bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure-function studies of human granulocyte-macrophage colony-stimulating factor. Identification of residues required for activity.
Authors: Clark-Lewis I , Lopez A F , To L B , Vadas M A , Schrader J W , Hood L E , Kent S B
Published: December 2022

Human granulocyte-macrophage colony stimulating factor (hGM CSF), a protein containing 127 amino acids, was chemically synthesized by using automated stepwise solid-phase methods. The unpurified synthetic hGM-CSF had the same range of actions on hemopoietic cells as the purified recombinant protein. The structural requirements for the activities of synthetic hGM-CSF were examined by the design and synthesis of fragments and analogs. The synthetic fragment, hGM-CSF (54-127), containing all four of the cysteine residues found in the intact protein, lacked detectable activity. Assays of fragments shortened at the N terminus showed that the residues 1-13 were not required for activity, but that the integrity of residues 14-25, particularly residues 16, 17, and 18, was critical for biologic activity. The 14-25 region is predicted to form the first alpha-helix in hGM-CSF. Synthetic peptides within the N-terminal 53 residue region lacked detectable activity. The synthetic analog hGM-CSF (1-121), which lacks the C-terminal 6 residues, had similar activity to hGM-CSF (1-127) indicating that residues 122-127 are not required for activity. An analog, [Ala88] hGM-CSF (14-96), which lacks the hydrophobic C-terminal region and 2 cysteine residues, had low but readily detectable activity suggesting that residues 14-96 are sufficient for detectable synthetic hGM-CSF activity, although the presence of residues 97-121 are required for full activity. No dissociation of the multiple biological activities of hGM-CSF was detected.
Evidence for a C4b binding site on the C2b domain of C2.
Authors: Oglesby T J , Accavitti M A , Volanakis J E
Published: December 2022

We raised murine mAb against human C protein C2. The representative mAb 3A3.3 (IgG1 kappa) recognized an epitope on the C2b domain of C2, as determined by binding and inhibition of binding radioassays. The hemolytic activity of purified human C2 and of C2 in normal human serum was inhibited by the mAb. The rate of decay of the C3-convertase at 30 degrees C was not affected by the mAb. C2 binding to EAC4b was inhibited by intact IgG and the Fab fragment of the mAb; 50% inhibition required 1 microgram/ml of either. The data suggest the presence of a C4b-binding site on the C2b domain of C2 and that the mAb recognizes an epitope at, or adjacent to, this site. The C2b portion of the C2 molecule may be important in assembly of the classical pathway C3-convertase.
IgE-dependent activation of human lung mast cells is not associated with increased phospholipid methylation.
Authors: Benyon R C , Church M K , Holgate S T
Published: December 2022

Enhanced phospholipid methylation has been suggested to be an obligatory process in IgE-dependent stimulus-secretion coupling in human lung mast cells. Our studies with mast cell-enriched lung preparations do not support this hypothesis, demonstrating no increased 3H-methyl radiolabeling of chloroform/methanol-extracted lipids or chromatographically separated phospholipids accompanying anti-IgE-dependent histamine secretion. Inhibitors of transmethylation, 3-deazaadenosine, and homocysteine thiolactone inhibited histamine secretion by both anti-IgE and calcium ionophore A23187, reflecting a requirement of secretion for overall integrity of cellular transmethylation. These agents induced small increases in cAMP concentration which are considered to make at most a minor contribution to this inhibition. The inability of methylation inhibitors to diminish anti-IgE-dependent increases in lung mast cell cAMP levels would suggest that not only does phospholipid methylation have no role in histamine secretion but also it does not participate in the activation of adenylate cyclase by this stimulus.
Eosinophils from granulomas in murine schistosomiasis mansoni produce substance P.
Authors: Weinstock J V , Blum A , Walder J , Walder R
Published: December 2022

Granulomas are chronic, usually focal, tissue-destructive inflammatory reactions that usually form around slowly degradable, poorly soluble substances. They are dynamic lesions, regulated by complex immune mechanisms. Tachykinins are a family of neuropeptides characterized by the common C-terminal amino acid sequence -Phe-X-Gly-Leu-Met-NH2. One such tachykinin, substance P, has been reported to modulate immunologic responses. In this investigation, granulomas were examined for substance P. Granulomas were isolated from the livers of mice infected with murine schistosomiasis, and substance P was extracted. Immunoreactive substance P was detected by RIA. The authenticity of the molecule was confirmed by elution profile on HPLC. Immunoreactive substance P, identified by immunostaining, localized to eosinophils derived from collagenase-dispersed granulomas. Granulomas were then probed for expression of the gene for substance P (preprotachykinin). Preprotachykinin mRNA was localized to granuloma eosinophils by in situ oligonucleotide hybridization. It is concluded that substance P is present within the granuloma as a result of preprotachykinin production by eosinophils.
Regulation of ornithine decarboxylase activity by IL-2 and cyclic AMP.
Authors: Farrar W L , Vinocour M , Cleveland J L , Harel-Bellan A
Published: December 2022

