bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipoxin A-induced inhibition of human natural killer cell cytotoxicity: studies on stereospecificity of inhibition and mode of action.
Authors: Ramstedt U , Serhan C N , Nicolaou K C , Webber S E , Wigzell H , Samuelsson B
Published: December 2022

Human leukocyte-derived lipoxin A (LXA; 5S,-6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid) inhibits the cytotoxic activity of human natural killer (NK) cells. LXA and three of its isomers were prepared by total organic synthesis and assayed for activity with human NK cells. Dose-response studies showed that biologically derived LXA and synthetic LXA were equally effective in inhibiting NK cell cytotoxicity. 6S-LXA, with its 6S-OH group in an (S) configuration, proved to be approximately half as potent as LXA. In contrast, 6S-11-trans-LXA and 11-trans-LXA displayed virtually no inhibitory activities. The methyl esters of both LXA and 6S-LXA proved to be more potent than their corresponding free acids. Thus, LXA inhibition of NK cells displays clear-cut stereochemistry. In the absence of putative inhibitors, NK cells bind to their targets to form conjugates. This event is followed by polarization of the NK Golgi apparatus, which moves towards the plasma membrane that is in contact with the target cell. However, in the presence of either the methyl ester or free acid of LXA, the Golgi apparati of NK cells bound to their targets were randomly oriented. In contrast, neither 6S-11-trans-LXA nor the potent NK inhibitor prostaglandin E2 affected the polarization. Furthermore, although prostaglandin E2 resulted in a decrease in NK-target cell binding efficiency, LXA and its isomers failed to affect conjugate formation. Together these results indicate that LXA-induced inhibition of NK cytotoxicity does not act on NK cell binding but may block cytotoxicity by disrupting "signals" involved in the specific orientation of the Golgi. Thus, this latter event may appear to be important in cytotoxicity.
Stimulation of T cells via the TAP molecule, a member in a family of activating proteins encoded in the Ly-6 locus.
Authors: Yeh E T , Reiser H , Daley J , Rock K L
Published: December 2022

T-cell activating protein, TAP, is a Ly-6 encoded 12,000 dalton glycoprotein involved in the activation of murine T cells. TAP is distinct from other known surface activating structures, such as the T cell receptor/T3 complex and Thy-1. This study investigates the mechanism of activation via the TAP molecule. Soluble alpha TAP monoclonal antibody (MAb) is sufficient for T cell activation. This, however, requires cross-linking because F(ab) monovalent antibody is not stimulatory unless cross-linked by a second antibody. Immediately after cross-linking, there is a rapid influx of calcium, which is comparable with concanavalin A or T cell receptor triggered responses. Subsequently, interleukin 2 (IL 2) is produced, and IL 2 receptors (IL 2R) are expressed. TAP-stimulated T cell proliferation is driven by this autocrine pathway because it is inhibited by alpha IL 2R MAb. Thus TAP-mediated activation appears to share a common final pathway with other mitogenic stimuli. A nonactivating alpha TAP MAb was observed to stimulate T cells upon additional cross-linking. Given this observation, we asked whether other Ly-6 linked proteins might share similar activating potential. We show that at least three distinct Ly-6 linked molecules are capable of stimulating T hybrid clones and/or heterogeneous T lymphocytes. Thus it appears that the Ly-6 locus encodes a family of activating cell surface molecules.
Amplification of human B cell activation by a monoclonal antibody to the B cell-specific antigen CD22, Bp 130/140.
Authors: Pezzutto A , Dörken B , Moldenhauer G , Clark E A
Published: December 2022

The B cell-specific antigen CD22 is a 130/140-Kd complex and is unique among human B cell antigens, since its surface expression is restricted to a subpopulation of Ig+ B cells. Here the function of the CD22 antigen was evaluated by using the mAb HD6, directed against one of the epitopes on the molecule. The HD6 antibody was constimulatory with anti-Ig in inducing small, dense tonsillar cells to proliferate; however, the antibody by itself was devoid of stimulatory activity. Anti-CD22 antibody also induced more anti-Ig-treated B cells to leave G0 and enter the G1 phase of the cell cycle. It also was constimulatory with low-m.w. BCGF and with an antibody to a 50-Kd polypeptide, Bp50, which mediates a BCGF-like activity. Results of kinetic experiments and analysis of different B cell fractions suggested that anti-CD22 acts during an early phase of B cell activation, probably by amplifying the anti-Ig signal. F(ab')2 fragments of anti-CD22 HD6 were as effective as the whole antibody in inducing augmentation of B cell proliferation, showing that the Fc portion of the molecule was not required for the activity. The results of these experiments, together with the intriguing distribution of the Bp 130/140 antigen in B cell ontogeny, suggest that this molecule plays an important role in the process that leads to B cell activation and proliferation.
Human T lymphocyte activation by tumor promoters: role of protein kinase C.
Authors: Isakov N , Altman A
Published: December 2022

