bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification of a lamprey complement protein homologous to the third component of the mammalian complement system.
Authors: Nonaka M , Fujii T , Kaidoh T , Natsuume-Sakai S , Nonaka M , Yamaguchi N , Takahashi M
Published: January 2023

A lamprey protein homologous to the third component of mammalian complement was isolated from lamprey plasma and was tentatively designated lamprey C3. Lamprey C3 is a major protein in lamprey serum with electrophoretic mobility of beta-globulin and with m.w. of 190,000. It consists of three polypeptide chains (84,000-alpha, 74,000-beta, and 32,000-gamma chains) linked by disulfide bonds. The protein retains a unique internal thiolester bond on the alpha-chain that is cleaved on methylamine treatment or on limited proteolysis with trypsin. Lamprey C3 by itself could not bind to zymosan or rabbit red cells, but it could covalently bind to these substances when activated by other factors present in lamprey serum. The binding of lamprey C3 to activating surfaces is mediated by covalent bonds and is accompanied by limited proteolysis of the alpha-chain. A fragment with an m.w. of 35,000 containing the internal thiolester site was isolated from methylamine-treated lamprey C3 bound to activated thiol-Sepharose by extensive tryptic digestion followed by dithiothreitol treatment. Lamprey C3 functions as the essential factor in phagocytosis of rabbit red cells by lamprey phagocytes. However, it is not involved in naturally occurring hemolytic activity in lamprey serum.
Stereospecific receptors for substance P on cultured human IM-9 lymphoblasts.
Authors: Payan D G , Brewster D R , Goetzl E J
Published: January 2023

The neuropeptide substance P (SP), which has been demonstrated to bind specifically to human blood T lymphocytes and to stimulate their uptake of [3H]thymidine and [3H]leucine, now is shown to bind stereospecifically to cultured human lymphoblasts of the IM-9 line. The specific binding of [3H]SP by IM-9 lymphoblasts increases linearly with the concentration of IM-9 lymphoblasts, achieves a plateau after approximately 15 to 20 min at 4 degrees C and 4 to 6 min at 37 degrees C, and is rapidly reversible at both 4 degrees C and 37 degrees C. The binding of [3H]SP at steady-state conditions demonstrates a dissociation constant (KD) of 0.65 +/- 0.19 nM (mean +/- SD, n = 5) and 22,641 +/- 6143 receptors per IM-9 lymphoblast. Maximal specific binding of [3H]SP to IM-9 lymphoblasts is observed at pH 7.4 and is dependent on the presence of Mg2+, but not Ca2+, in the medium. The peptide structural determinants of the inhibition of binding of [3H]SP to IM-9 lymphoblasts by substituent peptides and homologs of SP indicate that the receptors recognize predominantly the carboxy-terminal portion of SP. The characteristics of the interaction of SP with IM-9 lymphoblasts suggests a receptor-directed mechanism by which neuropeptides may modulate specifically the contributions of lymphocytes to immunity.
Flow cytometric quantitation of oxidative product formation by polymorphonuclear leukocytes during phagocytosis.
Authors: Szejda P , Parce J W , Seeds M S , Bass D A
Published: January 2023

Stimulation of the oxidative metabolic burst of human polymorphonuclear leukocytes (PMNL) may occur by an all-or-none trigger mechanism or by a graded response to increasing stimulation of an individual cell. If the proposed all-or-none mechanism occurred during phagocytosis, a PMNL would expend all of its metabolic potential at once, yet PMNL can proceed to ingest multiple organisms. This study employed dual laser flow cytometry to correlate the number of cell-associated organisms with oxidative product formation in individual PMNL. Intracellular oxidation of nonfluorescent 2',7'-dichlorofluorescein (DCFH) to highly fluorescent 2',7'-dichlorofluorescein (DCF) provided a quantitative assay of H2O2-dependent oxidative product formation generated by the cell's oxidative metabolic burst. Staphylococcus aureus were fixed and stained with Texas red to allow simultaneous monitoring of bacteria (red fluorescence, greater than 580 nm) and DCF (green fluorescence, 510 to 550 nm) content of each cell. Computer correlation of bacterial and DCF fluorescence allowed determination of the DCF formation by PMNL containing specific numbers (0 to 15) of bacteria. Oxidative product formation was directly related to the number of bacteria ingested in a time-dependent manner (mean per cell of 6.4, 12.8, 19.1, and 24.4 attomoles (amol) DCF formed per cell per bacterium after 15, 30, 45, and 60 min, respectively. Opsonization of bacteria with fresh normal serum (primarily C3b opsonization) or with specific IgG demonstrated qualitatively similar responses, except that the response per IgG-opsonized organism was, on the average, more than twice the response to bacteria opsonized with serum. Thus, sequential phagocytosis of multiple bacteria elicits an incremental oxidative response of human PMNL.
The immune response of the mouse to lymphocytic choriomeningitis virus. IV. Enumeration of antibody-producing cells in spleens during acute and persistent infection.
Authors: Moskophidis D , Lehmann-Grube F
Published: January 2023

