bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytotoxic effector function of B lymphoblasts.
Authors: Bersani L , Colotta F , Peri G , Mantovani A
Published: December 2022

EBV-transformed B lymphoblastoid cell lines and clones were cytotoxic in vitro against Actinomycin D-pretreated WEHI 164 sarcoma cells, a system in which mononuclear phagocytes were previously identified as effectors. Similarly, unlike resting B cells, normal B lymphoblasts stimulated for 72 hr with Staphylococcus aureus Cowan I and purified according to the expression of the B cell surface marker B1, demonstrated appreciable cytolytic activity. B lymphoblastoid cells and derived supernatants were cytotoxic for Actinomycin D-pretreated WEHI 164 cells, and killing was inhibited by an anti-lymphotoxin but not an anti-tumor necrosis factor antiserum. Thus, B lymphoblasts have cytotoxic potential, mediated by lymphotoxin or a lymphotoxin-like soluble product.
Detection of receptors for murine B cell stimulatory factor 1 (BSF1): presence of functional receptors on CBA/N splenic B cells.
Authors: Nakajima K , Hirano T , Koyama K , Kishimoto T
Published: December 2022

The presence of receptors specific for murine B cell stimulatory factor 1 (BSF1) was demonstrated by utilizing an internally radiolabeled recombinant BSF1. Radiolabeled BSF1 was efficiently produced in Xenopus laevis oocytes injected with a cloned mRNA for BSF1 and 35S-methionine. The labeled BSF1 specifically bound to splenic B cells. A Scatchard analysis indicated the existence of one class of receptor sites. BSF1 receptors were found to be distributed on a wide range of hematopoietic lineage cells, including B cells, T cells, macrophages, and mast cells. B cells from CBA/N mice with the xid gene defect had a similar level of BSF1 binding capacity compared with BALB/c strain B cells, and responded well to insoluble anti-Ig and BSF1 in proliferation assays, indicating that CBA/N B cells express functional BSF1 receptors at normal levels. Pre-B cell lines showed low levels of BSF1 binding, suggesting that cells in the B cell lineage acquire BSF1 responsiveness early in development.
Interferon-alpha, -beta, and -gamma augment the levels of rRNA precursors in peritoneal macrophages but not in macrophage cell lines and fibroblasts.
Authors: Radzioch D , Clayton M , Varesio L
Published: December 2022

We have studied the ribosomal RNA content in murine peritoneal macrophages activated to cytotoxicity with interferon-alpha (IFN-alpha), interferon-beta (IFN-beta), and interferon-gamma (IFN-gamma). Total RNA was purified from IFN-alpha, -beta, or -gamma activated macrophages, and Northern blot analysis was performed by using subcloned fragments specific for precursor rRNA (pre-rRNA) as probes. All types of interferon (alpha, beta, gamma) at the doses normalized for induction of cytotoxic activity caused accumulation of 45S, 41S, and 36S pre-rRNA. In contrast, the levels of 32S pre-RNA and 28S, 5.8S, and 18S mature rRNA were not affected by IFN treatment. Therefore, the accumulation of pre-rRNA in macrophages activated to cytotoxicity by IFN was not paralleled by changes in total content of mature rRNA. No accumulation of pre-rRNA upon IFN treatment was found in a murine fibroblast cell line (L929 cells) or in a macrophage cell line (GG2EE) immortalized from bone marrow of C3H/HeJ mice. Neither of those cell lines become cytotoxic in response to IFN. Overall, our data support the concept that IFN acts selectively on the mechanism controlling the levels of some pre-rRNA and that the mechanism of IFN action involves the post-transcriptional control of ribosomal gene expression.
Purification and NH2-terminal sequence of a plasmacytoma growth factor derived from the murine macrophage cell line P388D1.
Authors: Nordan R P , Pumphrey J G , Rudikoff S
Published: December 2022

Plasmacytoma growth factor (PCT-GF), a putative macrophage-derived lymphokine essential for the in vitro viability and proliferation of early generation plasmacytomas, was purified from conditioned medium of the murine macrophage cell line P388D1. The purification of PCT-GF was accomplished by a batch concentration on trimethylsilyl-controlled pore glass beads, followed by: gel filtration chromatography; hydrophobic interaction HPLC; and reverse-phase HPLC. SDS-PAGE analysis of the purified PCT-GF revealed a single band of Mr 23,000. The amino terminal sequence of PCT-GF was established as NH2-Pro-Thr-Ser-Gln-Val-Arg-Arg-Gly-Asp-Phe-Thr-Glu-Asp-Thr-Thr-Pro-Asn- Arg-Pro-Val-Tyr-Thr. No significant homology was found between this sequence and proteins in the National Biomedical Research Foundation database, suggesting that PCT-GF is a new lymphokine unrelated to previously described growth and differentiation factors.
Fine specificity of HLA-B27 cellular allorecognition. HLA-B27f is a functional variant distinguishable by cytolytic T cell clones.
Authors: Aparicio P , Rojo S , Jaraquemada D , López de Castro J A
Published: December 2022

