bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in the expression profiles of CD45RB, Pgp-1, and 3G11 membrane antigens and in the patterns of lymphokine secretion by splenic CD4+ T cells from young and aged mice.
Authors: Ernst D N , Hobbs M V , Torbett B E , Glasebrook A L , Rehse M A , Bottomly K , Hayakawa K , Hardy R R , Weigle W O
Published: December 2022

Previous studies indicate that the 3G11, CD45RB, and Pgp-1 determinants are differentially expressed on CD4+ T cell subsets in the mouse. We used multicolor immunofluorescence staining and flow cytofluorometric analysis to examine the expression of each of these determinants on splenic CD4+ cells from young (age 3 to 6 mo) and aged (age 24 to 26 mo) C57BL/6 mice. The CD4+ pool from aged mice contained significantly reduced numbers of 3G11+ and CD45RBhi cells, but increased numbers of Pgp-1hi cells, in comparison with the young group. Analysis of the simultaneous expression of all three subset determinants on CD4+ cells revealed that, in young mice, the major fraction (greater than 50%) was 3G11+CD45RBhiPgp-1lo. Among the less prevalent cell phenotypes, reductions in 3G11 expression correlated with decreases in CD45RB levels and increases in Pgp-1 levels. The phenotype that dominated the young group (3G11+CD45RBhiPgp-1lo) was approximately fivefold less represented in the aged group. The CD4+ pool from aged mice was characterized by increases in the 3G11-CD45RBvariablePgp-1hi and the 3G11+CD45RBloPgp-1hi phenotypes. To evaluate possible age-associated differences in cytokine secretion patterns by splenic CD4+ cells, purified CD4+ cells from each age group were stimulated in vitro with immobilized anti-CD3 epsilon mAb and accessory cells. At various times thereafter, supernatants from cultures were tested for IL-2 and IL-4 content by using the CTLL.6 and 11.6 bioassays, respectively, and the CD4+ cells were assayed for [3H]TdR uptake. Cell cultures from the aged group exhibited similar peak IL-2 accumulation and lower peak [3H]TdR uptake, but greatly increased peak IL-4 accumulation, as compared with cell cultures from the young group. The expression patterns of subset determinants, in conjunction with cytokine secretion profiles, indicate that, in aged mice, marked alterations occur in the subset composition of the splenic CD4+ cell pool. These findings are discussed in the context of previous findings on changes in T cell reactivity with advancing donor age.
Differentiation and functional maturation of bone marrow-derived intestinal epithelial T cells expressing membrane T cell receptor in athymic radiation chimeras.
Authors: Mosley R L , Styre D , Klein J R
Published: December 2022

The thymus dependency of murine intestinal intraepithelial lymphocytes (IEL) was studied in an athymic F1----parent radiation chimera model. IEL, although not splenic or lymph node lymphocytes, from athymic chimeras displayed normal levels of cells bearing the class-specific T cell Ag, CD4 and CD8; the TCR-associated molecule, CD3; and the Thy-1 Ag. Moreover, two-color flow cytometric analyses of IEL from athymic mice demonstrated regulated expression of T cell Ag characteristic of IEL subset populations from thymus-bearing mice. In immunoprecipitation experiments, surface TCR-alpha beta or TCR-gamma delta were expressed on IEL, although not on splenic lymphocytes, from athymic chimeras. That IEL from athymic chimeras constituted a population of functionally mature effector cells activated in situ, similar to IEL from thymus-bearing mice, was demonstrated by the presence of CD3-mediated lytic activity of athymic lethally irradiated bone marrow reconstituted IEL. These data provide compelling evidence that intestinal T cells do not require thymic influence for maturation and development, and demonstrate that the microenvironment of the intestinal epithelium is uniquely adapted to regulate IEL differentiation.
Inhibition of type 1 diabetes in BB rats with recombinant human tumor necrosis factor-alpha.
Authors: Satoh J , Seino H , Shintani S , Tanaka S , Ohteki T , Masuda T , Nobunaga T , Toyota T
Published: December 2022

We previously reported that streptococcal preparation (OK-432), which is a TNF inducer, inhibits insulitis and development of autoimmune diabetes in nonobese diabetic (NOD) mice and Bio-Breeding (BB) rats, as animal models of insulin-dependent diabetes mellitus. We have recently shown that recombinant human (h)TNF-alpha also suppresses development of diabetes in NOD mice. In this study we have extended our observation on TNF to BB rats in order to see whether TNF generally inhibits autoimmune diabetes. A total of 5 x 10(4) U of rhTNF-alpha was administered i.p., twice a week to male and female BB rats from 4 to 27 wk of age. The cumulative incidence of diabetes by 27 wk of age in nontreated rats was 36.4% (8/22), whereas that in hTNF-alpha-treated rats was 0% (0/21) (p less than 0.001). The hTNF-alpha-treated rats did not lose body weight and maintained normal blood glucose concentrations. Immunologic and histologic examinations were performed at the end of the experiment. Spleen cell cytotoxicities for NK-sensitive YAC-1 and rat insulinoma (RINm5F) cells in hTNF-alpha-treated rats significantly decreased in comparison with nontreated and nondiabetic BB rats. Intensity of insulitis was also inhibited in hTNF-alpha-treated rats. Interestingly, a huge hepatomegaly and splenomegaly was found in two of the 21 hTNF-alpha-treated rats. The latter consisted of W3/13dull+ and W3/25dull+ cells, which did not exhibit cytotoxicity for either YAC-1 or RINm5F cells. These results indicate that the chronic and systemic administration of TNF has a regulatory role in autoimmune diabetes in BB rats as well as in NOD mice, and that these animals may have a defect in TNF-mediated immunoregulation.
Down-regulation of IL-2 production by activation of T cells through Ly-6A/E.
Authors: Codias E K , Rutter J E , Fleming T J , Malek T R
Published: December 2022