IL-2 regulates the expression and activation of the principal rate-limiting enzyme of polyamine synthesis, ornithine decarboxylase (ODC). The apparent activation of ODC occurs by two independent steps. Rapid activation of enzyme occurs within the first 10 min of lymphokine treatment of cloned IL-2-dependent murine cell lines. The initial rapid rise in enzyme activity is insensitive to protein or mRNA synthesis inhibitors. The early rise in ODC activity is followed by a protracted increase in enzyme activity, apparent protein measured by [3H]difluoromethylornithine binding, and by accumulation of steady state ODC mRNA. Stable analogues of cAMP which inhibit IL-2-stimulated proliferation, suppressed only the mRNA-dependent increase in ODC activity. Phorbol esters were shown to increase steady state levels of ODC mRNA whereas cAMP analogue clearly inhibited the growth factor-induced increase in ODC mRNA. These studies show that IL-2 regulates the expression of ODC at multiple levels, including transcription and accumulation of steady state mRNA, and post-translational activation of an enzyme important for DNA synthesis. Furthermore, anti-proliferative signals such as cAMP may effect the regulation of ODC enzyme production at the level of mRNA accumulation and stability.
High strength binding of P815 mastocytoma cells is not necessary for their lysis by macrophages which have been primed and triggered in vitro.
Authors: Lu C Y , Lombardi M J , Shea C M , Dustin L B
Published: December 2022

We have examined the hypothesis that binding of P815 mastocytoma cells is a necessary step in lysis of these cells by macrophages which are both "primed" and "triggered" in vitro, Macrophages "primed" by conditioned media containing IFN-gamma, or by rIFN-gamma have an increased ability to bind P815. However, adding either heat-killed Listeria or endotoxin to trigger the primed macrophages has opposite effects on lysis and binding of P815. Lysis is increased. Binding is dramatically decreased. This is true when centrifugal forces of 200 x g, 400 x g, and 800 x g are used to disrupt P815-macrophage binding. Although 100% of P815 cells bound by cytotoxic macrophages are lysed, a large additional population of unbound P815 is also lysed. Detailed kinetic studies indicate that macrophages do not rapidly bind and lyse several cycles of P815. There is an initial lag period of 4 to 6 h before P815 lysis can be detected, and completion of lytic events then occurs within 12 to 14 h. Lysis of P815 bound to cytotoxic macrophages is slightly slower than lysis of the total population of bound and unbound P815. In contrast, D3.1, a cloned CD4+ T cell line, is tightly bound to macrophages but not lysed efficiently. When macrophages are simultaneously confronted with P815 and macrophage-bound D3.1, only the former are lysed. Altogether, the data indicate that P815-macrophage binding, as operationally defined by our assay, is not a necessary step for lysis. These results, by use of macrophages primed and triggered in vitro, are in contrast to previously reported experiments examining P815 binding and lysis by macrophages activated in vivo by infection with bacillus Calmette-Guérin.
IL-2 and IL-4 stimulate different subpopulations of double-negative thymocytes.
Authors: Takei F
Published: December 2022

Double-negative (CD4-/CD8-) thymocytes from young adult mice can be separated into two distinct subpopulations on the basis of the binding of mAb 7D4 directed against the receptor for IL-2. The 7D4+ cells have predominantly nonrearranged TCR beta-chain genes and express incomplete 1.0-kb beta-messages, whereas the 7D4- cells have rearranged beta-genes and express complete 1.3-kb as well as incomplete 1.0-kb beta-messages. These two populations of double-negative thymocytes also differ in their responses to IL-2 and IL-4. The 7D4+ cells are nonresponsive to IL-2 alone or IL-2 plus PMA but they are stimulated to proliferate by the combination of IL-4 and PMA. In contrast, the 7D4- cells vigorously proliferate in response to IL-2 alone or IL-2 plus PMA but they respond poorly to IL-4 alone or IL-4 plus PMA. These results suggest that IL-2 and IL-4 may be involved in the stimulation of immature thymocytes at distinct steps of their differentiation. IL-4 together with PMA stimulate immature thymocytes which seem to express the IL-2R but do not respond to IL-2.
A synthetic peptide from myelin proteolipid protein induces experimental allergic encephalomyelitis.
Authors: Tuohy V K , Lu Z J , Sobel R A , Laursen R A , Lees M B
Published: December 2022

Immunization of animals with proteolipid protein, the major protein constituent of central nervous system myelin, produces experimental allergic encephalomyelitis. The goal of the present study was to identify an encephalitogenic determinant of this protein. For this purpose, SWR mice were immunized with five groups of pooled synthetic peptides corresponding to various regions of the myelin proteolipid protein sequence. Clinical EAE was observed in only one group. Inguinal lymph node cells from animals in this group responded ([3H]thymidine incorporation) to a peptide within the pool containing residues 103-116 YKTTICGKGLSATV. Mice subsequently immunized with 50 nmol of this peptide developed severe EAE within 3 wk, and their T cell-enriched inguinal lymph node cells responded specifically to this peptide. Control mice immunized to proteolipid peptide 202-217 DARMYGVLPWNAFPGK did not develop experimental allergic encephalomyelitis, and their inguinal lymph node cells were unresponsive to either peptide. Thus, a peptide corresponding to a sequence within the proteolipid protein can produce classical acute experimental allergic encephalomyelitis. This is the first report of a synthetic encephalitogenic peptide from myelin proteolipid protein.
HLA-DR is involved in the HIV-1 binding site on cells expressing MHC class II antigens.
Authors: Mann D L , Read-Connole E , Arthur L O , Robey W G , Wernet P , Schneider E M , Blattner W A , Popovic M
Published: December 2022