Protein kinase C (PKC) has a major role in a ligand-receptor-mediated signal transduction system in a variety of cell types including T lymphocytes. One of the early phenotypic changes associated with T cell activation is the expression of cell surface receptors for interleukin 2 (IL 2). To test the role of PKC in regulation of IL 2 receptor (IL 2-R) expression and T cell activation in general, we used tumor promoters (TP) as modulators of PKC and compared their effects on intact human T cells and on the enzymatic activity of T cell-derived PKC in a cellfree system. In T cells, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) induced IL 2-R expression and proliferation associated with cytosol-to-membrane PKC translocation. A dose of TPA (1 to 4 ng/ml) that induced about 50% of the maximal activation of PKC in the enzymatic assay also induced half-maximal effects on cell proliferation, IL 2-R expression, and PKC redistribution in intact T cells. Structure-function studies with several phorbol ester analogs and non-phorbol ester TP directly correlated tumor promotion activity with the ability to activate PKC and induce IL 2-R. An inhibitor of PKC, chlorpromazine, was found to suppress TPA-mediated proliferation and IL 2-R expression, and inhibited T cell-derived PKC by competing with the phospholipid. Ca2+ ionophore, which synergizes with TPA in induction of T cell proliferation, facilitated the TPA-induced PKC translocation to the membrane. The results thus demonstrate a direct correlation between the effects of various chemicals on: subcellular redistribution of PKC in T cells; induction of T cell proliferation and IL 2-R expression; and activation of T cell-derived PKC in vitro. These data provide further support for the role of PKC in transduction of activation signals in T cells and in regulation of IL 2-R expression.
Comparative functional analysis of helper T lymphocyte responses to soluble and particulate antigens.
Authors: Krowka J F , Guidos C , Sinha A , Lee K C , Diener E , Pilarski L M
Published: December 2022

An adaptable and sensitive assay to analyze the roles of helper T lymphocytes (TH) which recognize soluble or cell-surface bound antigens in the induction of cytotoxic T lymphocyte precursors (CTLp) is described. Long-term T cell lines that recognize purified protein derivative, keyhole limpet hemocyanin, or Corynebacterium parvum were used in these studies. The ability of T cells from these lines to induce cytotoxic T lymphocyte or antibody responses were compared with their ability to proliferate or release interleukin 2 (IL 2). The results demonstrate that these T cell lines are able to react to soluble antigen by proliferation and IL 2 release. Moreover, the same cell lines are able to interact with CTLp or with the precursors of antibody-secreting B cells to induce a response. In the induction of CTLp we observed an inverse correlation between the number of TH cells required and the concentration of antigen used to pulse the antigen presenting cells. However the correlation between the ability of TH lines to proliferate specifically in response to antigen and to act as helpers for CTLp and B cells was not absolute as cells with compromised proliferative capacity were able to efficiently deliver inductive signals.
Induction of macrophage Ia expression by lipopolysaccharide and Listeria monocytogenes in congenitally athymic nude mice.
Authors: Wentworth P A , Ziegler H K
Published: December 2022

Experiments were performed to analyze the mechanism by which lipopolysaccharide (LPS) modulates the expression of Ia by murine peritoneal macrophages in vivo. We investigated the effect of LPS on Ia expression in T cell deficient mice by using the congenitally athymic nude mouse model. Injection (i.p) of LPS into athymic (nu/nu) mice resulted in a dramatic increase in the expression and biosynthesis of Ia by peritoneal macrophages 7 days after injection. The magnitude and kinetics of this induction were equivalent to increases observed after LPS injection of euthymic (nu/+) mice. Viable Listeria monocytogenes also increased Ia expression in athymic mice, but in contrast to the induction observed in euthymic mice at 3 and 7 days after injection, increased Ia expression was not seen until 7 days. Ia induction by either LPS or L. monocytogenes in athymic mice was not due to the presence or development of mature T cell function as defined by assays for T cell mitogenesis and interleukin 2 production. We conclude that increased macrophage Ia expression by LPS and L. monocytogenes in vivo can occur in the absence of mature functioning T cells.
Glucocorticoids enhance the gamma-interferon augmentation of human monocyte immunoglobulin G Fc receptor expression.
Authors: Girard M T , Hjaltadottir S , Fejes-Toth A N , Guyre P M
Published: December 2022

At physiologic and therapeutic concentrations, glucocorticoids decrease the number of Fc receptors for IgG (Fc gamma R) on human monocyte-like cell lines. In comparison, gamma-interferon (IFN-gamma) increases Fc gamma R expression on both human monocytes and monocyte-like cell lines. In this study, we examined the combined effects of glucocorticoids and IFN-gamma on human monocyte expression of the high affinity (72 kDa) Fc gamma R. Mononuclear cells prepared from heparinized venous blood of normal donors were treated for up to 90 hr with or without recombinant IFN-gamma and/or steroids. Monocyte Fc gamma R were measured by Scatchard analysis of the binding of human monomeric 125I-IgG1; indirect immunofluorescence plus flow cytometry, utilizing a monoclonal antibody (MoAb 32) which is specific for the high affinity Fc gamma R; and direct immunofluorescence using fluorescein isothiocyanate-labeled human monomeric IgG1 and flow cytometry quantitated using U-937 cells as a standard. Cultured monocytes incubated in the presence of both glucocorticoids and IFN-gamma for 18 hr had significantly higher (p less than 0.01) Fc gamma R levels than monocytes treated with IFN-gamma alone. The effect of combined treatment reached a plateau by 42 hr of incubation without increasing expression of other surface markers tested. Treatment with glucocorticoids alone did not consistently decrease monocyte Fc gamma R levels after either 18 or 42 hr of culture. Only glucocorticoids augmented the IFN-gamma increase in Fc gamma R; other steroids tested had no effect on IFN-gamma action. Furthermore, the effect was observed after treatment with only one type of interferon, IFN-gamma. These results describe a glucocorticoid immunoregulatory effect that may explain why combined IFN-gamma plus glucocorticoid treatment enhances mononuclear phagocyte Fc-mediated functions.
Dissociation between lymphocyte activation for proliferation and for the capacity to adoptively transfer uveoretinitis.
Authors: Fox G M , Redmond T M , Wiggert B , Kuwabara T , Chader G J , Gery I
Published: December 2022