The solid-phase immunoenzymatic technique for the enumeration of single rat cells producing antibodies against ovalbumin has been adapted to mouse cells producing antibodies against lymphocytic choriomeningitis (LCM) virus. After intravenous (i.v.) infection with 10(3) infectious units, IgM- and IgG-producing cells appeared on days 4 and 5, respectively. They rose to high numbers on days 7, 8, and 9 and were still detectable on day 38. After a second i.v. inoculation of 10(7) infectious units, antibody-producing cells (APC), most of which made IgG, appeared faster and reached much higher numbers. Mutatis mutandis, very similar results were obtained with mice inoculated with vesicular stomatitis virus. Antibodies were specific as to virus, but probably corresponded to more than one viral antigen. Relatively low numbers of APC were also detected in the spleens of NMRI strain carrier mice, which develop severe immune complex disease in later life, but not in spleens of persistently infected young mice of strains that remain free of late immune complex disease, namely CBA/J, C3H/HeJ, and gray house mice; APC were detected in spleens of aging CBA/J but not gray house mice.
Studies on the mechanism of natural killer cell-mediated cytotoxicity. V. Lack of NK specificity at the level of induction of natural killer cytotoxic factors in cultures of human, murine, or rat effector cells stimulated with mycoplasma-free cell lines.
Authors: Wright S C , Bonavida B
Published: January 2023

We have proposed that lysis of target cells by NK cells is mediated by NK cytotoxic factors (NKCF). According to our model, for a target cell to be NK-sensitive, it must be recognized by the NK cell, it must stimulate the release of NKCF, and it must be sensitive to lysis by these factors. This report examines whether the ability to stimulate release of NKCF is a characteristic restricted to NK-sensitive tumor cells or whether it is also a property of NK-resistant target cells. Many different types of cell lines were tested for their ability to stimulate release of NKCF in the human, rat, and murine systems. It was found that mycoplasma-free NK-sensitive cell lines, resistant cell lines, and Con A could stimulate the release of NKCF. Many different types of cell lines grown in suspension or in monolayers were found to be effective stimulators, including T or B lymphoid, myeloid, and those of histiocytic origin. Cells cultured in the absence of serum stimulated NKCF release, thus ruling out the possible involvement of serum components in stimulation. NKCF was also produced by xenogeneic combinations of effector and stimulator cells, demonstrating lack of species specificity in NKCF production. Factors stimulated by NK-resistant cell lines or by Con A exhibited the same NK target specificity as supernatants stimulated by NK-sensitive tumor cells. The finding that many different NK-resistant cell lines can stimulate the release of NKCF indicates that there is no apparent NK specificity at the level of induction of NKCF release from human, rat, or murine effector cells. Therefore, the NK specificity of a target cell is determined ultimately by its sensitivity to lysis by NKCF.
Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. I. Impairment of mitogen responses and suppressor phenomena.
Authors: Holda J H , Maier T , Claman H N
Published: January 2023

In our laboratory, we have developed a murine model to examine GVHD across minor histocompatibility antigens. In our model, GVHD is induced by injecting B10.D2 spleen cells into irradiated BALB/c recipients. Seven to 10 days after irradiation and injection of cells, there are significant changes in cell function in the recipient spleens. In the B10.D2----BALB/c (600 rad) model, recipient spleen cells are profoundly unresponsive to Con A and LPS stimulation but show increased B cell activity measured by Staphylococcus aureus protein A plaque-forming activity. Spleen cells from such GVH mice profoundly suppress the mitogenic responses of normal BALB/c or B10.D2 spleen cells to Con A and LPS. The degree of impairment of the mitogenic response and the ability to suppress normal cells is proportional to the dose of cells used to induce GVH reactions. Both the inability to respond to mitogens and the capacity to suppress are also related to the dose of irradiation given to the recipients. In addition, immunosuppression across minor histocompatibility antigens shows an unevenhandedness. If we inject parental B10.D2 or BALB/c cells into F1 recipients (P----F1), there is greater inhibition of mitogenic responses when B10.D2 parental cells are given than when BALB/c cells are given to the irradiated F1 recipients. These experiments show that significant immunosuppression occurs during GVH reactions across minor histocompatibility barriers. The degree of suppression varies according to the dose of cells used to induce GVH, the dose of irradiation to the recipient and the "strength" of the GVH recognition system. Such experiments provide models for GVH disease seen in humans who receive treatment for leukemia or other diseases that involves recipient irradiation and infusion of HLA-identical bone marrow.
Delayed reduction in T cell precursor frequencies accompanies diet-induced lifespan extension.
Authors: Miller R A , Harrison D E
Published: January 2023

Recent experimentation has suggested that a decline in the proportion of lymphocytes that can respond to antigenic stimulation may contribute to the loss in immune function with increasing age. If a diminution of precursor frequency is a fundamental element of immunosenescence, then one would expect that manipulations which extend lifespan would also lead to a retarded decline in the measured proportion of reactive cell. Food restriction to about two-thirds of normal intake, initiated at weaning, has long been known to extend the lifespan of mice and rats. We show here that food restriction from weaning causes old mice to retain high levels of T cell precursors in both the helper and cytotoxic lineages. This association of delayed precursor cell loss with extended lifespan supports the hypothesis that changes in T cell precursor frequency may be a key component of age-related immunodeficiency.
Development and distribution of a human B cell subpopulation identified by the HB-4 monoclonal antibody.
Authors: Tedder T F , Clement L T , Cooper M D
Published: January 2023