The primary interaction of HIV-1 with the target cell involves the viral large envelope protein (gp120) and the cellular CD4 molecule. mAb reacting with portions of CD4 have been shown to block HIV-1 attachment and infection. In one of the early reports describing HIV-1 cell interaction, some mAb reacting with MHC class II Ag were also found to block infection. To investigate further a possible role for MHC class II in HIV-1 binding, a cultured T lymphocyte cell line (H-9) that expresses MHC class II molecules and PHA-stimulated PBL was exposed for various time periods to concentrated viral particles and individual HIV-1 proteins. A decrease in the ability to detect the CD4a epitope and HLA-DR was observed after the cells were exposed to virus for 15, 30, and 60 min whereas HLA-DP and HLA-DQ Ag increased or remained unchanged. After 120 min of virus exposure, the CD4a epitope remained diminished whereas HLA-DR was detected at levels found on cells not exposed to virus. mAb detecting the CD4a epitope and HLA-DR, as well as alloantisera detecting the specific HLA-DR Ag on the target cell, blocked HIV-1 binding. When immunopurified gp120 was added to PHA-stimulated and unstimulated PBL, the CD4a epitope decreased in the same manner as was observed with whole virus preparations. In contrast to exposure to the intact virus, HLA-DR expression appeared to increase. Other viral proteins, p17, p24, and a portion of the small envelope protein, gp41, had no effect on the ability to detect cell surface Ag. Thus, although CD4 is the primary receptor for HIV-1 binding, HLA-DR appears to be involved in the binding site, probably by virtue of its close proximity to the CD4 molecule on the cell surface.
Functional modulation of hepatitis B core antigen-specific T lymphocytes by an autoreactive T cell clone.
Authors: Ferrari C , Chisari F V , Ribera E , Penna A , Mondelli M U
Published: December 2022

Hepatitis B core (HBc)Ag-specific T cells present in the peripheral blood of a patient with chronic active hepatitis B were expanded by co-cultivation for 7 days with rHBcAg. After cloning at 1 cell/well in the presence of PHA and IL-2, five HBcAg-specific CD4+ cloned lines were obtained. All five lines proliferated and produced IL-2, IFN-gamma, and TNF in a dose-dependent fashion in response to HBcAg, but not to HBV envelope Ag. The cloned lines and derivative clones were HLA class II (DR1) restricted. All T cell clones were able to induce anti-HBc production by autologous B cells in response to HBcAg (helper effect). The proliferative response and the helper effect of the HBcAg-specific T cell lines and clones were augmented by co-cultivation with an autologous, autoreactive (HLA-DQ1 specific) T cell clone, even in the absence of HBcAg, and the autoreactive T cells directly stimulated anti-HBc secretion by autologous B cells, presumably due to the release of Ag-nonspecific factors. These findings define a model immunoregulatory circuit the physiologic significance of which remains to be determined.
Evidence for two discrete phases of IL-2 production in bovine lymphocytes.
Authors: Weinberg A D , Magnuson N S , Reeves R , Wyatt C R , Magnuson J A
Published: December 2022

Regulation of IL-2 production in Con A-stimulated bovine lymph node cells was studied by following the time course of IL-2 synthesis and secretion over a 24-h period. A biphasic time course curve of IL-2 secretion was observed after stimulation with Con A alone or with Con A plus PMA. Both phases of IL-2 production were confirmed by three separate biochemical techniques: IL-2 protein bioassay, mRNA Northern blots, and anti-IL-2 antibody detection. Bovine IL-2 transcripts were detected as early as 2 h after Con A stimulation. The early phase of IL-2 protein secretion was initially detected 3 h after Con A stimulation, and the late phase occurred between 10 and 16 h. Both phases of IL-2 production were enhanced when PMA was added to the Con A stimulation. Each phase of IL-2 protein secretion was preceded by the accumulation of IL-2 mRNA.
Fc-receptor cross-linking induces rapid secretion of tumor necrosis factor (cachectin) by human peripheral blood monocytes.
Authors: Debets J M , Van der Linden C J , Dieteren I E , Leeuwenberg J F , Buurman W A
Published: December 2022

In this study it was demonstrated that cross-linking of FcR on human monocytes induces the secretion of the cytotoxic and immunoregulatory cytokine TNF. Both soluble and insoluble immune complexes, solid-phase antibody and antibody-coated phagocytizable particles were used to cross-link FcR on monocytes. It was observed that monocytes secreted large amounts of TNF in each of these instances. Kinetic studies performed with soluble immune complexes showed that TNF was secreted very rapidly, e.g., within 2 h after addition of immune complexes to monocytes. These findings are relevant for the understanding of FcR-mediated immune responses by monocytes and macrophages, for example antibody-dependent cellular cytotoxicity and phagocytosis, and for disease states associated with circulating or tissue-fixed immune complexes.
Lateral diffusion measurements give evidence for association of the Tac peptide of the IL-2 receptor with the T27 peptide in the plasma membrane of HUT-102-B2 T cells.
Authors: Edidin M , Aszalos A , Damjanovich S , Waldmann T A
Published: December 2022