Three HLA-B27 allospecific cytolytic T lymphocyte (CTL) clones were isolated by limiting dilution of HLA-B27-negative responder cells stimulated with HLA-B27.1-positive lymphoblastoid cells. These clones displayed three distinct reaction patterns when tested for their lytic ability against target cells expressing various structurally defined HLA-B27 subtypes. One of the clones was specific for HLA-B27.1; a second CTL clone reacted only with B27.1 and, less efficiently, with B27.2; the third clone recognized both B27.1 and B27f targets but not cells expressing any other B27 subtype. These results indicate that HLA-B27f is a functional variant amenable to differential recognition by alloreactive CTL. A correlation of the structure of the HLA-B27 subtypes with the reactivity of these clones revealed that multiple B27-specific alloreactive CTL are activated against epitopes of the HLA-B27.1 molecule sharing common structural features. This illustrates the complexity and fine specificity of the allogeneic CTL response against class I HLA antigens and suggests that their immunodominant regions are those which are capable of eliciting a diverse polyclonal response against each of these regions, rather than inducing the selective expansion of a single T cell clone.
Relationship between human IgE-binding factors (IgE-BF) and lymphocyte receptors for IgE.
Authors: Nakajima T , Sarfati M , Delespesse G
Published: December 2022

This study indicates that human IgE-binding factors (IgE-BF) found in the cellfree culture supernatant (CSN) of Fc epsilon R-bearing B cells are breakdown products of the surface Fc epsilon R. This conclusion is suggested by the following observations. 1) Fc epsilon R and IgE-BF share several antigenic determinants as shown by immunoprecipitation with several Mab to Fc epsilon R (MabER) and SDS-PAGE analysis of the precipitates. 2) Upon incubation at 37 degrees C, normal tonsillar lymphocytes lose their Fc epsilon R and this is associated in a time-related manner with the release in the CSN of molecules reacting with two MabER. 3) Surface radioiodinated tonsillar lymphocytes or RPMI 8866 cells release labeled IgE-binding molecules displaying the same antigenic composition and the same migration on SDS-PAGE as purified IgE-BF. 4) Peptide mapping of highly purified IgE-BF and Fc epsilon R reveals the presence of several identical fragments after digestion with either alpha-chymotrypsin, trypsin, or papain. Moreover, papain digestion of the 25-27 kD IgE-BF and of the affinity-purified Fc epsilon R, generated a 15 kD fragment reacting with two MabER and that is known to bind IgE. Although these data strongly suggest that IgE-BF may be directly derived from cell surface IgE receptors, they do not exclude the possibility that some IgE-BF may also be secreted without being first anchored in the cell membrane.
Inhibition of cytolytic T lymphocyte maturation with ornithine, arginine, and putrescine.
Authors: Susskind B M , Chandrasekaran J
Published: December 2022

L-Ornithine was shown to inhibit the development of cytolytic T lymphocytes (CTL) in mixed lymphocyte cultures (MLC). Lymphokines were unable to reverse the suppressive effect, and cytotoxic activity was not revealed by coupling ornithine-inhibited MLC cells to target cells with phytohemagglutinin (PHA). If addition of ornithine to MLC were delayed, sensitivity of CTL to inhibition was reduced after 24 hr and lost by 48 hr. Suppression of CTL development was not due to a toxic effect. MLC washed free of ornithine after 3 days produced detectable cytolytic activity within 24 hr of secondary culture, and to the same degree as the uninhibited MLC control within 48 hr. Cytotoxic cells generated in secondary cultures were Lyt-2+, did not kill the natural killer-sensitive YAC-1 cell line, and were shown to be antigen-specific by virtue of the findings that cytolysis and cold target inhibition were observed only with cells carrying the original, inducing H-2 haplotype. Cytolysis of target cells by normal CTL effector cells was not inhibited by L-ornithine. MLC depleted of accessory cells so that CTL activation was dependent upon addition of lymphokines remained susceptible to inhibition by ornithine. Our findings indicate that in the ornithine-inhibited MLC, CTL precursors undergo clonal expansion, but their maturation is arrested at a precytolytic stage. L-Arginine and putrescine also suppressed generation of CTL in primary MLC, and cells recovered from arginine- and putrescine-inhibited MLC developed control levels of CTL within 48 hr of secondary culture. Inhibition by putrescine was observed in tissue culture medium supplemented with human serum but not with fetal calf serum, presumably due to the presence of diamine oxidase activity in fetal calf serum. Similar to ornithine, the suppressive effects of arginine and putrescine on T lymphocytes were apparently selective for CTL because they did not inhibit mitogen activation with concanavalin A or the production of interleukin 2 and interleukin 3. These findings are consistent with a hypothesis that the inhibitory effects of ornithine, arginine, and putrescine are mediated by polyamines, and exerted on the differentiative stage of CTL development.
Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. VI. Detection of a non-MHC gene(s) in the E alpha-bearing RIIIS mouse strain that is associated with a specific lack of T cell responses to the M. arthritidis soluble mitogen.
Authors: Cole B C , Tuller J W , Sullivan G J
Published: December 2022