We have shown previously that immunization with bovine interphotoreceptor retinoid-binding protein (IRBP) induces in rats severe eye disease, experimental autoimmune uveoretinitis (EAU). This study examined the uveitogenic capacity of IRBP of another species, the monkey, and tested the cross-antigenicity between these two proteins by a battery of immunological assays. Monkey IRBP was found to be approximately 20 times less uveitogenic in Lewis rats than bovine IRBP. High levels of cross-reactivity between bovine and monkey IRBP were demonstrated by antibodies as measured by the enzyme-linked immunosorbent assay, and by the radiometric ear test of delayed-type hypersensitivity, by using rats immunized with either one of the IRBP. On the other hand, lymphocytes from these rats failed to detect the cross-reactivity between the two IRBP by the proliferation response in culture. Yet, such lymphocytes did recognize the nonimmunizing IRBP when activated in culture for acquiring the capacity to adoptively transfer EAU into naive recipients. The data are discussed with regard to the limited usefulness of the lymphocyte proliferation assay for detection of immunopathogenic processes and the role of cross-reacting antigens in initiation of autoimmune responses.
Murine monoclonal antibodies to the myelin-associated glycoprotein (MAG) recognize Leu-7-reactive molecules on human mononuclear cells.
Authors: Arai M , Nishizawa M , Inuzuka T , Tanaka M , Baba H , Sato S , Miyatake T
Published: December 2022

It is known that the antibody to human myelin-associated glycoprotein (MAG) reacts with a subset of human mononuclear cells (MNC) mediating a natural killer (NK) activity. The properties of the target molecule of the anti-MAG antibody, however, have not yet been elucidated. Three (GC-J4, MC-P2, and MC-P4) of five murine monoclonal antibodies (mAb) to MAG bound to human MNC. Moreover, MC-P2 and MC-P4 inhibited the binding of 125I-labeled anti-Leu-7 to MNC in a dose-dependent fashion. Conversely, anti-Leu-7 inhibited the binding of MC-P2 and MC-P4 to MNC, but did not inhibit the binding of GC-J4. Therefore, it is possible that MC-P2 and MC-P4 bind directly to or close to the Leu-7 epitope, and that GC-J4 binds to the epitope which is distinct from the Leu-7 epitope. The electrophoretic patterns of immunoprecipitates with GC-J4, MC-P2 and anti-Leu-7 from detergent lysates of surface-labeled human MNC were very similar. The target molecules of anti-Leu-7 and anti-MAG mAb have apparent m.w. of 205, 170, 150, 135, 110, 85, 65, and 55 kDa. All of the molecules precipitated by these mAb are monomeric or noncovalently associated proteins, because the electrophoretic mobilities of the proteins remained unchanged whether the samples were reduced or not. MC-P4 may have a higher affinity for the 65 kDa molecule than the other mAb, and precipitates the 58 kDa molecule as well. Therefore, the fine antigenic specificity of MC-P4 is slightly different from those of anti-Leu-7 or MC-P2. The implication of these results is that mAb, whose specificity is directed to the carbohydrate part of human MAG, reacts with the Leu-7 reactive molecules on human MNC, and that at least two epitopes detected by anti-MAG mAb coexist on the surface molecules with various apparent m.w.
Human antibodies to phosphocholine. IgG anti-PC antibodies express restricted numbers of V and C regions.
Authors: Scott M G , Briles D E , Shackelford P G , Smith D S , Nahm M H
Published: December 2022

We examined the IgG subclass composition and isoelectric focusing (IEF) spectrotype pattern of naturally occurring human IgG antibodies that bind phosphocholine (PC) and found direct evidence for restricted expression of both V and C regions among these antibodies. In most individuals, the isotype of these IgG anti-PC antibodies was primarily IgG2. However, serum from some individuals contained significant amounts of IgG1 and IgG3 anti-PC antibodies. We also found that in individual sera, anti-PC antibodies are pauciclonal, as demonstrated by restricted spectrotypic patterns of the anti-PC antibodies. The IEF pattern of these antibodies were for the most part unique for each individual. In some sera, certain anti-PC antibodies with isoelectric points of basic pH bound PC conjugated to bovine serum albumin (PC-BSA) but did not bind pneumococcal C-carbohydrate bearing PC determinants. In two individuals, we found that the spectrotypes that bound only PC-BSA were of the IgG1 subclass. Taken together, these findings demonstrate that within individual sera, human antibodies to PC are quite restricted in both V and C region expression, and furthermore, these V and C regions of human Ig may not randomly associate.
Monoclonal antibody specific for T cell-derived human IgE binding factors.
Authors: Kisaki T , Huff T F , Conrad D H , Yodoi J , Ishizaka K
Published: December 2022