Monoclonal antibodies have been successfully used to identify B cell differentiation antigens, few of which mark discrete B cell subpopulations. We have produced a monoclonal antibody, HB-4, against a cell surface antigen on the human B cell line, BJAB, which has an unusual distribution on normal lymphoid cells. HB-4, an IgM antibody, was found to react with an antigen that is expressed by a subpopulation of B cells, approximately 50% of natural killer cells, and not by other types of cells in bone marrow, blood, and lymphoid tissues. In two-color immunofluorescence assays, the HB-4-reactive antigen was found on less than 5% of immature IgM+ B cells in fetal liver and bone marrow and on 25% of B cells in fetal spleen. The HB-4 antibody reacted with 40% of IgM+ cells in newborn blood and 60% of B cells in adult blood. In contrast, only 2 to 26% of IgM+ B cells in the peripheral lymphoid tissues of adults were HB-4+. HB-4+ B cells could be induced to proliferate by cross-linkage of their surface immunoglobulins but not by T cell-derived growth factors. The subpopulation of activated B cells that is responsive to T cell-derived differentiation factors was HB-4-, as were plasma cells. The HB-4 antibody was reactive with some but not all B cell malignancies and cell lines, and not with malignancies or cell lines of other lineages. The HB-4 antigen may therefore serve as a useful nonimmunoglobulin marker for the identification of a subpopulation of mature resting B cells that are present in the highest frequency in the circulation.
Activation of a human T cell line: a two-stimulus requirement in the pretranslational events involved in the coordinate expression of interleukin 2 and gamma-interferon genes.
Authors: Wiskocil R , Weiss A , Imboden J , Kamin-Lewis R , Stobo J
Published: January 2023

In the resting state, the T3-positive, human T cell line Jurkat does not synthesize detectable amounts of either interleukin 2 (IL 2) or gamma-interferon (IFN-gamma). Activation of Jurkat as measured by the secretion of substantial amounts of both lymphokines requires two distinct signals. One signal is produced by the phorbol ester, phorbol myristate acetate, and the other by either phytohemagglutinin or antibodies to T3. To elucidate the molecular events by which these activation signals lead to the synthesis of IL 2 and IFN-gamma activity we used cDNA probes to follow the appearance of IL 2 and IFN-gamma-specific transcripts after activation of Jurkat. These studies demonstrate that both signals are required for the appearance of IL 2 or IFN-gamma-specific transcripts and that the appearance of IL 2 and IFN-gamma RNA is coordinate with regard to a) the signals required for their production, b) the kinetics of their appearance, and c) the inhibition of their appearance by cyclosporin A. These studies suggest that distinct T cell-activation signals may operate through a common regulatory pathway involved in the expression of both IL 2 and IFN-gamma genes.
Monoclonal antibodies to murine gamma-interferon which differentially modulate macrophage activation and antiviral activity.
Authors: Schreiber R D , Hicks L J , Celada A , Buchmeier N A , Gray P W
Published: January 2023

Four monoclonal IgG antibodies to purified, recombinant murine gamma-interferon (rIFN-gamma) have been produced by fusion of immune hamster splenocytes with HAT-sensitive murine myeloma cells. Specificity was confirmed either with an enzyme-linked immunosorbent assay (ELISA) that used immobilized rIFN-gamma or with a radioimmunoassay that employed soluble 125I-rIFN-gamma and heat-killed, fixed Staphylococcus aureus-bearing Protein A. Competition binding experiments suggested that the monoclonal antibodies (MoAb) displayed two distinct epitope specificities: one displayed by H1 and H2, and the other displayed by H21 and H22. By using murine-human recombinant IFN-gamma hybrid molecules, the H1/H2 epitope was shown to depend on the amino-terminus of IFN-gamma, whereas the H21/H22 epitope was formed by the carboxy-terminal amino acid sequence. The MoAb also reacted with natural IFN-gamma. When bound to a surface, all four MoAb, but not normal hamster IgG, removed 100% of the antiviral and MAF activities present in supernatants of cultures of the murine 24/G1 T cell hybridoma. In free solution, all four antibodies inhibited IFN-gamma dependent antiviral activity, but with different efficiencies. Soluble H21/H22 also blocked all of the 24/G1-derived activity that induces nonspecific tumoricidal activity in macrophages (MAF) while H1/H2 enhanced MAF activity. The differential inhibitory or enhancing activities of H21 or H1 reflected their ability to inhibit or enhance binding of 125I-rIFN-gamma to macrophages, respectively. Soluble H21/H22 and solid-phase H1/H2 inhibited 100% of the MAF, microbicidal, and Ia-inducing activities from lymphokine preparations produced by mitogen stimulation of normal murine splenic cells. These results help to establish definitive structure-function relationships for the IFN-gamma molecule, and indicate that IFN-gamma is the primary lymphokine responsible for inducing nonspecific tumoricidal activity and Ia antigen expression, and for enhancing microbicidal activity in macrophages.
Ontogeny of proliferative and cytotoxic responses to interleukin 2 and concanavalin A in murine fetal thymus.
Authors: Teh H S , Ho M
Published: January 2023

The ontogeny of proliferative and cytotoxic responses to concanavalin A (Con A) and interleukin 2 (IL 2) in C57BL/6J (B6) fetal thymus (FT) was investigated. Embryonic thymocytes were either taken from embryos at different times of gestation or from 14 day B6 FT that were maintained as organ cultures for various times. It was found that the B6 FT could proliferate to Con A and EL4 SN (an IL 2 containing culture supernatant) in a synergistic fashion. This synergy between Con A and EL4 SN was first observed at the 16th to 17th day of gestation. A similar differentiation process took place in 14-day FT that had been maintained as organ cultures; the synergy between Con A and EL4 SN was first observed after 3 days in organ culture. This synergy increased with increasing time of organ culture, and was most evident after 10 days. The synergy between Con A and EL4 SN was also observed when the EL4 SN was replaced with IL 2 which had been purified from crude EL4 SN to apparent homogeneity. B6 FT could also form cytotoxic T lymphocytes (CTL) on stimulation with Con A and EL4 SN. Con A-activated CTL (polyspecific) were detected by including phytohemagglutinin in the assay medium. CTL response was first detected in the 17-day fetal thymus by using this assay. In organ cultures, CTL responses were first detected after 4 days in organ culture, and reached peak levels after 12 to 14 days. The CTL precursor (CTL-P) frequencies in the B6 FT after 2, 5, 10, and 14 days in organ culture were less than 1/10,000, 1/2232, 1/297, and 1/70, respectively; the corresponding CTL-P frequency in adult thymus was 1/60. After 6 days in organ culture, B6 FT could also form CTL in response to Con A and pure IL 2. This finding suggests that the ability to synthesize other differentiation factors that are required for CTL responses is acquired at an early time of thymic differentiation.
The roles of T cell factors in activation, cell cycle progression, and differentiation of human B cells.
Authors: Jelinek D F , Lipsky P E
Published: January 2023