Fluorescence photobleaching recovery measurements show that the F1-IgG-labeled Tac peptide of the IL-2R can diffuse in the plane of the membrane of HUT-102-B2 T lymphocytes, with a mean diffusion coefficient of 2 to 3 x 10(-10) cm2s-1. Although only a fraction (mean 37%) of the Tac peptides is mobile on any given cell, lateral diffusion of the Tac peptide can be measured in 94% of cells examined. In contrast, the 95-kDa peptide, T27, is 90 to 100% immobilized in cells labeled with OKT27. Immobilization of T27 also affects the lateral diffusion of the Tac peptide, because the Tac peptide is immobile in more than 30% of cells pretreated with OKT27 and then labeled with anti-Tac IgG. The effect is specific for OKT27 to the extent that pretreatment with an anti-HLA mAb does not immobilize the Tac peptide. It appears, then, that Tac and T27 peptide not only are in proximity on HUT-102-B2 lymphocyte membranes but also interact physically in situ.
Activated T cells and monocytes have characteristic patterns of class II antigen expression.
Authors: Robbins P A , Maino V C , Warner N L , Brodsky F M
Published: December 2022

The expression of human histocompatibility class II Ag was measured on activated T cells and monocytes by quantitative mAb binding in direct two-color immunofluorescence. Monocytes activated by IFN-gamma bound an average of 2 x 10(6) DR-specific mAb, 3 x 10(5) DQ-specific mAb, and 7 x 10(5) DP-specific mAb per cell. For T cells activated by anti-CD3, a subpopulation bound 1 x 10(5) DR-specific mAb, 5 x 10(4) DQ-specific mAb and 5 x 10(4) DP-specific mAb per cell. These measurements were obtained after establishing a base line of class II Ag expression on resting B cells and monocytes. Resting B cells and those monocytes that were positive for class II Ag bound identical amounts of mAb; 3 x 10(4) DR-specific mAb, 3 x 10(3) DQ-specific mAb and 2 x 10(4) DP-specific mAb. However, most resting monocytes (75%) expressed only DR Ag. In the process of studying the expression of class II Ag on T cells, it was necessary to define and analyze the activated T cell state. Cell cultures activated with 0.3 ng/ml anti-CD3 had the highest expression of class II Ag on T cells, whereas those activated with 3.0 ng/ml anti-CD3 had the highest expression of IL-2R on T cells. Addition of IL-2 had no further effect on DR Ag expression on T cells but did up-regulate IL-2R expression. Reducing the initial monocyte concentration before activating T cells increased class II Ag expression on T cells without affecting IL-2R expression. The results obtained on T cell activation suggest that perhaps a lymphokine may be made by CD3-activated T cells which induces class II Ag expression on T cells.
In vitro release of histamine from murine mast cells by block co-polymers composed of polyoxyethylene and polyoxypropylene.
Authors: Atkinson T P , Smith T F , Hunter R L
Published: December 2022

A series of block co-polymers composed of polyoxyethylene and polyoxypropylene were investigated for their ability to induce in vitro activation of mouse mast cells. We found that six of these co-polymers could cause histamine release from mouse mast cells in vitro. At low concentrations, the most efficacious co-polymer, T130R2, caused rapid and extensive concentration-dependent release of histamine from mouse mast cells. The release process was not cytotoxic; it required metabolic energy and was not accompanied by release of lactate dehydrogenase. Optimal release of histamine was dependent on both calcium and sodium ions in the extracellular medium. The degree of in vitro histamine release correlated with in vivo inflammation and in vitro ionophore activity. We believe that this represents the first report of the activation of mediator-containing cells by an ionophore selective for monovalent cations. These copolymers may therefore represent new reagents for investigations of cellular excitation.
Aging of the murine immune system is reflected by declining ability to generate antibodies that promote elimination of Trypanosoma musculi.
Authors: Albright J W , Matusewicz N M , Albright J F
Published: December 2022

Trypanosoma musculi established extracellular infections in aged BC3F1 and C57BL/6 mice that were approximately 10 times greater and twice as long in duration as those in young adult mice. Elimination of T. musculi infections was found to be an antibody-dependent, cell-mediated process involving effector (presumably phagocytic) cells in the liver and, to a lesser extent, the spleen. A major difference between young and aged mice of the C57BL/6 strain was the deficiency in the ability of aged animals to generate antibodies of appropriate specificity and/or isotype in sufficient amount to promote trypanosome elimination. Indeed, at the time when infected young-adult mice began to produce antibodies that facilitated rapid trypanosome clearance (in young adult, but not in aged animals), the serum of aged mice was found to contain substances that inhibited parasite clearance. Overall, however, the development of antibodies of different isotypes, capable of reacting with intact trypanosomes, was about the same in young and aged animals. Hepatic and splenic effector cells of old mice were at least as efficient as those of young adults. The immunoblotting procedure was used to try to detect trypanosome Ag against which aged animals failed to generate antibodies of one or more isotypes. The complexity of the reactions of infected mouse serum antibodies with the spectrum of trypanosome Ag precluded a precise analysis. However, it was apparent that a delay in the appearance of antibodies of IgG2a and IgG2b isotypes, against Ag of relatively high m.w., was typical of aged, in comparison to young, mice. More exacting analyses, involving fractionated trypanosome extracts and mAb of varying isotypes, are underway to identify key trypanosome Ag that elicit antibodies of isotypes that can facilitate hepatic and splenic clearance of the parasites. This line of investigation will provide information that, in addition to its intrinsic interest, may be vital in judging the future impact of endemic parasitic infections on the emerging cohort of elderly persons in developing tropical nations.
Accessory cells, dendritic cells, or monocytes, are required for the lymphokine-activated killer cell induction from resting T cell but not from natural killer cell precursors.
Authors: Silvennoinen O , Vakkila J , Hurme M
Published: December 2022