A B cell hybridoma secreting monoclonal antibody against human IgE binding factors was obtained by immunization of BALB/c mice with partially purified IgE binding factors, and fusion of their spleen cells with SP-2/0-AG14 cells. The monoclonal antibody bound all of the 60,000, 30,000, and 15,000 dalton IgE binding factors from two T cell hybridomas and those from activated T cells of a normal individual. The antibody bound both IgE-potentiating factors, which had affinity for lentil lectin, and IgE-suppressive factors, which had affinity for peanut agglutinin. However, the monoclonal anti-IgE-binding factor bound neither Fc epsilon R on RPMI 8866 cells nor IgE binding factors from the B lymphoblastoid cells. A monoclonal antibody against Fc epsilon R on B cells (H107) bound the 60,000 and 30,000 dalton IgE binding factors from both T cell hybridomas and RPMI 8866 cells but did not bind the 15,000 dalton IgE binding factors from either T cells or B cells. The results indicate that T cell-derived IgE binding factors have a unique antigenic determinant that is lacking in both Fc epsilon R on B cells and B cell-derived IgE binding factors. The anti-IgE binding factor and anti-Fc epsilon R monoclonal antibodies both failed to stain cell surface components of IgE binding factor-producing T cell hybridomas. However, both antibodies induced the T cell hybridoma to form IgE binding factors. The results suggest that the T cell hybridomas bear low numbers of Fc epsilon R that share antigenic determinants with IgE binding factors secreted from the cells.
A collagen-like immunodeterminant on the surface of Streptococcus sanguis induces platelet aggregation.
Authors: Erickson P R , Herzberg M C
Published: December 2022

The basis of similarities in the mechanism of human platelet aggregation induced by soluble collagen and the dental plaque bacterium Streptococcus sanguis was analyzed. Structural and functional comparisons were made by using molecular probes, including rabbit antibody fractions reactive with components on S. sanguis and a synthetic, collagen-like octapeptide mimicking segments from cyanogen bromide fragments 6 and 4 of types I and III collagen, respectively. When platelets were pretreated with tryptic peptides or class II antigen of S. sanguis or with the synthetic, collagen-like octapeptide, the onset of aggregation in response to S. sanguis and collagen was prolonged. When compared to other peptides of similar size and charge, the collagen-like peptide's action towards platelets was shown to be selective. Indeed, absorption of antiserum to S. sanguis cells with particulate type I collagen removed specificities directed at a single S. sanguis antigen. These observations suggested that a common platelet-interactive immunodeterminant on soluble types I and III collagens, particulate type I collagen, and S. sanguis cells was present. Selective inhibition by antibody was used to show structural similarities between the S. sanguis surface proteins and collagen. When either agonist was pretreated with anti-S. sanguis IgG or Fab fragments, the lag time to onset of platelet aggregation was increased. Greater increases in the lag time to aggregation was seen when S. sanguis cells or collagen were pretreated with anti-S. sanguis IgG or Fab fragments made relatively specific for the class II antigen. Neutralization of the platelet-interactive action of the octapeptide by anti-S. sanguis antibody fractions showed that the immunodeterminant common to S. sanguis and collagen triggered platelets in plasma to aggregate. Although the anti-S. sanguis antibodies could inhibit fibrillogenesis, this action was apparently independent of interactions with platelets. In contrast, S. sanguis could bind or adhere to platelets by different determinants. Our data suggest that platelets have at least two distinct sites that bind collagen or S. sanguis. One of these may be a common site for collagen and S. sanguis agonists.
Quantitative analysis of the T cell response to antigen and planar membranes containing purified Ia molecules.
Authors: Fox B S , Quill H , Carlson L , Schwartz R H
Published: December 2022

Planar lipid membranes containing the purified Ia molecule E beta k:E alpha k can present a peptide antigen derived from cytochrome c to the T cell hybridoma 2B4.11. The incorporation of E beta k:E alpha k into planar membranes was linear over a 120-fold range of Ia molecule concentrations, permitting the dependence of the T cell response on the Ia molecule concentration to be examined. As the Ia molecule concentration was increased in the planar membranes, two parameters changed: less antigen was needed to stimulate the T cells, and the plateau response seen at functionally saturating concentrations of antigen increased. The antigen sensitivity was analyzed by plotting the antigen concentration (log2) required to stimulate the release of 10 U of IL 2 from the T cells as a function of the Ia molecule concentration (log2). If the T cell recognized a simple unit of one antigen molecule and one Ia molecule, this plot should have generated a straight line with a slope of -1. Surprisingly, a line with a slope of -2.04 X/divided by 1.12 was observed, suggesting that the T cell might recognize one antigen molecule and two Ia molecules. This complexity, however, resulted from changes in the maximal response achieved at different Ia molecule concentrations. A similar phenomenon was observed when the Ia molecule concentration was decreased in cultures containing splenic antigen-presenting cells (APC) by the addition of an anti-E beta k:E alpha k monoclonal antibody, or the use of [B10.A(4R) X B10.PL]F1APC. The Ia molecule concentration can therefore be limiting for T cell hybridomas in cultures containing normal APC and functionally saturating amounts of antigen. When the planar membrane data were normalized to the maximal response to eliminate the effect of the changing plateau response, the resulting plot generated a line with a slope of -1.17 X/divided by 1.11. These results suggest that the sole stimulatory signal for this T cell hybridoma consisted of a 1:1 ratio of antigen and Ia molecules.
Monoclonal antibodies to glucose-6-phosphate dehydrogenase (G6PDH) form cyclic 1:1 complexes with G6PDH and act as regulatory subunits.
Authors: Skold C , Gibbons I , Gould D , Ullman E F
Published: December 2022

