i.p. injection of virus is an efficient method of producing these particular myeloid tumors. Although both McML and MML tumors require the inflammatory environment for their development, treatment of mice with a nonsteroid anti-inflammatory drug, indomethacin, has no effect on McML monocyte/macrophage tumors but completely prevents the development of the MML promonocyte tumors.
Effects of transforming growth factor-beta on human lymphokine-activated killer cell precursors. Autocrine inhibition of cellular proliferation and differentiation to immune killer cells.
Authors: Kasid A , Bell G I , Director E P
Published: December 2022

With subpopulations of human lymphoid cells that were enriched for lymphokine-activated killer (LAK) cell precursors, studies were performed to examine the effects of transforming growth factor-beta (TGF-beta) on their IL-2-dependent growth and differentiation to killer cells. The majority of the LAK precursor cells appeared to reside in nonadherent, non-T, and non-B lymphocyte populations that expressed CD11 and CD16 Ag. These cells were induced to proliferate and become LAK cells by high concentrations of rIL-2 alone in the apparent absence of any prior activation with mitogen or Ag. The partially purified lymphocyte subpopulations generated varying but several-fold greater levels of LAK killing on a per cell basis than did unfractionated lymphocytes. The exogenous addition of TGF-beta to the LAK precursor cultures, markedly inhibited IL-2-stimulated growth as well as the development of LAK activity in a dose-dependent manner. The antimitotic effect of TGF-beta was reversible; inhibition of proliferation could be largely restored by increasing the concentration of IL-2 in culture. In contrast, TGF-beta inhibition of cytotoxicity was relatively independent of the concentration of IL-2. Further, LAK precursors constitutively expressed TGF-beta mRNA and high affinity receptor for TGF-beta. Activation of LAK precursors with IL-2 alone, resulted in a three- to fivefold up-regulation of intracellular TGF-beta mRNA and TGF-beta biologic activity secreted in the culture media. Furthermore, Northern blotting revealed that the resting LAK precursors did not express the Tac-mRNA. Receptor binding studies with 125I-IL-2 suggested the presence of a single class of IL-2R with an apparent Kd of intermediate range (beta-chain of IL-2R) on the unstimulated cells. Stimulation with high concentrations of Il-2 induced Tac-mRNA (both the 3.5- and 1.5-kb transcripts) and resulted in the expression of high affinity IL-2R (Kd approximately 10(-11) M) on these cells. Suppression of IL-2-dependent responses by TGF-beta was accompanied by a selective down-regulation of the 1.5-kb Tac-mRNA as well as by reduction in high affinity IL-2R. The results suggest a negative autocrine control of TGF-beta on IL-2-dependent growth and differentiation of human LAK cells, possibly related to regulate the killer activation function.
Thymocyte subpopulations during early fetal development in the BALB/c mouse.
Authors: Husmann L A , Shimonkevitz R P , Crispe I N , Bevan M J
Published: December 2022

Phenotypic analysis of thymocytes during murine fetal development may be of use in determining the pathways of thymocyte differentiation. The expression of the functionally significant molecules Lyt-2 (CD8), L3T4 (CD4), and the TCR has already been described. However, mAb specific for several other murine lymphocyte surface markers are now available and, although these have been used to characterize adult thymocytes, a detailed analysis of fetal thymocytes with these antibodies has not previously been undertaken. In this study, we have used mAb specific for Thy-1, J11d, Pgp-1, and the IL-2R, in addition to those for Lyt-2 and L3T4, to identify subpopulations of early fetal thymocytes. By using two-color flow cytometric analysis of cells obtained from fetal thymuses on sequential days of gestation, we have been able to follow the development of various subpopulations through early fetal ontogeny. Our data indicate that the earlier thymocytes are found in the J11d+/Pgp-1+ subset which is abundant at fetal day 14 but constitute a numerical minority by day 16.
Augmentation by anti-T3 antibody of the lymphokine-activated killer cell-mediated cytotoxicity.
Authors: Ting C C , Hargrove M E , Yun Y S
Published: December 2022