Previous work using inbred, congenic and recombinant mouse strains showed a positive association with expression of E alpha and the ability of splenic cells to bind to and undergo proliferation in response to a T cell mitogen present in culture supernatants of Mycoplasma arthritidis (MAS). Studies described in the present manuscript confirm this association because lymphocytes from mice expressing H-2a, H-2d, H-2j, H-2k, H-2p, H-2u, and H-2v all of which possess E alpha responded to MAS, whereas those expressing H-2b, H-2f, H-2q, and H-2s, which lack E alpha, failed to respond. One exception was noted in that the inbred RIIIS mouse (H-2r) that expresses E alpha failed to respond to MAS but responded normally to concanavalin A, and phytohemagglutinin. In contrast, the congenic B10.RIII (H-2r) mouse did respond to MAS, suggesting the presence of an MAS nonresponsive, non-major histocompatibility complex (MHC) gene(s) in the RIIIS mouse. MAS nonresponsiveness in the RIIIS mouse was recessive because the lymphocytes from F1 crosses with responder B10.RIII (H2r) and C3H (H2k) mice responded to MAS. Analysis of (RIIIS X B10.RIII)F1 X RIIIS or B10.RIII parental test cross progeny confirmed that nonresponsiveness to MAS was associated with a recessive, non-MHC gene(s). Evidence was also found that a non-MHC, MAS-nonresponsive gene(s) is also present in the inbred SWR (H-2q) and SJL (H-2s) strains, because lymphocytes from F1 crosses between these strains and the RIIIS mouse failed to respond to MAS. Both RIIIS and B10.RIII splenic cells bound the mitogen in MAS to a similar degree, confirming the presence of the binding site in both mice. In contrast, C3H.SW (H-2b) splenic cells that do not express E alpha failed to bind the mitogen. The nonresponsiveness of RIIIS lymphocytes to MAS was exercised at the level of the T cell rather than the accessory cell. Thus RIIIS T cells failed to respond to MAS presented by RIIIS, B10.RIII, or (RIIIS X B10.RIII)F1 accessory cells. In contrast, B10.RIII and (RIIIS X B10.RIII)F1 T cells responded to MAS when presented by RIIIS, B10.RIII, or F1 accessory cells. Similar observations were made using SWR and SJL T cells, which failed to respond to MAS irrespective of the source of accessory cells.(ABSTRACT TRUNCATED AT 400 WORDS)
T cell specificity for class II (I-A) and the encephalitogenic N-terminal epitope of the autoantigen myelin basic protein.
Authors: Zamvil S S , Mitchell D J , Moore A C , Schwarz A J , Stiefel W , Nelson P A , Rothbard J B , Steinman L
Published: December 2022

The role of class II restriction in T cell recognition of an epitope of the autoantigen myelin basic protein (MBP) has been investigated. Encephalitogenic PL/J(H-2u) and (PL/J X SJL/J(H-2s))F1 ((PLSJ)F1) clones, isolated after immunization with intact MBP, recognize the N-terminal 11 amino acid residues of MBP in association with I-Au class II molecules. The synthetic peptide MBP 1-11 has been tested in vivo for induction of EAE. Clinical and histological EAE occurs in PL/J and (PLSJ)F1 mice but not SJL/J. The class II restriction of T cells primed with MBP 1-11 has been examined in primary cultures in vitro. Similar to encephalitogenic T cell clones, isolated after continuous selection in vitro, the population of MBP 1-11-specific proliferative PL/J and (PLSJ)F1 T cells, recognize this epitope in association with I-Au class II molecules. Not all MBP-specific T cell clones which are restricted to I-Au class II molecules cause autoimmune encephalomyelitis. The specificity of these non-encephalitogenic clones has been examined in this report. These clones also recognize MBP 1-11. Thus recognition of an encephalitogenic T cell epitope is not sufficient for induction of EAE.
Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells.
Authors: Morrissey P J , Bressler L , Park L S , Alpert A , Gillis S
Published: December 2022

Purified, recombinant-derived murine granulocyte-monocyte colony-stimulating factor was found to enhance the primary in vitro immune response to SRBC by murine spleen cells. In determining the mechanism of this augmentation, it was found that only splenic adherent cells and neither resting nor activated T cells nor B cells expressed specific receptors for GM-CSF. When splenic adherent cells were pulsed briefly with GM-CSF before addition to macrophage-depleted cultures, they reconstituted the PFC response to a significantly greater degree than did control macrophages. Splenic adherent cells incubated overnight with SRBC plus GM-CSF were also more efficient antigen-presenting cells than splenic adherent cells incubated with antigen alone. The mechanism of this enhanced antigen presentation was found to be due to a GM-CSF-dependent increase in the level of IL 1 secretion and Ia antigen expression. Consistent with these data was the finding that GM-CSF augmented IL 2 production by splenic T cells in response to suboptimal concentrations of Con A. Finally, the day 5 in vivo antibody response (as measured by serum titers) of mice immunized with a low dose of SRBC was enhanced by two daily inoculations of GM-CSF. Thus, the role that GM-CSF plays in augmenting immune responses may not be solely accounted for by its ability to cause the proliferation or differentiation of macrophages, but more than likely includes its ability to enhance the function of antigen-presenting macrophages.
Interleukin 1 release by human monocytes treated with liposome-encapsulated lipopolysaccharide.
Authors: Bakouche O , Koff W C , Brown D C , Lachman L B
Published: December 2022