A number of IgG monoclonal antibodies against L. mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) have been prepared. Four of the antibodies form 1:1 enzyme-antibody complexes which are stabilized in the presence of glucose-6-phosphate (G6P) and have greatly reduced enzyme activity. In the absence of G6P, the 1:1 complexes convert gradually to a more active multimeric form. Reduction of the IgG inter-heavy chain disulfides partially relieves inhibition and removes the G6P requirement for stability. F(ab')2 fragments of one of the antibodies behave similarly to the intact IgG. Reduction of the disulfides in the G6PDH-F(ab')2 complex leads to complete recovery of activity. The activity of complexes of G6PDH with reduced antibodies or Fab with digoxin bound to the antibody or Fab sulfhydryl groups can be modulated with antibodies to digoxin. The anti-G6PDH antibodies bridge two identical epitopes of this two subunit enzyme and simulate the function of regulatory subunits in which anti-digoxin acts as an activator. The system can be used to provide a sensitive homogeneous immunoassay for digoxin.
Synergism between eosinophil cationic protein and oxygen metabolites in killing of schistosomula of Schistosoma mansoni.
Authors: Yazdanbakhsh M , Tai P C , Spry C J , Gleich G J , Roos D
Published: December 2022

To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.
A purified 28,000 dalton protein from Schistosoma mansoni adult worms protects rats and mice against experimental schistosomiasis.
Authors: Balloul J M , Grzych J M , Pierce R J , Capron A
Published: December 2022

We have purified a 28,000 dalton (P28) protein from Schistosoma mansoni adult worms and used it to immunize Fischer rats. Immunofluorescence assays demonstrated that the P28 antigen was mainly located in the parenchyma of the schistosomulum and of the adult worm, including the dorsal spines of the parasite. Western blot analysis revealed that this antigen was present in three species of schistosomes: S. mansoni, S. japonicum, and S. bovis. The antibody response raised against this protein was able to kill S. mansoni schistosomula in in vitro cytotoxicity assays in the presence of rat eosinophils. The inhibition of this cytotoxic activity by an aggregated myeloma IgG2a indicated that one of the major isotypes involved in this in vitro model is IgG2a. The passive transfer of P28 antisera induced a significant level of protection against experimental infection. Moreover, we have immunized Fischer rats and BALB/c mice with the purified 28,000 dalton protein and observed a marked decrease (up to 70%) in the parasite burden in both experimental infection models.
Structure function relationships for the IL 2 receptor system. III. Tac protein missing amino acids 102 to 173 (exon 4) is unable to bind IL 2: detection of spliced protein after L cell transfection.
Authors: Neeper M P , Kuo L M , Kiefer M C , Robb R J
Published: December 2022

High affinity receptors for interleukin 2 (IL 2) contain the Tac protein as one ligand-binding subunit. Localization of the IL 2-binding site on this molecule, as well as localization of the complementary site on IL 2, should provide insight into the design of IL 2 analogs. In this report, we examine the ability of normal and modified Tac protein to bind IL 2 and several antibodies that recognize the native Tac molecule. Using a transient L cell expression system, we have determined that transfection with cDNA-missing Tac exon 4 resulted in expression of spliced protein that had no measurable binding to IL 2 or the monoclonal anti-receptor antibodies, anti-Tac, and 7G7/B6. This protein was detected, however, by rabbit polyclonal antibodies prepared against synthetic Tac peptides. Thus, one or more amino acids encoded by exon 4 is important, either for direct ligand contact or for the proper folding of critical segments of the Tac molecule. In addition, insertion of stop codons at a unique restriction enzyme site near the beginning of exon 5 resulted in cellular secretion of truncated Tac molecules that were capable of binding IL 2, anti-Tac, and 7G7/B6. Amino acids encoded by exons 5 to 8 thus play no critical role in IL 2 binding. The ligand association demonstrated for truncated Tac protein produced by exons 2 to 4 should guide attempts to define the IL 2-binding segment of the Tac molecule.
Cell lineage segregation during bursa of Fabricius ontogeny.
Authors: Houssaint E
Published: December 2022