This study showed that a mAb (145-2C11) against the T3 epsilon-chain of the TCR complex augmented the cytotoxic activity of the lymphokine-activated killer (LAK) effectors. The LAK cells were induced by culturing normal spleen cells with purified human rIL-2. Adding alpha T3 at the effector phase of the cytotoxic reactions augmented the LAK-mediated cytotoxicity. The alpha T3-augmented LAK killing was seen only with tumor targets, and there was no increase of killing against Con A-induced lymphoblasts. The augmentation effect was dose dependent on both the amounts of alpha T3 and the number of LAK cells added. A very low concentration of alpha T3 (1/10,000 dilution of culture supernatants) was sufficient to induce alpha T3-augmented LAK-mediated cytotoxicity. Human rIL-2 at 10 to 30 U/ml was sufficient to generate LAK cells for maximal alpha T3 augmentation, whereas 300 to 1000 U/ml of IL-2 were needed to generate maximal LAK activity when tested in the absence of alpha T3. LAK cells generated for longer periods of time showed a progressive increase of alpha T3-augmented cytotoxicity. For some targets, the alpha T3-augmented LAK killing was FcR dependent as evidenced by the ability of alpha FcR mAb 2.4G2 to inhibit, and for others it was not inhibited. The alpha T3-augmented killing did not correlate with the FcR expression on target cells as defined by 2.4G2. The LAK cells were both Lyt-2+ and Lyt-2-, but the LAK cells involved in alpha T3-augmented killing were exclusively Lyt-2+. Preincubation of LAK cells with alpha T3, but not preincubation of targets with alpha T3, resulted in augmented killing suggesting that the alpha T3 effect was unrelated to an antibody-dependent cell-mediated cytotoxicity. Our findings indicate that alpha T3 is a potent reagent to augment the cytotoxic reaction of LAK cells. These results suggested that a relationship might exist between the T3 complex and the cytotoxic activity of a subpopulation of Lyt-2+ LAK cells.
Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes.
Authors: Gromkowski S H , Brown T C , Masson D , Tschopp J
Published: December 2022

It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed.
Massive proteinuria induced in rats by a single intravenous injection of a monoclonal antibody.
Authors: Orikasa M , Matsui K , Oite T , Shimizu F
Published: December 2022

A single i.v. injection of a mAb 5-1-6 to rats was found to cause massive though transient proteinuria. This mAb 5-1-6, IgG1 was produced by immunization of BALB/c mice with collagenase-treated Wistar rat glomeruli and was highly organ and species specific. Immunoelectron microscopy using immunoperoxidase with the avidin-biotin complex and immunogold staining indicated mAb 5-1-6 to bind in vitro to the surface of glomerular epithelial foot processes, mainly to slit diaphragms. The recognized antigenic molecule was not susceptible to neuraminidase treatment and its Mr was about 51 kDa by immunoprecipitation. A one-shot i.v. injection of this mAb induced proteinuria in rats starting immediately, reaching the peak on day 8 (mean value of 150 mg/24 h), then gradually decreasing to normal level on day 18. The in vivo localization of administrated mAb 5-1-6 changed with time. Linear binding along glomerular capillary walls was observed 2 h after injection. However, 3 days later, it partially shifted to a fine granular pattern. The linear pattern disappeared and the size as well as intensity of the fluorescent granules decreased on day 12 to trace positive on day 18. Immunoelectron microscopy revealed the binding pattern of in vivo injected mAb 5-1-6 after 2 h to be similar to that in vitro. Three days later, injected mAb was observed within multivesicular bodies in glomerular epithelial cells as well as along the surface of foot processes and around slit diaphragms. Twelve days after injection, mAb along the surface of the foot processes and around slit diaphragms decreased but those in multivesicular bodies were observed more frequently. Rat IgG and C3 could not be detected throughout the period of observation. No histologic abnormalities were noted except for partial retraction of epithelial foot processes at the peak of proteinuria on day 8. This mAb thus provides a valuable means for examining the mechanism of proteinuria.