The relationship of the T cell influences involved in human B cell activation and differentiation into immunoglobulin-secreting cells (ISC) was investigated. T cell supernatants (T supt) generated by stimulating T cells with phytohemagglutinin and phorbol myristate acetate contained activities capable of augmenting DNA synthesis and the growth of mitogen-stimulated B cells and supporting the differentiation of ISC. To examine the role of T supt in B cell activation and the progression through the cell cycle, T cell- and monocyte-depleted B cells were stimulated with formalinized Cowan I strain Staphylococcus aureus (SA), and the percentages of cells in G1, S, and G2 + M were determined by acridine orange staining and analysis. In all experiments, a similar percentage of cells entered G1 during the first 24 to 36 hr of culture when stimulated with SA or SA + T supt. Similar results were seen when B cell activation was analyzed by acquisition of a number of other markers of cell activation. Analysis of cell cycle progression with mithramycin staining of cellular DNA in the presence or absence of vinblastine to arrest mitosis indicated that SA-activated B cells were able to complete S and divide in the absence of T supt. Although an effect of T supt on the progression of B cells through the S phase was evident during the first cell cycle, the major effect only became apparent after the first round of cell division. Although T supt was not necessary for initial B cell activation, T cell influences were absolutely necessary for the differentiation of ISC. T supt did not need to be present during the initial 24 to 36 hr of incubation to permit subsequent generation of ISC. However, when T supt was present initially, an increased number of ISC were produced. Hydroxyurea elimination of cells traversing the G1-S interphase indicated that reception of the differentiation signal occurred before the S phase, but that the generation of ISC required subsequent DNA synthesis and/or cell division. Although precursors of ISC were entirely contained within the population triggered to divide by SA alone, there was no preferential expansion of such precursors as a result of SA stimulation. These results indicate that T cell signals are not absolutely necessary for initial B cell activation and progression through the first cell cycle, although T cell factors promote DNA synthesis by some activated B cells. In contrast, differentiation into ISC is completely dependent on T cell influences.(ABSTRACT TRUNCATED AT 400 WORDS)
Pregnancy-associated growth factor. II. A T-dependent polyclonal activator of human adult peripheral blood lymphocytes (PBL).
Authors: Carlino J A , Morse J H
Published: January 2023

This study examined the ability of pregnancy-associated growth factor (PAGF), a substance found in crude human chorionic gonadotropin (hCG), to induce plaque-forming cells (PFC) in cultured human peripheral blood lymphocytes (PBL). PAGF, 0.25 to 1 mg/ml, induced maximal PFC at 6 to 7 days as measured by the staphylococcal protein A-coupled SRBC reverse hemolytic plaque assay with a rabbit anti-human Ig antiserum. PAGF-induced PFC/culture ranged from 1800 to 39,000 with a mean of 11,524 in unfractionated PBL (N = 24), as compared to 540 to 77,840 with a mean of 17,303 for pokeweed (PWM) (N = 22). Comparison of PAGF- and PWM-induced PFC showed that both induced specific IgG, IgA, and IgM PFC. In most individuals, PAGF induced more IgM and PWM more IgG PFC. The kappa: lambda ratio was 1.5 for unstimulated PBL, and approximately 3.5 for PAGF and PWM. To see if PAGF was a T-dependent polyclonal activator of B cells, T and non-T populations were obtained by SRBC rosettes and negatively selected T4 and T8 cells by complement-mediated lysis of SRBC+(T) cells. Only the recombined subsets which included T4 cells and non-T cells supported PAGF- and PWM-induced PFC. These data indicate that PAGF, a substance derived from commercial extracts of pregnancy urine, is a T4-dependent polyclonal activator of normal human B cells.
Induction of cAMP-dependent protein kinase I during human monocyte differentiation.
Authors: Wenger G D , O'Dorisio M S
Published: January 2023

Differentiation of human peripheral blood monocytes into macrophages was accompanied by induction of the regulatory subunit of cAMP-dependent protein kinase I as determined by photoaffinity labeling of cytosol proteins with 8-N3-[32P]cAMP and DEAE-Sephacel chromatography. The appearance of cAMP-dependent protein kinase I in macrophages was not due to translocation from the particulate fraction of monocytes. The regulatory subunit of cAMP-dependent protein kinase II was present in both monocytes and in vitro-differentiated macrophages. Protein kinase I in macrophages demonstrated higher affinity for 8-N3-cAMP (KD = 0.7 nM) than did protein kinase II from either monocytes (KD = 14.5 nM) or macrophages (KD = 4.9 nM). These studies demonstrate induction of the regulatory subunit of cAMP-dependent protein kinase I during the differentiation of a normal human cell and support the hypothesis that cAMP may regulate some stages of differentiation.
Oxidant membrane injury by the neutrophil myeloperoxidase system. I. Characterization of a liposome model and injury by myeloperoxidase, hydrogen peroxide, and halides.
Authors: Sepe S M , Clark R A
Published: January 2023