The population dynamics of myeloid and lymphoid lineages during bursa of Fabricius ontogeny were analyzed by immunofluorescence by using two monoclonal antibodies (mAb). CL-1 mAb reacts with all chicken hemopoietic cells, except mature erythrocytes. L22 mAb reacts with bursa and bursa-derived lymphocytes, with a minor subset of macrophages and with some cells of the thymic medulla. The staining of embryonic bursas by these antibodies helps to distinguish between two different lineages of hemopoietic cells: CL-1+/L22+ cells represent B lymphocytes and a minor subset of macrophages, while CL-1+/L22- cells correspond to most of the macrophages and to the granulocytes, which disappear at the end of the embryonic life. CL-1+/L22- as well as CL-1+/L22+ cells were first observed outside the bursal rudiment. This indicates that there is a pre-bursal segregation between these two hemopoietic lineages and that two different kinds of precursors colonize the bursal rudiment at about the same time (day 9 for CL-1+/L22- cells and days 9 or 10 for CL-1+/L22+ cells). Moreover our data show that the colonization of the bursal epithelium by hemopoietic precursors is a two-step phenomenon. The first cells which enter belong to the CL-1+/L22- lineage, express Ia-like antigens at a high level, are dendritic in morphology, and represent cells of the macrophage/dendritic cell lineage. They are responsible for the formation of the epithelial bud which are then colonized by a small number of lymphoid precursors which belong to the CL-1+/L22+ lineage. Quail-chick bursa grafting experiments were also performed and the grafts were examined for CL-1 (restricted to chicken hemopoietic cells) and L22 reactivity. These observations confirmed our previous findings about the kinetics of the colonization of bursal rudiment by hemopoietic precursors and give support for a pre-bursal segregation between two hemopoietic pathways.
Characterization of dual-reactive H-2Kb-restricted anti-vesicular stomatitus virus and alloreactive cytotoxic T cells.
Authors: Sheil J M , Bevan M J , Lefrancois L
Published: December 2022

Cross-reactive recognition of alloantigen by "self + X"-reactive cytotoxic T lymphocytes (CTL) has been documented in a variety of systems. It has been shown previously that the H-2Kb-restricted CTL response of C57BL/6 (B6) mice to vesicular stomatitis virus (VSV) infection is partially cross-reactive on uninfected target cells expressing the H-2Kbm8 mutation. In this report, we describe the isolation and detailed characterization of such dual-reactive CTL. By employing EL4 tumor lines transfected with genes encoding various VSV proteins, we demonstrated that the majority of dual-reactive CTL recognize the internal N protein of VSV and are also reactive against uninfected bm8 targets. Although the response of normal B6 mice to bm8 stimulators shows no measurable cross-reactivity on VSV-infected targets, the response of VSV-primed B6 mice to bm8 stimulation is almost entirely cross-reactive, lysing VSV-B6 targets and uninfected bm8 targets roughly equally. Furthermore, about 70% of CTL clones isolated from such mice by bm8 stimulation are dual-reactive with respect to effector function. Analysis at the population and clonal levels with cold target competition and antibody blocking suggests that the bulk of dual-reactive CTL have a higher avidity for VSV-B6 targets than for bm8 targets. The extreme case of this is illustrated by a fraction of CTL clones, isolated and maintained on bm8 stimulators, which lyse VSV-B6 targets but do not lyse bm8 targets. One such CTL clone is shown to be specific for the bm8 antigen in proliferation assays. These results demonstrate that: the specificity of an alloreactive CTL response may be dramatically altered by previous antigenic encounters; and dual-reactive CTL display a significant difference in affinity of the CTL receptor-determinant interaction, depending on the target which is recognized.
Depletion of T-4+ lymphocytes with monoclonal antibody reactivates toxoplasmosis in the central nervous system: a model of superinfection in AIDS.
Authors: Vollmer T L , Waldor M K , Steinman L , Conley F K
Published: December 2022

Central nervous system toxoplasmosis causes disability and death in up to 30% of patients with acquired immune deficiency syndrome (AIDS). The source of the toxoplasma infection in these patients and the specific immune deficit that allows for this virulent form of the infection are unknown. By using a mouse model of toxoplasmosis, we found that selective depletion of T-4+ lymphocytes (CD4+ T cells) produces overwhelming infection and death in both acute and chronic toxoplasmosis. However, the pattern of infection is remarkably different in chronically infected mice as compared with acutely infected mice when the mice are depleted of CD4+ T cells. During acute infection loss of the CD4+ T cell population leads to severe systemic infection with only mild disease in the brain. In chronically infected mice depleted of CD4+ T cells, death follows severe CNS damage due to toxoplasma infection with only minor systemic involvement. In chronically infected mice treated with anti-CD4 monoclonal antibody, reactivation of the toxoplasma infection occurs despite high titers of circulating antitoxoplasma antibody. In parallel with these results in mice, CNS toxoplasmosis in AIDS patients may be due to reactivation of infection acquired much earlier in life.
Complement dependence of antibody-induced mesangial cell injury in the rat.
Authors: Yamamoto T , Wilson C B
Published: December 2022