In this study we have investigated the role of accessory cells in the development of lymphokine-activated killer cells (LAK) from highly purified human NK and small resting T cell progenitors. As accessory cells we used autologous, as well as allogeneic, monocytes, and dendritic cell enriched cells. Both NK and T cells were able to generate LAK activity, but their activation requirements were different. NK cells were activated merely by IL-2, and accessory cells did not enhance their lytic activity in the presence or absence of IL-2. Conversely, T cells were practically unresponsive to even high concentrations of IL-2 having a strict requirement for accessory cells for the development of lytic activity and proliferation. Accessory cells differed in their ability to activate T cells presumably depending on their ability to induce IL-2 synthesis, allogeneic dendritic cells being the most effective accessory cells and IL-2 synthesis stimulators. Allogeneic accessory cells could induce lytic activity in T cells even in the absence of exogenous IL-2. Thus, accessory cells play a central role in expanding the LAK effector cell population.
T cell receptor/CD3 negative variants are unresponsive to stimulation through the Ly-6 encoded molecule, TAP.
Authors: Bamezai A , Reiser H , Rock K L
Published: December 2022

TAP is a phosphatidylinositol-anchored Ly-6-encoded, glycoprotein that is involved in the activation of murine T-inducer cells. Anti-TAP antibodies can stimulate T cells directly and also synergize with Ag stimulation. To determine the relationship between TAP and TCR-mediated activation, we have derived TCR/CD3 loss variants of functional T hybrid clones. T-T hybrid variants that have lost TCR expression are not responsive to anti-TAP stimulation. Isolation and analysis of revertant clones, obtained from one of the TCR/CD3-negative mutant cell lines, demonstrate a concordant re-expression of the TCR/CD3 complex and responsiveness to anti-TAP stimulation. A similar loss of responsiveness to TAP stimulation is observed after antibody-induced modulation of the TCR/CD3 complex from the surface of T cell hybrids. In contrast, both the TCR/CD3 loss variants and TCR/CD3-modulated T-T hybrids remain fully responsive to stimulation with a calcium ionophore and phorbol esters. This structure-function correlation has been observed repeatedly in independent isolations of variants, and with TCR/CD3 modulated cells, from two different T-T hybridomas. Given the apparent functional interrelationship between TAP and the TCR/CD3 complex, we have also analyzed if these molecules were significantly associated on the T cell surface. Antibody-induced modulation of the TCR/CD3 complex does not affect the cell surface expression of TAP molecules. Moreover, the expression of these two sets of proteins is also independent as evidenced by the selective loss of either set of these proteins in the TCR/CD3 expression mutants.
Artificial T cell:B cell conjugation. A unique approach to analyze weak cell-cell interactions.
Authors: Cassatt D R , Kaplan A M , Cohen D A
Published: December 2022

An antibody response against a thymic-dependent Ag requires cognate recognition of the Ag by B and T cells. Functional T-B cell (T-B) interaction involves binding of Ag by B cell surface Ig, internalization and processing of Ag, expression of an Ag fragment in the context of Ia, binding of Ag/Ia by the TCR and binding of T cell-derived lymphokines by B cell lymphokine receptors. It is becoming increasingly evident that B and T cell accessory molecules also are involved in T-B interactions. To determine the role of accessory molecules in T-B collaboration, we have designed a system in which T-B interaction was artificially induced in the absence of carrier protein. TNP-modified, turkey gamma-globulin-specific, Th cells were allowed to form conjugates with TNP-specific B cells in the absence of hapten-carrier complex. Both B and T cells were induced to proliferate and B cells partially differentiated into antibody-secreting cells when B cells were cultured with TNP-modified but not unmodified T cells. The activation of B cells by TNP-modified T cells was not MHC restricted but was blocked by anti-Ia antibodies, suggesting a role for Ia distinct from Ag presentation. Furthermore, B cell proliferation was also inhibited by antibodies to L3T4 and LFA-1, suggesting a functional accessory role for these molecules in induction of B cell proliferation/differentiation.
T cell activation by purified, soluble, class I MHC molecules. Requirement for polyvalency.
Authors: McCluskey J , Boyd L F , Highet P F , Inman J , Margulies D H
Published: December 2022

To examine the nature of the interaction of the TCR with the MHC class I Ag, we have studied the stimulation requirements of an H-2Dd-reactive T cell hybridoma, using a homogeneous, purified preparation of a molecularly engineered soluble counterpart of the class I Ag, H-2Dd/Q10b. We demonstrate that this monovalent, soluble MHC Ag is incapable of stimulating the release of IL-2 from this T cell hybridoma. However, the same preparation of the purified protein can elicit a dose-dependent response when made multivalent either by covalent coupling to soluble, high m.w. dextran or to agarose beads, or by adsorption to polystyrene tissue culture plates.
In vivo administration of IL-1 induces thymic hypoplasia and increased levels of serum corticosterone.
Authors: Morrissey P J , Charrier K , Alpert A , Bressler L
Published: December 2022