Liposome therapy offers a unique method of delivering antifungals, antiprotozoans, and macrophage-activating factors directly to reticuloendothelial cells. Although liposomes are entering clinical trials, their effect on Il 1 synthesis and release has yet to be determined. The beneficial, as well as possible pathological, effects of liposome therapy may be due to IL 1 released by reticuloendothelial cells. This study examined the effects of multilamellar phospholipid vesicles on the synthesis and release of IL 1 from human peripheral blood monocytes. Liposomes consisting of phosphatidylcholine and phosphatidylserine in molar ratio of 7:3 did not stimulate IL 1 release, and did not diminish the level of IL 1 release when monocytes were subsequently stimulated with LPS or Staphylococcus aureus. Surprisingly, LPS encapsulated in liposomes failed to stimulate IL 1 release from monocytes. This defect was limited to the release of IL 1, because monocytes stimulated with liposomes containing LPS had control levels of intracellular (cytosolic) and membrane IL 1. By contrast, LPS associated with lyophilized liposomes induced the secretion of IL 1 and behaved similarly to free LPS. These findings indicate that LPS activation of monocytes may involve not only the synthesis of IL 1 but activation of the secretory mechanism for IL 1, and that these two events could be independent, that the density of LPS molecules at the surface of the liposome may influence the degree of monocyte activation as measured by intracellular IL 1 synthesis, membrane accumulation, and secretion into the medium, and that intracellular lipids may not interfere with the secretory mechanism, because liposomes made with phospholipids differing in acyl chain length or the headgroup behaved identically.
Down regulation of the receptors for tumor necrosis factor by interleukin 1 and 4 beta-phorbol-12-myristate-13-acetate.
Authors: Holtmann H , Wallach D
Published: December 2022

Binding of radiolabeled tumor necrosis factor (TNF) to cell surface receptors was markedly reduced in human foreskin fibroblasts and cells from SV-80 and HeLa cell lines subsequent to treatment with interleukin 1 (IL-1) or 4 beta-phorbol-12-myristate-13-acetate (PMA). The decrease in TNF binding was initiated within minutes of application of IL-1 or PMA and could not be blocked by cycloheximide, suggesting that it is independent of protein synthesis. Scatchard plot analysis of TNF binding to the SV-80 cells indicated that its decrease in response to IL-1 and PMA reflects a reduced amount of TNF receptors, with no change in their affinity. IL-1 and PMA together had an additive effect on TNF binding. Treatment with TNF did not result in decreased binding of IL-1 to its receptors nor did TNF and IL-1 compete directly for their respective receptors. Human U937 cells on which receptors for IL-1 were below detectable levels exhibited no decrease in TNF binding when treated with IL-1, but did so in response to PMA. In addition to a decrease in TNF receptors, cells treated with IL-1 or PMA exhibited a lesser vulnerability to the cytolytic effect of TNF. The two kinds of changes were not completely correlated. A particularly notable dissimilarity was evident when comparing the rate of their reversal: the TNF receptor level was fully recovered within a few hours of removal of IL-1 or of the water-soluble analogue of PMA, 4 beta-phorbol-12,13-dibutyrate, from pretreated SV-80 cells; yet at that time resistance to the cytotoxicity of TNF was still prominent. These findings indicate that IL-1 as well as tumor-promoting phorbol diesters can down regulate cellular response to TNF by inducing a decrease in the number of receptors for TNF, and apparently through some other effect(s) as well.
The murine lymphocyte receptor for IgE. V. Biosynthesis, transport, and maturation of the B cell Fc epsilon receptor.
Authors: Keegan A D , Conrad D H
Published: December 2022

The post-translational processing and maturation of the receptor for IgE (Fc epsilon R) on murine hybridoma B cells were studied to determine the carbohydrate content and the importance of processing events in cell surface expression and ligand (IgE) binding ability. Endo and exoglycosidase treatment demonstrated that the mature receptor is composed of two to three complex-type N-linked oligosaccharides and contains sialic acid. Pulse-chase experiments indicated that the receptor is synthesized as a 44,000 dalton precursor that begins to be processed by 1 hr to the mature 49,000 dalton form, and the latter is expressed at the cell surface by 2 hr. It was determined that the processing included the conversion of N-linked oligosaccharides to the complex type as well as an additional processing event, because in the presence of tunicamycin, the receptor is synthesized as a 36,000 dalton precursor that is processed to a 38,000 dalton species. Analysis of the effects of tunicamycin treatment and endo F digestion on soluble Fc epsilon R isolated from cell supernatants demonstrated the existence of several m.w. species of Fc epsilon R fragments, and indicated that only the higher m.w. fragments were N-glycosylated. The use of several inhibitors of the N-linked carbohydrate processing pathway demonstrated that the addition of core N-linked side-chains, but not their processing to the complex type, is required for cell surface expression of Fc epsilon R. Also, processing of N-linked carbohydrate is not required for ligand binding activity. Finally, IgE affinity chromatography indicated that the 49,000 and 38,000 dalton (tunicamycin) Fc epsilon R bind IgE more effectively than their precursor forms, 44,000 and 36,000 daltons, respectively, indicating that a processing event independent of N-linked glycosylation is necessary for optimal ligand binding activity.
Species-specific antibodies to Leishmania tropica (minor) recognize somatic antigens and exometabolites.
Authors: Jaffe C L , Sarfstein R
Published: December 2022

Monoclonal antibodies were raised against membranes of Leishmania tropica (formerly L. tropica minor). These antibodies do not react with membranes or homogenates prepared from any other species of Leishmania examined. The antigens identified by all four monoclonal antibodies reside on the external surface of the promastigote and are released into the culture medium. Immunoprecipitation of metabolically ([3H]glucose, -glucosamine, or [35S]methionine)-labeled, [125I]lactoperoxidase-labeled, or Western blot analysis of the parasites identified species-specific determinants residing on several different molecules with Mr between 12,000 and 140,000. All of the monoclonal antibodies appear to recognize determinants on a common [3H]sugar-labeled glycoconjugate, Mr = 32,000 to 44,000, but also react with additional components of 55,000, 80,000, 92,000, and 130,000 m.w. Antibody binding to the parasites is very sensitive to mild oxidation with periodate, suggesting that the species-specific determinants are carbohydrate in nature.
Maleylated bovine serum albumin triggers cytolytic function in selected populations of primed murine macrophages.
Authors: Somers S D , Hamilton T A , Adams D O
Published: December 2022