Intravenous administration of rabbit anti-rat thymocyte serum (ATS) reactive with Thy-1-like antigens present on rat mesangial cells induces almost immediate (1-hr) mesangial cell injury in rats followed by sequential mesangiolytic and mesangial-proliferative/infiltrative lesions. To determine the role of complement in these ATS-induced glomerular lesions, ATS was given to Lewis rats that had been depleted of C3 by cobra venom factor (CVF). CVF treatment prevented the degenerative changes in mesangial cells and accumulation of even the few polymorphonuclear leukocytes (PMN) seen in the glomeruli (2.67 PMN/glomerulus) 1 hr after ATS-treatment in rats not given CVF. In addition, CVF prevented the mesangiolysis and mesangial hypercellularity seen at day 4. Rat C3 and late complement components identified in the mesangial of ATS-treated rats in close association with the deposition of rabbit immunoglobulin G was also absent as a result of CVF treatment. CVF treatment did not affect binding of ATS to glomeruli as studied by immunofluorescence or paired label radioisotope techniques. The depletion of leukocytes and/or PMN by irradiation or treatment with anti-I-MN serum had no effect on the induction of the acute mesangial cell damage or the mesangiolytic lesion. Irradiation did diminish the 4-day proliferative/infiltrative lesion. Complement depletion normalized the ATS-induced increase in mesangial uptake of heat-aggregated human gamma-globulin (655.0 +/- 35.2 micrograms in ATS-treated vs 20.3 +/- 2.9 micrograms/5 X 10(4) glomeruli in ATS plus CVF-treated rats; mean +/- SEM). Small immune deposits present in the mesangial areas of kidneys 4 to 5 days after CVF treatment represented CVF-anti-CVF antibody-C3 complexes. The model of mesangial cell damage induced by ATS in the rat is complement-dependent and may relate, at least in part, to complement-mediated mesangial cell lysis.
Effect of UV irradiation on expression of membrane IL 1 by rat macrophages.
Authors: Lange-Wantzin G , Rothlein R , Kahn J , Faanes R B
Published: December 2022

The effect of UV-B irradiation on the expression of membrane-associated IL 1 (mIL 1) by rat pulmonary alveolar macrophages (PAM) was studied. We found that although there was an increase in secreted IL 1 by PAM exposed to UV-B, the expression of mIL 1 was inhibited in a dose-dependent manner. Furthermore, PAM that were allowed to express mIL 1 before UV-B irradiation had a faster decay of mIL 1 activity than unirradiated cells. These data suggested that mIL 1 expression is inhibited by UV-B irradiation, and that under normal circumstances, mIL 1 synthesis and degradation is at a steady state, with the half-life of mIL 1 activity being 24 hr when assayed in an IL 1-dependent cell line proliferation assay. These data indicate that secreted forms of IL 1 and mIL 1 are differentially regulated and that the therapeutic effects of UV irradiation may be due to its inhibition of mIL 1 activity.
Reexpression of a T15 idiotope on variant immunoglobulins after the binding of another anti-idiotopic antibody.
Authors: Strickland F M , Gleason J T , Cerny J
Published: December 2022

The phosphorylcholine (PC)-binding myeloma protein TEPC15 (T15) contains several distinct idiotopic determinants that are detectable with monoclonal anti-idiotopic antibodies. This study focuses on one of these anti-idiotopes, designated B24-44, which binds to a site near the T15 paratope. Another anti-idiotope, B36-82, recognizes an idiotope that is distant from the paratope. Two PC-binding immunoglobulins, 7-22 and 140.7C6, that differ from T15 by one to three amino acids have selectively lost their reactivity with the anti-idiotope, B36-82. However, the B36-82 binding was restored when B24-44 was first allowed to react with these immunoglobulins. The binding of B24-44 as well as the restoration of the B36-82 site was specifically inhibited by PC-protein conjugates. Competition experiments suggested that the newly induced B36-82 determinant is in the same location as the B36-82 binding site on T15. These data indicate that the binding of anti-idiotopic determinants to an immunoglobulin can alter the protein structure and create new determinants.
Structural and functional characteristics of the CD3 (T3) molecular complex on human thymocytes.
Authors: Tsoukas C D , Landgraf B , Bentin J , Lamberti J F , Carson D A , Vaughan J H
Published: December 2022

The CD3 (T3) molecular complex is noncovalently associated with the antigen receptor molecule on T cells. The mitogenic properties of anti-CD3 antibodies have suggested that this complex may be the transducer of the antigenic signal to the intracellular environment. In the present investigation, we studied some of the structural and functional characteristics of the CD3 complex on human thymocytes. In 11 specimens tested, we found that anti-CD3 antibodies react with 50 to 76% of the thymocytes. Two-color immunofluorescence analysis revealed that the majority (greater than 50%) of thymocytes express both CD3 and CD1 on their surfaces. The latter is a marker of immature thymocytes. However, a distinct subpopulation comprising 13 to 19% of the total cells displays only CD3, while an approximately equal percentage of cells expresses only CD1. The mitogenic potential of anti-CD3 antibodies on peripheral T cells is dependent on the presence of monocytes. Anti-CD3 antibodies by themselves cannot activate thymocytes, indicating that functionally active monocytes are absent from the thymocyte population. Even the addition of peripheral monocytes does not allow a response of thymocytes to anti-CD3 antibodies. However, when the anti-CD3 antibody 64.1 is added in the presence of exogenous rIL 2, a strong antibody and lymphokine dose-dependent response ensues. Only CD1- CD3+ thymocytes are stimulated by the addition of antibody and IL 2. The mere expression of CD3 on the CD1+ CD3+ subpopulation of thymocytes apparently is not sufficient to render the cells responsive to the signals of anti-CD3 and IL 2.
The effect of LPS on expression of the early "competence" genes JE and KC in murine peritoneal macrophages.
Authors: Introna M , Bast R C , Tannenbaum C S , Hamilton T A , Adams D O
Published: December 2022