Administration of IL-1 alpha or IL-1 beta to normal mice induces a decrease in thymic cellularity, the magnitude of which depends on the number of injections and dose of IL-1. Twice daily injections of 200 ng of IL-1 alpha or -beta for 4 days results in a 90% decrease in thymic cellularity, which regenerated after cessation of treatment. Study of thymocyte subpopulations revealed that the number of CD4+/CD8+ thymocytes was dramatically decreased in IL-1-treated mice. Functional assessment of the CD4-/CD8- population from treated animals showed that these cells had adequate mitogenic responses in vitro and that the proportion of these cells in cycle was not different from control CD4-/CD8- cells. IL-1 treatment also prevented the regeneration of thymic cellularity after irradiation. The use of strains of mice differing genetically at the Ly 1 locus to construct radiation bone marrow chimeras demonstrated that bone marrow-derived thymocyte precursors were able to seed the thymus in the IL-1-treated animals. Again, however, the CD4+/CD8+ thymocyte population was significantly decreased. Thymic repopulation occurred upon cessation of IL-1 therapy. Finally, we determined that a single i.p. injection of IL-1 caused a three-fold increase in serum corticosterone levels, which peaked approximately 3 h after IL-1 administration. Thus, an IL-1-dependent increase in serum corticosterone levels may be responsible for the observed thymic hypoplasia.
Signal transduction of gamma/delta T cell antigen receptor with a novel mitogenic anti-delta antibody.
Authors: Wu Y J , Tian W T , Snider R M , Rittershaus C , Rogers P , LaManna L , Ip S H
Published: December 2022

Human T lymphocytes express either alpha/beta- or gamma/delta-TCR in association with the CD3 complex. We have isolated a mAb, delta TCS1, that immunoprecipitated the gamma/delta-TCR heterodimer from cell lysates of Peer and Molt-13 leukemia cell lines. After dissociation of the gamma- and delta-chains of TCR by treatment with SDS, delta TCS1 specifically immunoprecipitated the delta-chain. This antibody bound to the surface of other gamma/delta-positive T cell lines and clones and was able to stimulate the proliferation of a minor cell population (0.9 to 4.0%) of resting human PBL. Upon binding to gamma/delta-TCR-bearing Molt-13 cells and PBL, delta TCS1 elicited a fura-2 Caa+ signal indicating that the gamma/delta-receptor is functionally similar to the alpha/beta-heterodimer. These data indicate that the delta TCS1 antibody recognizes an epitope on TCR delta-chain and its mitogenic activity should be useful in characterizing the functional properties of human gamma/delta-positive T lymphocytes.
Oral administration of cholera toxin-Sendai virus conjugate potentiates gut and respiratory immunity against Sendai virus.
Authors: Liang X P , Lamm M E , Nedrud J G
Published: December 2022

Successful oral immunization to prevent infectious diseases in the gastrointestinal tract as well as distant mucosal tissues may depend on the effectiveness of an Ag to induce gut immune responses. We and others have previously reported that cholera toxin possesses strong adjuvant effects on the gut immune response to co-administered Ag. To explore further adjuvant effects of cholera toxin, the holotoxin or its B subunit was chemically cross-linked to Sendai virus. The resulting conjugates, which were not infectious, were evaluated for their capacity to induce gut immune responses against Sendai virus after oral administration to mice. Conjugating cholera toxin to virus significantly enhanced the adjuvant activity of cholera toxin compared to simple mixing. Cholera toxin B subunit, however, did not show an adjuvant effect either by itself or conjugated with the virus. Oral administration of the Sendai virus-cholera toxin conjugate was also able to prime for protective anti-viral responses in the respiratory tract. Mice that were orally immunized with the conjugate and intra-nasally boosted with inactivated virus alone showed virus-specific IgA titers in nasal secretions that correlated with protection against direct nasal challenge with live Sendai virus. For comparison, s.c. immunization was also studied. Systemic immunization with the virus-cholera toxin conjugate induced virus-specific antibody responses in serum as well as in the respiratory tract but failed to protect the upper respiratory tract against virus challenge. Systemic immunization plus an intra-nasal boost did, however, confer a variable degree of protection to the upper respiratory tract, which correlated primarily with bronchoalveolar lavage (lung) antibody titers.
Cooperation between an anti-T cell (anti-CD28) monoclonal antibody and monocyte-produced IL-6 in the induction of T cell responsiveness to IL-2.
Authors: Baroja M L , Ceuppens J L , Van Damme J , Billiau A
Published: December 2022

CD28 is an Ag of 44-kDa Mr that is expressed on the membrane of the majority of human T cells and that is recognized by mAb 9.3. The functional effects of mAb 9.3 on peripheral blood T cells were studied. mAb 9.3 was not mitogenic, unless it was combined with PMA. When CD28 was cross-linked after binding of mAb 9.3 to the T cell by immobilized or soluble anti-mouse IgG, T cells proliferated in response to rIL-2, provided that monocytes were also present. The additional signal required for IL-2 responsiveness after cross-linking of CD28 could also be delivered in cultures of purified T cells by a cellfree monocyte culture supernatant. Expression of IL-2R on about 10% of the T cells was demonstrated by staining with an anti-IL-2R mAb, and was found to be largely restricted to CD4+ cells. The active compound responsible for the helper signal in the monocyte culture supernatant was identified as IL-6 because purified IL-6 (but not IL-1 beta) had similar activity and because an antiserum to IL-6 (but not an antiserum to IL-1 beta) neutralized the activity of the monocyte supernatant and blocked T cell proliferation. An anti-IL-2R antibody also completely inhibited T cell proliferation induced by the combination of mAb 9.3, IL-2, and IL-6. Our results provide evidence that cross-linking of CD28 induces functional IL-2R and that this activity is dependent on a helper signal provided by monocytes, more specifically IL-6. Moreover, our results indicate that IL-6 (previously called B cell stimulatory factor-2) is active on T cells. If a natural ligand for CD28 can be identified, the mechanism of induction of IL-2 responsiveness described here might explain how T cells become nonspecifically involved in an ongoing cellular immune reaction.
Recombinant IFN-gamma synergizes with lipopolysaccharide to induce macrophage membrane procoagulants.
Authors: Moon D K , Geczy C L
Published: December 2022