The complex algal polysaccharide fucoidan has been reported as serving as a second signal for activation of macrophages primed in vivo by BCG. To assess the potential utility of this observation in analyzing biochemical mechanisms involved in macrophage activation, we examined the triggering effects of maleylated bovine serum albumin (maleylated-BSA), a defined molecule that clears via similar mechanisms. Cytolysis of P815 mastocytoma targets was triggered by maleylated-BSA, in a dose-dependent manner, in murine peritoneal macrophages primed in vivo by BCG. Unlike bacterial LPS, which triggered cytolysis when used to pretreat the macrophages, maleylated-BSA was only effective if present throughout the period of macrophage-target cytolytic interaction. Maleylated-BSA alone did not lyse the P815 targets and did not affect the binding of such targets by macrophages. Maleylated-BSA was equally effective in triggering cytolysis in BCG-primed macrophages from C3H/HeJ or C3H/HeN mice. Macrophages primed in vitro with IFN-gamma, however, could not be triggered by maleylated-BSA, even though these macrophages bound maleylated-BSA comparably to the BCG-primed macrophages. When responsive macrophages were fully activated in vitro by IFN-gamma and LPS and then allowed to decay to the primed state, maleylated-BSA was then as effective as LPS in triggering cytolysis. Taken together, the results indicate that maleylated-BSA can trigger cytolysis in certain populations of primed macrophages but not in others.
Bi-specific monoclonal antibodies: selective binding and complement fixation to cells that express two different surface antigens.
Authors: Wong J T , Colvin R B
Published: December 2022

A new dimension of immunotherapeutic selectivity might be achieved if antibodies could distinguish cells that co-express two different surface antigens. Bi-specific monoclonal antibodies (BSMAB) with two different antigen combining sites that share a common Fc region theoretically might have such a potential. Two such BSMAB were produced by hybrid-hybridoma clones prepared by fusion of pre-existing hybridomas and were purified by isoelectric focusing. CD3,4 (IgG2a, IgG2b) recognizes the T cell surface antigens CD3 and CD4, and CD3,8 (IgG2a, IgG2a) recognizes CD3 and CD8. These BSMAB promote complement-mediated lysis of target cells that bear both surface antigens 25 to 3125 times more efficiently than those that express only one of the antigens. This selectivity results from the increased avidity of these antibodies for cells with both antigens, as reflected by the increased surface immunoglobulin concentration detected by flow cytometry. It was also demonstrated that there exists a threshold surface immunoglobulin density necessary for antibody-dependent complement-mediated cytotoxicity microtiter assays for the various IgG antibodies tested in both bivalent and monovalent binding. These results support the associative model of IgG-mediated complement fixation.
Interleukin 2 mediates an alteration in the T200 antigen expressed on activated B lymphocytes.
Authors: Cook R G , Landolfi N F , Mehta V , Leone J , Hoyland D
Published: December 2022

We have examined the effect of exogenous IL 2 on cell surface antigen expression in LPS/dextran sulfate-activated murine B cells with the use of a panel of fluorescein-conjugated lectins. Elevated binding of the lectins PNA and SBA to activated B cells was found to be mediated by IL 2-containing supernatants from stimulated EL4 cells as well as by recombinant IL 2. These lectins have specificity for terminal beta-(1-3)-N-acetylgalactosaminyl residues; thus, the quantity or accessibility of these moieties is mediated by IL 2 in activated B lymphocytes. PNA binding in all strains tested, regardless of MHC or background genes, was found to be elevated fivefold to 15-fold by exogenous IL 2. To observe this effect, IL 2 must be added during the first 24 hr of culture. Based on anti-Thy-1 + complement depletion studies, T cells were not required, suggesting a direct effect of IL 2 on B cells. The glycoprotein responsible for this elevated binding of PNA has an Mr of approximately 220K and by immunodepletion was shown to belong to the T200 (Ly-5) family of cell surface antigens. These data demonstrate that exogenous IL 2 can mediate alterations in T200 expression on activated B cells that may be related to IL 2-driven modulation of B cell proliferation and/or differentiation.
T cell growth without serum.
Authors: Herzberg V L , Smith K A
Published: December 2022

Most in vitro T cell proliferation experiments are performed by using serum-supplemented medium, yet the actual contributions of serum components to cell cycle progression remain ill-defined, thus complicating attempts to fully define requirements for cell division. By utilizing a functional separation between T cell receptor-triggered "competence" and IL 2-promoted "progression" to independently assess serum requirements during each cell cycle stage, it was shown that serum serves an essential, active role only during the early events of the competence phase (G0-G1 transition) of T cell activation. Serum is required for optimal IL 2 production and the cell surface expression of IL 2 receptors after the stimulation of the T3/Ti antigen receptor complex. In contrast, serum does not function actively during IL 2-mediated progression through the G1 phase of the cycle. Serum proteins serve only a passive role at this stage, preventing the adsorption of IL 2. This same effect can be provided by any number of proteins including IL 2 itself, or even a high cell concentration. Supplementation of serum-free T cell cultures solely with IL 2 and transferrin is sufficient for maximal T cell proliferation, although the time of the peak response is delayed owing to a suboptimal rate of IL 2 receptor expression. Accordingly, the realization that serum is only necessary for the earliest stage of T cell activation will now enable studies designed to identify the critical individual serum components and to define their mechanism of action.
Selective elimination of non-lpr lymphoid cells in mice undergoing lpr-mediated graft-vs-host disease.
Authors: Perkins D L , Michaelson J , Glaser R M , Marshak-Rothstein A
Published: December 2022