Neutrophils and other phagocytes can injure cells by means of oxygen-dependent mechanisms, particularly the myeloperoxidase (MPO)-H2O2-halide system. The extent of such damage depends in part on the antioxidant defenses of the target cell. To facilitate the study of this phenomenon, we developed a model system in which we employed liposomes as targets for the myeloperoxidase system. The most useful species of liposomes employed 51Cr as the aqueous space marker and phosphatidyl choline with or without dicetyl phosphate and cholesterol as the structural lipid. Marker entrapment was established on the basis of 1) resolution of free from lipid-associated 51Cr by gel exclusion chromatography, 2) latency of 51Cr on rechromatography of detergent-treated liposomes, and 3) a correlation between entrapment and surface charge density. Exposure of liposomes to the complete MPO system resulted in release of 50 to 75% of the entrapped 51Cr. Release was abrogated by omission of myeloperoxidase or H2O2, heating of MPO, or addition of azide, cyanide, or catalase. Reagent H2O2 could be replaced by glucose plus glucose oxidase. Kinetic studies indicated a rapid process, lysis reaching half-maximal levels in less than 2 min. The addition of cyanide at various times interrupted lysis at once, indicating a requirement for ongoing myeloperoxidase-dependent reactions. Liposome disruption by the MPO system was pH dependent, increasing dramatically as pH was decreased from neutrality to 6.0. In the absence of halides, no lysis was observed. Maximum lysis was found with chloride at 10 to 100 mM, although at 1 mM concentrations, iodide, bromide, and thiocyanate were more active than chloride. Fluoride was inactive. Antagonism between halide species was demonstrated in that low concentrations of iodide or bromide inhibited the effect of optimal concentrations of chloride. Using 125I, we found that exposure of liposomes to the MPO system resulted in an association between iodide and liposomes; moreover, there was a close correspondence between this phenomenon and 51Cr release, suggesting that halogenation may be one mechanism of injury. These studies establish the usefulness of the liposome as a model of oxidant injury by a physiologically relevant system. They bear a striking parallel to work being done on MPO-mediated injury to eukaryotic and prokaryotic cells. By using this simplified model system, it should be possible to explore a number of determinants of target cell injury at a biochemical and molecular level.
Reactivity of Leu-1+ tonsillar B cells to a high molecular weight B cell growth factor.
Authors: Richard Y , Leprince C , Dugas B , Treton D , Galanaud P
Published: January 2023

This work was focused on the responsiveness of B cells from blood and tonsils to typical B cell growth factors (BCGF) in the absence of anti-mu antibody. This direct responsiveness was not observed with small dense B cells from either organ. Large tonsillar B cells but not large blood B cells did respond directly to a high m.w. BCGF (m.w. 50,000 BCGF), whereas large B cells from both organs responded directly to the low m.w. BCGF (m.w. 20,000 BCGF). The tonsillar B cells directly responsive to the m.w. 50,000 BCGF express the 4F2 marker and thus belong to a population of in vivo-preactivated B cells. Moreover, this direct responsiveness to the m.w. 50,000 BCGF is localized in Leu-1+ tonsillar B cells. The Leu-1+ and Leu-1- tonsillar B cell subsets do not differ in their responsiveness to the m.w. 50,000 BCGF upon costimulation with anti-mu antibody and to the m.w. 20,000 BCGF regardless of the presence of anti-mu antibody. Thus, the Leu-1+ subset present in tonsils shows a particular reactivity to a high m.w. BCGF.
Brugia malayi antigens associated with lymphocyte activation in filariasis.
Authors: Lal R B , Lynch T J , Nutman T B
Published: January 2023

Filarial parasites induce immune response in humans which are still poorly characterized. To define the antigens responsible for inducing lymphocyte responses, fast protein liquid chromatography was used to fractionate the antigens of Brugia malayi adult worms which were then tested on lymphocytes from patients with filariasis and normal controls. From an anion exchange column (Mono Q), three peaks of lymphocyte-stimulating activity were eluted which were further fractionated by gel filtration (Superose-12). Peak I induced both a proliferative response as well as the production of filaria-specific antibody in patient lymphocytes. Peak II, capable of inducing only a proliferative response (without antibody production) in patient lymphocytes, was a glycoprotein with phosphocholine as one of the antigenic determinants. Peak III induced proliferative responses in both patient and normal lymphocytes and thus appears to be mitogenic. Two-dimensional gel electrophoresis was then used to identify changes in the major cellular proteins associated with the activation of patient lymphocytes by these partially purified antigens. Stimulation of patient lymphocytes with peak I resulted in increased synthesis of immunoglobulin heavy, light, and J chains. Further, these were the only major secreted proteins found in the culture supernatants. Peak II resulted in quantitative changes in proteins associated with T and not B lymphocyte stimulation. Further analysis of these antigens should help to elucidate the mechanism of host-parasite interaction at both the cellular and molecular levels.
Recovery of activated cytotoxic T cells from minor H incompatible tumor graft rejection sites.
Authors: Ksander B R , Streilein J W
Published: January 2023