Fibrin deposition is an important histopathologic feature of inflammation and is mediated, in part, by monocyte/macrophage procoagulants. rIFN gamma acted in synergy with suboptimal levels of bacterial LPS by priming thioglycollate-induced mouse peritoneal exudate cells (TG-PEC) to express high levels of surface procoagulant. TFN-alpha beta, TFN-alpha, IL-1, either alone or in combination with LPS or IFN-gamma, had no effect on macrophage procoagulant activity expression. In contrast to the dramatic increases of macrophage procoagulant activity induced by IFN-gamma/LPS, on exudate macrophages, normal peritoneal macrophages, or peripheral blood monocytes were unresponsive suggesting that the state of activation of the macrophage determines reactivity. IFN-gamma induced a Factor VIIa-like activity detected only after cell disruption. Synergy between LPS and IFN-gamma-induced procoagulants may occur as the result of the assembly of the thromboplastin (induced by LPS), Factor VII/VIIa complex on the macrophage surface. RNA synthesis was required for procoagulant induction. Procoagulant expression may, as for other cytokines involved in inflammatory responses, be regulated by short lived repressor proteins as low dose cycloheximide superinduced procoagulant responses to both LPS and IFN-gamma and caused the extracellular expression of procoagulant in response to IFN-gamma. This study suggests an important role for IFN-gamma in the assembly of components of the extrinsic coagulant cascade on the macrophage surface. The synergy between IFN-gamma and LPS may moderate macrophage-initiated fibrin deposition characteristic of inflammatory responses.
Interferon-beta 2/interleukin 6 is a co-stimulant for human T lymphocytes.
Authors: Tosato G , Pike S E
Published: December 2022

IFN-beta 2/IL-6 promotes the proliferation of human peripheral blood T cells in the presence of either PHA or Con A. This effect is observed with highly purified T cells and is masked by the addition of as few as 2% monocytes, suggesting that accessory cells are not required and that IFN-beta 2/IL-6 acts directly on the T cells. Both T4-positive and T8-positive cells respond to IFN-beta 2/IL-6 plus PHA. Studies of the time requirements of IFN-beta 2/IL-6 and of PHA in the response of T cells show that optimal co-stimulation occurs when both IFN-beta 2/IL-6 and PHA are added together at the outset of culture, suggesting that IFN-beta 2/IL-6 acts predominantly on resting T cells. Unlike T cell proliferation induced by IL-2, T cell proliferation induced by IFN-beta 2/IL-6 is not blocked by antibodies to the IL-2 R, suggesting that IFN-beta 2/IL-6 does not act by stimulating IL-2 production. Thus, in addition to its previously reported properties, IFN-beta 2/IL-6 stimulates T cells in the presence of mitogen, and may prove to be of considerable importance in the physiologic activation of T cells.
Activation of human C1r: Western blot analysis reveals slow and dose-dependent activation.
Authors: Hosoi S , Borsos T
Published: December 2022

Spontaneous activation of C1r in the presence of EDTA was examined by a Western blot. Partially purified native C1r was prepared by ultracentrifugation of fresh serum in 10 to 30% sucrose gradient; final concentration of C1r was one-sixth of the original serum. C1(-)-INH was not detectable by a single radial immunodiffusion (less than 0.5% of serum). The results demonstrated that 1) the rate of spontaneous activation of C1r was slow (less than 10% in 30 min); 2) it was concentration-dependent; 3) it was enhanced by activated C1r; and 4) it was almost completely suppressed by serine protease inhibitors up to 1 h. These results were inconsistent with an intramolecular autoactivation model of C1r in the fluid phase and suggested intermolecular activation by contaminating protease or activated C1r.
Synthesis of complement factor B by the human monocyte U-937 cell line. Augmentation by immunostimulatory agents.
Authors: Minta J O
Published: December 2022

The human pre-monocytic cell line U-937 was shown to synthesize and to secrete increasing amounts of factor B in short term cultures in serum-free medium containing BSA. The kinetics of factor B production were higher on day 2 than on days 1 and 3. The production of factor B was reversibly inhibited by cycloheximide, indicating de novo synthesis. Metabolic labeling with [35S]-methionine and SDS-PAGE analysis revealed that both intracellular and secreted factor B were single-chain proteins with similar m.w. (90,000), which co-migrated with purified plasma factor B. Incubation of U-937 cells with the immunostimulants PMA, LPS, IFN-gamma, and IL-1 resulted in a dose-dependent augmentation of factor B production. A 24-h exposure to IL-1 was shown to be required for maximal stimulation. A combination of suboptimal doses of LPS and IFN-gamma was shown to exert a synergistic effect on factor B production. The U-937 cell line is thus a valuable model for the study of the regulation of the factor B gene expression.
Activation of the respiratory burst in macrophages. Phosphorylation specifically associated with Fc receptor-mediated stimulation.
Authors: Brozna J P , Hauff N F , Phillips W A , Johnston R B
Published: December 2022