The transfer of lpr BM stem cells into lethally irradiated non-lpr recipients (including the congenic MRL/+ differing only at the lpr locus) causes GVHD characterized by a wasting syndrome. In this study we investigated the interaction between the autoimmune (lpr) and normal (A-Thy) B, T, and RBC cell lineages in two types of radiation chimeras: MRL/lpr plus A-Thy----(MRL/lpr X A-Thy)F1 and MRL/+ plus A-Thy----(MRL/lpr X A-Thy)F1. Analysis of B cell repopulation by competitive RIA of serum Igh-1 allotype showed that both the MRL and the A-Thy donor cells initially engrafted. However, by 2 to 4 mo post-transplantation the normal A-Thy allotype was barely detectable (reduced greater than 2 orders of magnitude), whereas the autoimmune MRL/lpr allotype persisted at normal levels. Similarly, investigation of the donor origin of peripheral blood T cells by two-color flow cytometry showed that by 8 mo post-transplantation normal A-Thy T cells had been eliminated and only MRL/lpr T cells were present in the circulation. In contrast, erythrocytes from both the MRL/lpr and A-Thy donor strains successfully engrafted the F1 recipients and persisted until the termination of the study. Control chimeras transplanted with a mixture of MRL/+ plus A-Thy BM were stably engrafted with both donor strains in both the erythroid and lymphoid populations. Additional experiments in which either B6/lpr or MRL/lpr (and B6/+ or MRL/+ control) BM cells were transferred into (MRL/lpr X B6/+)F1 and (MRL/lpr X B6/lpr)F1 recipients demonstrated that the development of GVHD was not simply due to increased alloreactivity by the lpr donor cells. In these chimeras only the recipients heterozygous (but not homozygous) for the lpr gene developed lpr-GVHD, although both types of recipients had identical genotypes except at the lpr locus.
Mechanism of cell contact-mediated inhibition of natural killer activity.
Authors: Heiskala M K , Carpén O , Saksela E , Timonen T
Published: December 2022

Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells.
Concomitant immunoglobulin E and immunoglobulin G1 formation in Nippostrongylus brasiliensis-infected mice.
Authors: Lebrun P , Spiegelberg H L
Published: December 2022

The immunoglobulin (Ig)E, IgG subclass, and IgM formation in vivo and in vitro were measured by radioimmunoassays in normal and immunodeficient mice infected with the parasite Nippostrongylus brasiliensis (Nbr). High IgE responder mice had higher IgE serum levels than low responder mice both before (100 to 250 vs 2 to 20 ng of IgE/ml) and after (17,000 to 27,000 vs 1,000 to 5,000 ng of IgE/ml) infection. In vitro, mesenteric lymph node cells formed 5- to 10-fold more IgE than the spleen cells. Nu/nu mice had less than 1 ng before and 5 ng of IgE/ml serum after Nbr infection; no IgE was detectable in cell supernatants. There was no difference in the in vivo and in vitro IgE formation between C57BL/6 and C57BL/6-beige mice. Both in vivo and in vitro Nbr-infected Xid+ mice (CBA/N; (CBA/N X BALB/c) F1 male) formed approximately twice the amount of IgE than Xid- mice. Concomitant with the rise of the IgE serum levels, the IgG1 serum level increased by 100 to 1,500 micrograms/ml over preinfection levels in all strains except nu/nu+ mice. The IgM serum level increased by 100 to 500 micrograms/ml. In contrast, the IgG2a and IgG2b levels decreased in most strains, and the IgG3 levels remained unchanged. Nu/nu+ mice showed an increase of all IgG subclasses after Nbr infection. The antibodies to an Nbr extract were predominantly of IgM and IgG1, except in SJL mice which also had IgG2b and nude mice which only had IgM antibodies. The concomitant increase of IgE and IgG1 formation in all but the nude strains suggests that the regulation of these two Ig isotypes may be linked via regulatory T helper cells, which corroborates recent data on the simultaneous induction of IgG1 and IgE synthesis in vitro by the B cell stimulatory factor 1.
Activity of a partially purified human BCGF on murine assays for B cell stimulatory factors. II. Synergy between two distinct BCGF II-like factors in promoting B cell differentiation.
Authors: Ennist D L , Elkins K L , Cheng S C , Howard M
Published: December 2022