The expression of early "competence" genes has been examined in murine peritoneal macrophages treated with bacterial lipopolysaccharide (LPS). This set of genes (e.g., c-myc, c-fos, r-fos, JE, and KC) were first described in BALB/c 3T3 cells treated with platelet-derived growth factor. We have previously reported that LPS induces the rapid and transient expression of both c-myc and c-fos in macrophages. In the present report, we present evidence demonstrating that the mRNA for JE and KC are also induced in macrophages after treatment of LPS. The r-fos gene was not detectably induced by LPS under the experimental conditions used in this study. The induction of JE and KC were dependent upon the dose of LPS and exhibited different time courses. mRNA for both KC and JE was induced within 30 min from the initiation of treatment. Although mRNA for JE continued to accumulate for up to 24 hr, mRNA for KC was optimally seen after 60 min and had disappeared by 4 hr. c-fos, JE, and KC mRNA were all inducible by a variety of structurally diverse but functionally similar agents (e.g., heat killed Listeria monocytogenes, maleyl-bovine serum albumin, and fucoidan). Interferon-gamma, a potent but functionally distinct stimulus of macrophage activation, did not effect the expression of JE or KC mRNA. The expression of mRNA for c-fos could be readily induced by treatment of macrophages with phorbol myristate acetate (PMA) alone and that for JE by PMA plus the inophore A23187; mRNA for KC was largely unaffected by these agents. These results suggest that expression of the c-fos and JE genes are regulated by products of polyphosphoinositide hydrolysis. The difference between c-fos or JE and KC raises the possibility that LPS may stimulate at least two independent routes of early gene expression. LPS does not promote macrophage proliferative activity alone, and in fact inhibits the proliferative response to the macrophage growth factor colony-stimulating factor 1. Taken together these findings suggest that the products of these genes may function in the acquisition of competence for highly differentiated functions in addition to that for cell division.
Opioid-mediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity.
Authors: Peterson P K , Sharp B , Gekker G , Brummitt C , Keane W F
Published: December 2022

Opiate addiction and stress have been associated with altered immune responses. In this study, we evaluated the influence of morphine and the stress responsive opioid peptide beta-endorphin (beta-END) on O-2 and H2O2 production by cultured human peripheral blood mononuclear cells. Exposure of these cells during 48 hr of culture to morphine and beta-END at pharmacologically (10(-8) M) and physiologically (10(-12) M) relevant concentrations, respectively, markedly suppressed peripheral blood mononuclear cell O-2 and H2O2 release in response to the respiratory burst stimuli opsonized zymosan and phorbol myristate acetate. Both opioids also induced a minimal, but statistically significant, increase in resting O-2 and H2O2 generation. The modulatory effects of morphine and beta-END on peripheral blood mononuclear cell oxygen metabolism appeared to involve a classical opioid receptor, because opioid activity was blocked by naloxone and was not observed with N-acetylated-beta-END. Using purified lymphocyte and monocyte preparations, we determined that although opioids directly increase monocyte-resting oxygen metabolism, lymphocytes are the primary target cell in opioid-mediated suppression of monocyte respiratory burst activity. The release of a suppressive product from opioid-triggered lymphocytes was inhibited by cyclosporine. Based on the results of this study, we propose that opioid-mediated suppression of mononuclear phagocyte respiratory burst activity is another factor to be considered in the immunodeficiency of opiate addiction and stress.
Effects of ADP-ribosylation of GTP-binding protein by pertussis toxin on immunoglobulin E-dependent and -independent histamine release from mast cells and basophils.
Authors: Saito H , Okajima F , Molski T F , Sha'afi R I , Ui M , Ishizaka T
Published: December 2022

Pretreatment of rat peritoneal mast cells, human basophils, bone marrow-derived mouse mast cells (BMMC) and mouse mast cell line PT-18 cells with 1 microgram/ml pertussis toxin (PT) failed to inhibit immunoglobulin E (IgE)-dependent histamine release from the cells. In BMMC and PT-18 cells, even 20-hr incubation of the cells with 1 microgram/ml PT, which ADP-ribosylates more than 97% of 41 kDa, alpha-subunit of Ni in the cells, failed to affect the IgE-dependent release of histamine or arachidonate. The results indicate that GTP-binding protein, Ni, is not involved in the transduction of triggering signals induced by cross-linking of IgE receptors. In contrast, pretreatment of rat mast cells with 1 ng/ml to 0.1 microgram/ml PT for 2 hr inhibited histamine release induced by compound 48/80 in a dose-dependent manner. A similar pretreatment with PT inhibited thrombin-induced histamine release from BMMC and N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced histamine release from human basophils in a similar dose-dependent fashion. However, even 20 hr of incubation of sensitized BMMC with 1 microgram/ml PT failed to inhibit either thrombin-induced or antigen-induced breakdown of phosphatidylinositides (PI), i.e., the formation of inositol triphosphate and diacylglycerol, Quin-2 signal, and the release of arachidonic acid. The results indicate that the inhibition of thrombin-induced histamine release by PT-treatment is not due to the inhibition of PI-turnover, and that Ni is not involved in thrombin-induced or antigen-induced (IgE-dependent) hydrolysis of phosphatidylinositides in mast cells.