In this study we determined whether minor H-specific cytotoxic T cells and their precursors (pTc) are present at the site of rejection of minor H disparate tumor allografts. Lymphocytes were retrieved from eyes of BALB/c mice that received subconjunctival injections of minor H-incompatible P815 tumor cells. The lymphocytes were then assayed for direct cytotoxic activity as well as precursor frequency by limiting dilution. Similar assays were conducted on cells obtained from the draining lymph nodes and from the spleen. As expected, tumor rejection was accompanied by significant clonal expansion of minor H-specific pTc within the draining lymph node and the spleen. A correspondingly high frequency of pTc was also detected at the graft site. More importantly, fully functional cytotoxic T cells were recovered from the tumor graft site during rejection, but no similarly active cells were found in either the draining nodes or spleen. We conclude that, after Ag stimulation, pTc are generated in draining central lymphoid compartments. From this generative site, the precursor cells then disseminate systemically, gradually reaching and infiltrating the tumor graft site. A further activation step, dependent upon Ag and T cell help, permits these cells to mature into fully active cytotoxic cells which can then effect tumor rejection. We propose that the terminal stage(s) of pTc activation is promoted by lymphokines released locally from TDH cells that are also generated during the alloimmune response and simultaneously infiltrate the site.
Arsonate-specific murine T cell clones. V. Antigen presentation by L cells transfected with normal and mutant class II genes.
Authors: Norton F L , Davis C B , Jones P P , Goodman J W
Published: January 2023

Class II-restricted murine T cell clones specific for the immunogenic determinant L-tyrosine-p-azobenzenearsonate failed to proliferate to Ag presented by L cell lines transfected with and expressing the appropriate class II genes, but are activated to kill the APC in an Ag-dependent, MHC-restricted manner. Inhibition of APC proliferation was used as an assay to determine the relative contributions of polymorphic sites on the class II alpha- and beta-chains to MHC-restricted activation of I-A beta k-restricted cloned T cells. Transfectants expressing A beta k in conjunction with the alpha chain of k, u, or d were equally effective APCs, whereas transfectants expressing A beta u were completely ineffective, implicating the beta-chain as more critical for the presentation of L-tyrosine-p-azobenzenearsonate. Site-directed mutagenesis of polymorphic positions in the beta chain revealed a remarkable stringency for the k haplotype, in contrast to the relaxed alpha-chain requirement. These results, in conjunction with others, indicate that the relative contribution of polymorphic sites on class II alpha- and beta-chains to T cell Ag recognition can differ markedly, and, furthermore, may vary as a function of the Ag.
Antibodies against a peptide representative of a conserved region of human IFN-alpha. Differential effects on the antiviral and antiproliferative effects of IFN.
Authors: Barasoain I , Portolés A , Aramburu J F , Rojo J M
Published: January 2023

mAb generated from mice immunized with a synthetic peptide representative of a conserved region (amino acids 133 to 147) of human IFN-alpha are described. Antisera from mice immunized with the peptide coupled to keyhole limpet hemocyanin were able to specifically bind to the peptide and also to bind and precipitate human IFN-alpha. Binding of the antibodies to IFN-alpha was inhibited by the immunizing peptide, but not by an unrelated peptide. Immunized mice were used to obtain three hybridomas producing mAb able to recognize both the immunizing peptide and human IFN-alpha, as determined by RIA and immunoprecipitation. These antibodies also neutralized the antiviral effect of human leukocyte IFN. In contrast, none of the mAb significantly affected the inhibition of Daudi cell proliferation induced by IFN-alpha.
A single amino acid substitution in HLA-A2 can alter the selection of the cytotoxic T lymphocyte repertoire that responds to influenza virus matrix peptide 55-73.
Authors: Shimojo N , Cowan E P , Engelhard V H , Maloy W L , Coligan J E , Biddison W E
Published: January 2023

Previous studies have demonstrated that certain amino acid substitutions in the alpha two domain at positions 152 and 156 in the alpha two helix of the HLA-A2 molecule can affect presentation of the influenza virus matrix peptide M1 55-73 without abolishing binding of the M1 peptide. HLA-A2.1-restricted M1 55-73 peptide-specific CTL lines obtained from almost all HLA-A2.1+ individuals fail to recognize the M1 peptide presented by site-directed mutants of HLA-A2 that have either a Val----Ala or Val----Gln substitution at position 152 or a Leu----Trp substitution at position 156. Only one HLA-A2+ individual (donor Q66, HLA-A2,-B53,-B63) has been found who is able to generate a unique repertoire of HLA-A2-restricted M1 peptide-specific CTL that can recognize peptide presented by HLA-A2 mutants with either an Ala or Gln substitution at position 152 or a Trp substitution at position 156. These Q66 M1 peptide-specific CTL could be selected by stimulation with M1 peptide-pulsed transfectants that express the mutant HLA-A2 gene with the Trp substitution at 156. To determine if the presence of the unique CTL repertoire could be attributed to a variant HLA-A2 molecule in Q66, sequences were determined from polymerase chain reaction-amplified segments of the HLA-A2 RNA. Two different HLA-A2 genes were found expressed in Q66 cells: one is identical to HLA-A2.1 and the other is identical to HLA-A2.2F (Gln----Arg at position 43, Val----Leu at position 95, and Leu----Trp at position 156). These results demonstrate that a different CTL repertoire specific for HLA-A2 plus the M1 55-73 peptide is generated in an individual that expresses both HLA-A2.1 and HLA-A2.2F compared to individuals who express HLA-A2.1 alone, and that the unique repertoire can be selected by the presence of an HLA-A2 molecule with a single amino acid substitution at position 156.
Neutrophil cathepsin G increases calcium flux and inositol polyphosphate production in cultured endothelial cells.
Authors: Peterson M W , Gruenhaupt D , Shasby D M
Published: January 2023