Inflammatory macrophages elicited from the peritoneal cavity of mice injected with endotoxin can avidly ingest E opsonized with IgG antibody (EIgG) or with IgM antibody and C (EIgMC). However, only ingestion of EIgG is associated with activation of the respiratory burst and release of superoxide anion. We compared the endogenous phosphorylation of proteins from macrophages stimulated by interaction with EIgG or EIgMC on the premise that proteins phosphorylated after stimulation by EIgG but not EIgMC could play a role in activating the enzyme (oxidase) responsible for the respiratory burst. Proteins were separated by one-dimensional and two-dimensional electrophoresis in polyacrylamide gels. We found that proteins with approximate Mr of 20 kDa, 23 kDa, 46 kDa, 48 kDa (three proteins), 67 kDa, and 130 kDa were more heavily phosphorylated after EIgG stimulation than after EIgMC stimulation. Exposure to PMA, which activates the respiratory burst oxidase, induced phosphorylation of the 23-kDa, 48-kDa group, and 130-kDa proteins that were phosphorylated after stimulation by EIgG. Activity of protein kinase C was found to be significantly increased in the particulate fraction of macrophages stimulated by EIgG but not in the particulate fraction of EIgMC-stimulated cells. These data are compatible with the hypotheses that phosphorylation of specific cellular proteins, especially with a Mr of approximately 48 kDa, is involved in activation of the respiratory burst oxidase, and that function of protein kinase C also plays a part in this activation process.
Induction of intercellular adhesion molecule 1 on primary and continuous cell lines by pro-inflammatory cytokines. Regulation by pharmacologic agents and neutralizing antibodies.
Authors: Rothlein R , Czajkowski M , O'Neill M M , Marlin S D , Mainolfi E , Merluzzi V J
Published: December 2022

The expression of intercellular adhesion molecule-1 (ICAM-1) on primary human fibroblasts, a human fibrosarcoma, chondrosarcoma, and adenocarcinoma cell line in response to IL-1, TNF-alpha, or IFN-gamma was studied using an ELISA with anti-ICAM-1 mAb. The induction of ICAM-1 by these cytokines was neutralized by cytokine-specific antisera as well as some steroids and the glycosylation inhibitor, tunicamycin. Cyclohexamide up-regulated the expression of ICAM-1 on chondrosarcoma cells but had little or no effect on carcinoma cells. These data indicate different mechanisms in the regulation and expression of ICAM-1 on the various cell types and provide some insight into the anti-inflammatory effects of some pharmacologic agents.
Analysis of T and B cell epitopes of the Schistosoma mansoni P28 antigen in the rat model by using synthetic peptides.
Authors: Auriault C , Gras-Masse H , Wolowczuk I , Pierce R J , Balloul J M , Neyrinck J L , Drobecq H , Tartar A , Capron A
Published: December 2022

The Schistosoma mansoni P28 molecule is an Ag inducing protective immunity in various experimental models. Three synthetic peptides, derived from the primary sequence of the recombinant P28 and comprising amino acids 24-43, 115-131, and 140-153, respectively, were synthesized according to their hydrophilicity, mobility, and accessibility profiles. The presence of B and T lymphocyte epitopes in these peptides has been examined in the rat model. The results showed that the 24-43 and the 115-131 peptides contained major epitopes for IgG but not for IgE. Moreover, the 24-43 peptide-specific IgG produced after injecting either the recombinant P28 Ag or the 24-43 peptide coupled to tetanus toxoid was essentially of the IgG2a subclass and to a lesser extent of the IgG1 subclass, whereas no IgG2c was detected. These 24-43 peptide-specific antibodies were cytotoxic in vitro for schistosomula in the presence of eosinophils as effector cells. The 24-43 and the 140-153 peptides contained major targets of T lymphocytes specific for the recombinant P28 Ag. T cell lines specific for the 24-43 peptide have been prepared. These cells proliferated in vitro when stimulated with various S. mansoni crude antigenic preparations or with the recombinant P28 Ag. Moreover, their passive transfer to rats immunized with the P28 Ag led to a significant increase in specific IgE without modifying the IgG response.
Genetic control of murine T cell proliferative responses to Mycobacterium leprae. V. Evidence for cross-reactivity between host antigens and Mycobacterium leprae.
Authors: Harris D P , Jones A G , Wade S , Krahenbuhl J L , Gillis T P , Watson J D
Published: December 2022

T cell proliferative responses to Mycobacterium leprae were measured by immunization of mice at the base of the tail with Ag and challenging lymphocytes from draining lymph nodes in culture with M. leprae. C57BL/10J and B10.BR mice were identified as low responder mice and the congenic strains B10.M, B10.Q, and B10.AKM as high responders whereas F1 (high x low) hybrid mice were found to be low responders. The cellular basis of low responsiveness did not appear to result from a defect in Ag-presenting cells or the activation of suppressor T cells by M. leprae. The influence of the environment in which T cells developed on responsiveness to M. leprae was analyzed in chimeric mice prepared by irradiating F1(C57BL/10J x B10.M) mice and reconstituting with bone marrow from C57BL/10J, B10.M, or F1 donors. Six weeks later, chimeric mice were immunized with M. leprae, lymph node cells were subsequently prepared, and H-2 phenotyped and challenged in culture with M. leprae Ag. T cell proliferative responses were found to be low in all cases, similar to those observed using lymph node cells from F1 hybrid mice. These results suggested that high responder B10.M lymphocytes developing in the irradiated F1 mice became tolerized to antigenic determinants found on M. leprae. This implied cross-reactive epitopes existed between some mouse strains and M. leprae. Low responsiveness to M. leprae in low responder and F1 hybrid mice may result from tolerance to H-2-encoded Ag that show cross-reactivity with M. leprae.