We have previously identified two BCGF II-like factors which can be distinguished by their differential reactivity in several murine BCGF II assays. Prototype sources of these two factors are a partially purified preparation derived from PHA-P-stimulated human peripheral blood T lymphocytes (designated human BCGF), and supernatant from antigen-stimulated D10.G4.1 murine T cells (designated D10 sup). Extending the characterization of these two factors, we show here that human BCGF and D10 sup both cause tritiated thymidine ([3H]TdR) incorporation and IgM secretion by T cell-depleted, in vivo-activated, large murine B cells. In contrast, only the human BCGF consistently induced proliferation and IgM secretion by T cell-depleted, small murine B cells. When simultaneously added to cultures, D10 sup and human BCGF synergized to produce optimal IgM secretion by large murine B cells and murine BCL1 B lymphoma cells. The same factors were tested in an IgM-specific plaque assay, and a similar synergistic response was observed for the large B cells, but not for the BCL1 cells. The combination of factors also produced maximal [3H]TdR incorporation by large murine B cells. In contrast, the addition of human BCGF totally abrogated D10 sup-induced BCL1 proliferation. Together, these data suggest that the synergies observed in IgM secretion result from an increased production of plaque-forming cells (PFC) in cultures of large B cells and an increase in IgM production per responding cell in BCL1 cells. Kinetic analysis of the time of action of the two BCGF II-like lymphokines in the induction of the PFC response by large B cells indicated that human BCGF was required within the first 24 hr of a 4-day culture period, while D10 sup could be added as late as the final 15 hr without significant diminution of the response. In summary, these data provide further support for the existence of two distinct B cell stimulatory factors which cause growth and differentiation of activated B cells, and indicate that these two factors synergize to produce optimal Ig secretion. For ease of discussion, the activity in the human BCGF preparation is referred to as BCGF IIA, and the activity in D10 sup is referred to as BCGF IIB.
A urine inhibitor of interleukin 1 activity affects both interleukin 1 alpha and 1 beta but not tumor necrosis factor alpha.
Authors: Seckinger P , Williamson K , Balavoine J F , Mach B , Mazzei G , Shaw A , Dayer J M
Published: December 2022

Urine from monocytic leukemia and other febrile patients contains an inhibitor of interleukin 1 (IL-1), as measured by prostaglandin E2 and collagenase production by human fibroblasts and synovial cells. With the use of recombinant IL-1, the IL-1 inhibitor was partially purified by using ammonium sulfate precipitation, anion-exchange, and gel filtration chromatographies. IL-1 inhibitory activity elutes with an 18,000 to 25,000 apparent molecular size. The same fractions also inhibit IL-1 assayed by the proliferation of murine thymocytes and human fibroblasts. Both forms of human recombinant IL-1, IL-1 alpha and IL-1 beta, which show only 26% homology, but nevertheless bind to the same receptor, are affected by this natural inhibitor to the same extent. In contrast, human recombinant tumor necrosis factor, which shares some of the biologic activities of IL-1, is not inhibited by the urinary IL-1 inhibitor. This study shows that the various biologic activities of both forms of human recombinant IL-1 are inhibited by a partially purified natural urine-derived factor.
Constitutive expression of Slp genes in mouse strain B10.WR directed by C4 regulatory sequences.
Authors: Rosa P A , Sepich D S , Robins D M , Ogata R T
Published: December 2022

The murine fourth component of complement (C4) and sex-limited protein (Slp) are two closely related serum proteins that exhibit very disparate patterns of gene expression: all mice constitutively express C4, whereas only adult male mice from a limited number of standard inbred strains express Slp. Several exceptional strains exhibit constitutive (C4-like) Slp expression, a phenotype that correlates with multiple copies of the Slp gene. To determine the molecular basis for constitutive Slp expression we have isolated genomic clones and compared the sequences of 1.5 kb of 5' flanking DNA from 1 C4 gene and three different Slp genes from the Slp-constitutive strain B10.WR. These sequence comparisons demonstrate C4-like regulatory sequences adjacent to two of the Slp genes. By analysis of cDNA clones isolated from a B10.WR liver library we demonstrate that the constitutive Slp phenotype is due primarily to expression of one of these C4/Slp hybrid genes. It appears likely that Slp gene duplication in strain B10.WR came about via homologous unequal crossover events between C4 and Slp genes; this would accommodate both the gene sequence data and the pattern of C4-like Slp expression in mouse strain B10.WR.
Functionally distinct agretopic and epitopic sites. Analysis of the dominant T cell determinant of moth and pigeon cytochromes c with the use of synthetic peptide antigens.
Authors: Fox B S , Chen C , Fraga E , French C A , Singh B , Schwartz R H
Published: December 2022

The dominant T cell determinant on moth and pigeon cytochromes c in B10.A (E beta k:E alpha k) mice is located in the C-terminal portion of the protein, contained within residues 93-103 or 93-104. Thirty-seven antigen analogs, containing single amino acid substitutions at positions 98, 99, 101, 102, 103, and 104, were synthesized. The effects of the substitutions on in vitro antigenicity and in vivo immunogenicity were determined. Functional assays with T cell clones identified residues 99, 101, 102, and 103 as critical, based on their effect on antigenic potency. Peptides containing substitutions at residues 99, 101, and 102 were capable of eliciting unique clones upon immunization of B10.A mice. This was consistent with the identification of these residues as part of the epitope, the site on the antigen that interacts with the T cell receptor. Immunization with peptides substituted at residue 103, however, failed to elicit clones with unique specificity for the immunogen. When these peptides were tested for their ability to stimulate the T cell clones with antigen-presenting cells from B10.A(5R) mice expressing the E beta b:E alpha k Ia molecule, a consistent change in the relative antigenic potency was observed with 50% of the peptides. The effect of the Ia molecule on the antigenic potency ruled out the possibility that residue 103 nonspecifically affected antigen uptake or processing and identified residue 103 as part of the agretope, the site that interacts with the Ia molecule. The locations of the agretope and the epitope on this antigenic determinant appear to be fixed, even in the presence of large numbers of amino acid substitutions. However, some substitutions were found to affect both the agretope and the epitope, placing limits on the functional independence of the two sites. The results are discussed in terms of the trimolecular complex model of T cell activation and the implications of these data for antigen-Ia molecule interactions.
Regulation of HLA class II antigen expression: intracellular signaling molecules responsible for the regulation by IFN-gamma and cross-linking of Fc receptors in HL-60 cells.
Authors: Ina Y , Koide Y , Nezu N , Yoshida T O
Published: December 2022