Exposure of endothelial cells (ENDO) to human neutrophil cathepsin G (CG) increases albumin flux across the endothelial monolayer. Since calcium influences cell shape and barrier function of ENDO monolayers, the current study was designed to determine if CG acted through alterations in Ca2+ homeostasis in ENDO. The role of Ca2+ in the increased permeability of ENDO monolayers to albumin after exposure to CG was studied by using ENDO monolayers cultured on polycarbonate filters. Exposure of ENDO monolayers to CG in the presence of the Ca2+-antagonist lanthanum partially prevented the increase in albumin flux, but exposure in the presence of agents that block voltage-regulated calcium channels did not block the increase in albumin flux. To monitor the effect of CG on Ca2+-flux, ENDO were labeled with 45Ca2+ and changes in Ca2+ flux were monitored by the release of 45Ca2+. From 1 to 15 minutes after exposure of ENDO to CG, there was increased release of 45Ca2+ compared with control cells. Calcium channel blocking agents did not inhibit the increased release of 45Ca2+, but lanthanum partially blocked the increase. The increased release of Ca2+ appeared to be due, at least in part, to activation of phospholipase C because there was an increase both in inositol polyphosphate species and in diglycerides after incubation of ENDO with CG. These studies support the hypothesis that CG increases the flux of calcium in ENDO, that this increase in Ca2+ flux may result from activation of phospholipase C, and that this system may be involved in the decreased barrier properties of the ENDO after CG exposure.
Comparative studies on FcR (FcRII, FcRIII, and FcR alpha) functions of murine B cells.
Authors: Tsujimura K , Park Y H , Miyama-Inaba M , Meguro T , Ohno T , Ueda M , Masuda T
Published: January 2023

Distribution of FcR II, FcRIII, and FcR alpha on murine splenic B cells was examined by using FITC-labeled heat-aggregated IgG of each subclass and IgA. Almost 60 to 80% of B cells expressed both FcRII and FcRIII. However, FcR alpha was expressed on only a small proportion (6%) of B cells that co-expressed FcRII. By inhibition assays with the use of cold IgG of each subclass and IgA in addition to anti-FcRII mAb (2.4G2), it was found that IgG1, IgG2a, and IgG2b utilized the same receptor (FcRII), whereas IgG3 and IgA bound only to their unique receptors, FcRIII and FcR alpha, respectively. Immune complexes IC prepared by IgG1, IgG2a, IgG2b, and IgA anti-TNP mAb with TNP-coupled SRBC inhibited the polyclonal Ig secretion and proliferative responses of B cells stimulated with either IL-4 or LPS. The inhibition of B cell activation was associated with the blockade of the membrane depolarization. Moreover, IC prepared by these antibodies caused production of suppressive B cell factor (SBF) as is the case with rabbit IgG antibody to SRBC, and SBF thus prepared regulated antibody responses in an isotype-nonspecific manner. In contrast, no inhibition for these responses or production of SBF was attained by the IC of IgG3 antibody. We concluded that FcRII and FcR alpha mediates a suppressive signal for B cells by acting on the initial step of activation, whereas FcRIII lacks this activity.
Stimulation of a T helper cell class 2 clone with immobilized anti-T cell receptor antibody activates a Ca2+ and protein kinase C-independent lethal signaling pathway.
Authors: Cherwinski H M , Semenuk G T , Ransom J T
Published: January 2023

Stimulation of an IL-2-dependent variant of the Th2 clone D10.G4.1 with antibodies (Ab) specific for CD3 epsilon or the TCR-alpha beta caused either activation of the clone to secrete the autocrine lymphokine IL-4, or lethal activation in which the cells secreted high quantities of IL-4 but then died within 2 days. High densities of immobilized Ab delivered a lethal signal, whereas soluble forms of Ab and low densities of immobilized Ab caused productive activation in which cell viability was maintained. Lethal activation was not prevented by accessory cells, IL-1, or IL-2, or by co-cross-linkage of CD4 and TCR. The lethal signal was not mediated via a soluble effector from the activated cells. Lethal signaling was insensitive to cyclosporin A or dexamethasone. Studies with activators of protein kinase C (PKC), and PKC inhibitors, indicated that direct activation of PKC was not sufficient for lethal signaling. Nor could direct activation of PKC prevent the lethal signal. The lethal signal was not caused by Ca2+ mobilization mediated by Ca2+ ionophore and there was no evidence of apoptosis. The combination of a PKC activator and Ca2+ ionophore was not lethal, thereby showing that together these events are not sufficient. That these signal pathways were not necessary for lethal activation was evidenced by their inability to lower the density of immobilized anti-CD3 required to cause cell death. In this model, ligation of the TCR specifically activates a Ca2+/PKC-independent lethal signal transduction pathway.
IL-12 receptor. II. Distribution and regulation of receptor expression.
Authors: Desai B B , Quinn P M , Wolitzky A G , Mongini P K , Chizzonite R , Gately M K
Published: January 2023

IL-12 is a heterodimeric lymphokine that induces IFN-gamma production by resting PBMC, enhances the lytic activity of NK/lymphokine activated killer cells, and causes the proliferation of activated T cells and NK cells. In this report, we have investigated the expression of IL-12R on mitogen- and IL-2-activated PBMC or tonsillar lymphocytes as well as on a variety of cell lines. The results of radiolabeled IL-12-binding assays indicated that high affinity IL-12R are present on PBMC activated by various T cell mitogens or by IL-2. High affinity IL-12R were also found to be expressed constitutively on a transformed marmoset NK-like cell line HVS.SILVA 40. At the time of peak IL-12R expression, mitogen- or IL-2-activated cells displayed approximately 1000 to 9000 IL-12 binding sites/cell with an apparent Kd of 100 to 900 pM. Kinetic studies revealed that maximum expression of IL-12R occurred earlier on PHA-activated PBMC as compared with PBMC activated by IL-2, and that expression of IL-12R on these cells correlated with their ability to proliferate in response to IL-12. Although IL-2 could up-regulate IL-12R expression on resting PBMC, the ability of mitogen-activated PBMC to up-regulate IL-12R was found to be independent of IL-2. Analysis of IL-12R expression by flow cytometry revealed that receptors for IL-12 are present on activated T cells of both the CD4+ and CD8+ subsets and on activated CD56+ NK cells. In contrast, neither resting PBMC or tonsillar B cells nor tonsillar B cells activated by anti-IgM/Dx, anti-IgM/Dx + IL-2, or SAC + IL-2 displayed IL-12R detectable by flow cytometry or by the radiolabeled IL-12-binding assay. In summary, these results indicate that activation of T cells or NK cells results in up-regulation of IL-12R expression; on the other hand, B cell activation, at least under some circumstances, appears not to be associated with enhanced expression of IL-12R.
IL-8 stimulates phosphatidylinositol-4-phosphate kinase in human polymorphonuclear leukocytes.
Authors: Pike M C , Costello K M , Lamb K A
Published: January 2023

IL-8 is a neutrophil-specific chemoattractant and cellular activator which exists in at least three forms, 69, 72, and 77 amino acids. The predominant monocyte product has 72 amino acids, whereas endothelial cells secrete the 77-amino acid form. The 72-amino acid form has been shown to increase intracellular calcium in neutrophils, but the exact biochemical pathways involved in stimulation of these cells is unknown. N-formyl peptide chemoattractants in neutrophils stimulate the formation of phosphatidylinositol-4,5-bisphosphate (PIP2), a reservoir for second messenger molecules and regulator of actin assembly through its association with the actin-binding proteins, profilin, and gelsolin. The present study examined whether IL-8 altered the enzyme which synthesizes PIP2, phosphatidylinositol-4-phosphate (PIP) kinase. Incubation of intact neutrophils with 10 nM IL-8 caused approximately a twofold increase in the activity of the enzyme. All forms of IL-8 stimulated PIP kinase activity in concentrations ranging from 1 to 50 nM, and the dose-response curves exactly correlated with the order of potency of these cytokines for interacting with the IL-8R on the surface of neutrophils. Lineweaver-Burk analysis of the kinetics of PIP kinase assayed in the presence of 0.03 to 0.7 mM ATP showed that 10 nM IL-8 increased the Vmax of the enzyme 38 to 70.5%, with no significant change in the apparent Km for ATP or for PIP. The stimulation of PIP kinase activity could not be explained by decreased degradation of PIP2 by phospholipase C or phosphomonoesterase activity in the membranes isolated from cells treated with IL-8 or by a decrease in the degradation of ATP. The microfilament disrupter, cytochalasin b, inhibited IL-8 induced stimulation of PIP kinase. These findings demonstrate that all forms of IL-8 stimulate PIP kinase in human neutrophils. This event may provide molecular signals to these cells that are necessary to maintain or change the state of microfilament assembly during cellular activation.
Biologic activity of synthetic analogues of C5a anaphylatoxin.
Authors: Ember J A , Sanderson S D , Taylor S M , Kawahara M , Hugli T E
Published: January 2023

The C activation fragment C5a is the most potent plasma-derived chemotactic factor known. This humoral factor induces both neutrophil and macrophage activation at low nanomolar concentrations. We have synthesized a series of C-terminal C5a analogues that exhibit all of the characteristic biologic activities of C5a. These peptides apparently contain the effector site for C5a receptor-mediated cellular activation, but express only a fraction of the potency of intact C5a. We have demonstrated the following in vitro activities for these C5a peptides: 1) ileal (guinea pig) contraction; 2) platelet (guinea pig) activation; and 3) neutrophil (human) polarization and chemotaxis. The effect of C5a peptides in vivo was evaluated by measuring enhancement in vascular permeability. Although potencies of the most effective synthetic C5a analogues were on the order of 0.01 to 0.1% that of the natural factor, our biologic data confirm that the C5a peptides are full agonists of the intact factor and may be useful substitutes for intact C5a. Furthermore, our results indicate that elongation of the C5a analogues from 10 to 19 residues in length contributes little toward enhancing or decreasing potency of the synthetic C5a analogues. Replacement of residues in the effector region by D-amino acids or by introduction of a cyclic group to reduce flexibility of the backbone decreased potency of the analogues. Substitution of His 67 by Phe in the decapeptide C5a 65-74 resulted in a significant increase in potency of the C5a analogue. The marked enhancement in potency from replacing His 67 by Phe in analogue C5a peptides identifies an important hydrophobic subsite. We conclude that site-specific amino acid modifications in or near the C-terminal effector site sequence can diminish or optimize potency of the model C5a peptides. However, there apparently are subsites on folded C5a, from regions other than the C-terminal portion of the molecule, that contribute significant receptor interactions. These subsites must be identified and incorporated into C5a model peptide designs before expression of full potency by synthetic analogues of this factor will be realized.