The cross-linking of Fc receptors (FcR) on HL-60 cells inhibited the ability of recombinant IFN-gamma to induce HLA class II antigens. This appeared to be correlated with intracellular mRNA level. HL-60 lacked detectable HLA class II mRNA. IFN-gamma led to appearance of these transcripts, which were canceled by the cross-linking of FcR. Therefore, experiments were designed to investigate the intracellular signaling molecules regulating the appearance of HLA class II molecules or transcripts. The expression of HLA class II antigen induced by IFN-gamma was blocked by a calmodulin antagonist, W-7, but not by a protein kinase C (PKC) inhibitor, H-7. Furthermore, a direct activator of PKC, phorbol myristate acetate, was not able to induce the HLA class II antigen expression. These results suggest that IFN-gamma induces HLA class II antigens on HL-60 cells via a calcium-calmodulin pathway and not via a PKC pathway. Calmodulin is activated by a transient rise in the cytosolic free calcium. In fact, the measurement of calcium influx into HL-60 cells showed that a remarkable and time-dependent calcium accumulation was caused by IFN-gamma, and that depletion of Ca2+ from culture medium resulted in failure of IFN-gamma to induce class II antigen expression. Furthermore, calcium ionophore, A23187, by itself induced HLA class II antigen expression. These results suggest that IFN-gamma stimulates calcium influx and activates the calmodulin branch of the calcium messenger system, resulting in the induction of class II antigen expression on HL-60 cells. On the other hand, cross-linking of FcR elicited the accumulation of intracellular cAMP, which appeared to suppress the IFN-gamma-induced calcium influx, resulting in annulling HLA class II antigen-inducing activity of IFN-gamma. These intracellular events of HL-60 regulate the expression of HLA class II transcripts and molecules.
Identification of a prominent nuclear protein associated with proliferation of normal and malignant B cells.
Authors: Feuerstein N , Mond J J
Published: December 2022

Experiments were undertaken to identify nuclear proteins that might be involved in regulation of the mitogenic process in B lymphocytes. Murine splenic B lymphocytes were purified and cultured with anti-Ig insolubilized onto Sepharose (anti-Ig/Sepharose) for 16 hr and labeled with [35S]methionine. Nuclei were isolated and the nuclear proteins were analyzed by two-dimensional gel electrophoresis. Anti-Ig/Sepharose induced a prominent increase in the synthesis and abundance of a 40 kDa/pI 5 nuclear protein (p40/pI-5). Inhibition of anti-Ig/Sepharose-induced mitogenesis by pretreatment of the cells with phorbol-12-myristate-13-acetate was associated with a specific inhibition (63%) of p40/pI-5. Subcellular fractionation experiments showed that p40/pI-5 is not detected in the soluble fraction of resting or activated B cells, indicating that this protein is located exclusively in the nucleus. Analysis of the expression of p40/pI-5 relative the cell cycle showed that the synthesis of this protein was increased during G1 phase and gradually reduced during S phase of the cell cycle. Abundant amounts of p40/pI-5 were also found in the rapidly proliferating B lymphoma cells, WEHI-231, and growth arrest of these cells by anti-mu was found to be associated with a marked inhibition (68%) of this protein. Taken collectively these results suggest that the nuclear protein p40/pI-5 may have an important role in regulation of the proliferation of normal and malignant B lymphocytes.
The role of interferon-beta 1 and the 26-kDa protein (interferon-beta 2) as mediators of the antiviral effect of interleukin 1 and tumor necrosis factor.
Authors: Van Damme J , De Ley M , Van Snick J , Dinarello C A , Billiau A
Published: December 2022

This study confirms our earlier finding that human interleukin (IL)-1 beta exerts an antiviral effect on diploid fibroblasts and on MG-63 osteosarcoma cells. It also extends the observation in that a similar effect was noted on aged but not freshly trypsinized HEp-2 cells, and that not only IL-1 beta but also IL-1 alpha and tumor necrosis factor (TNF)-alpha exerted similar antiviral effects on cells. The antiviral effects of these cytokines were neutralized by addition to the assay system of an antibody that was specific for interferon (IFN)-beta 1, indicating that IFN-beta 1 or a structurally or functionally related substance is involved in the antiviral activity observed. Both IL-1 and TNF were able to induce production of the 26-kDa protein, also known as IFN-beta 2, hybridoma/plasmacytoma growth factor (HPGF) or B-cell stimulatory factor-2 (BSF-2) and previously proposed as an alternative to IFN-beta 1 for mediating the antiviral effect of TNF. However, no good correlation was found between the antiviral effects of TNF and its potential to induce production of the 26-kDa protein. Furthermore, the anti-IFN-beta 1 serum which neutralized the antiviral activity of IL-1 and TNF did not cross-react with the 26-kDa protein. Conversely, the antiviral effect of IL-1 and TNF was only weakly neutralized by an antibody that did react with the 26-kDa protein and showed low cross-reactivity with IFN-beta 1. These observations, together with the low specific activity of the 26-kDa protein as an antiviral agent (less than 10(5) U/mg protein) provide strong arguments against this protein and in favor of IFN-beta 1 (or still another IFN-beta 1-related molecule) as the ultimate mediator of the antiviral effect of IL-1 and TNF.