Ly-6A/E molecules are expressed on the surface of T cells and have been shown to function in activation by the capacity of anti-Ly-6A/E mAb to induce T cell hybridomas or normal T cells to produce IL-2. Recent evidence suggests that activation through Ly-6A/E may be linked to the TCR signaling pathway. To further investigate the relationship between Ly-6- and TCR-induced T cell activation, we have examined whether an anti-Ly-6A/E mAb (D7) modulates TCR signaling in vitro. We now report that mAb D7 specifically inhibited IL-2 production by T cells also activated through TCR. Such inhibition was noted for normal T cells stimulated by soluble anti-CD3 or alloantigen and for T hybridomas stimulated by soluble anti-CD3. The ability of D7 to inhibit IL-2 production by T hybridomas was dependent on the nature of the TCR activating signal because IL-2 production was not inhibited when T hybridomas were stimulated with Ag or immobilized anti-CD3. Inhibition of IL-2 production by D7 apparently required cross-linking of the mAb because D7 F(ab')2 fragments were not effective for inhibition of IL-2 production. Similar to its ability to enhance anti-Ly-6A/E-induced activation of T and B cells, IFN-gamma enhanced the D7-induced inhibition of IL-2 production by alloantigen-activated normal T cells. These data further support the notion that Ly-6 and TCR signaling pathways are interrelated.
Stimulation of alpha-adrenergic receptor augments the production of macrophage-derived tumor necrosis factor.
Authors: Spengler R N , Allen R M , Remick D G , Strieter R M , Kunkel S L
Published: December 2022

Accumulating evidence supports the hypothesis that neuroendocrine hormones may participate in immunologic processes. In our study we have determined that UK-14304 (UK) and norepinephrine (NE), both alpha 2-adrenergic agonists, can augment LPS-stimulated TNF from elicited macrophages (MO). The increase in TNF production was concentration dependent with an EC50 for UK and NE of 8.1 +/- 2.6 and 0.52 +/- 0.17 nM, respectively. The concentration-effect curve for UK and NE was shifted to the right by the alpha 2-antagonist yohimbine (10(-6) M), with new EC50 of 49.7 +/- 12.2 (p less than 0.001) nM and 10.3 +/- 22 nM. The augmenting effect of UK on MO TNF production was assessed over a 7 log LPS response curve. Within a single population of MO 10 nM UK shifted the LPS-induced TNF curve eightfold to the left with the greatest increase in TNF production at lower LPS concentrations. At the transcriptional level, Northern blot analysis demonstrated that UK increased LPS-induced TNF mRNA accumulation. This augmentation in TNF mRNA accumulation was blocked by yohimbine. The presence of a MO alpha-adrenergic receptor was established by demonstrating binding of the alpha 2-adrenergic antagonist 3H-yohimbine to membranes prepared from MO. This binding was rapid, saturable, reversible, and blocked by UK, clonidine, and phentolamine. These investigations support the role of alpha 2 adrenergic agonists as immunostaining compounds that may regulate cytokine production during an inflammatory response.
Mast cell heterogeneity. Differential synthesis and expression of glycosphingolipids by mouse serosal mast cells as compared to IL-3-dependent bone marrow culture-derived mast cells before or after coculture with 3T3 fibroblasts.
Authors: Raizman M B , Austen K F , Katz H R
Published: December 2022

The synthesis and intracellular expression of glycosphingolipids by mouse serosal mast cells (SMC) have been characterized by radiolabeling and TLC and by immunodetection in situ. Chromatographic analysis of purified glycosphingolipids from SMC intrinsically labeled with [14C]galactose and [14C]glucosamine hydrochloride revealed the predominant synthesis of only the simplest neutral glycosphingolipid and ganglioside, glucosylceramide and ganglioside GM3, respectively. Intracellular indirect immunofluorescence staining of permeabilized SMC demonstrated the absence of the more complex neutral glycosphingolipids lactosylceramide, globotriosylceramide, globotetraosylceramide, and globopentaosylceramide, the absence of ganglioside GM1, and the presence of ganglioside GM3. By contrast, permeabilized mouse IL-3-dependent bone marrow culture-derived mast cells (BMMC) and mast cells recovered after 21 days of coculture of BMMC with mouse 3T3 fibroblasts expressed lactosylceramide, globotriosylceramide, globotetraosylceramide, ganglioside GM1, and ganglioside GM3, but not globopentaosylceramide intracellularly as determined by immunofluorescence. The findings indicate a loss of biosynthetic capacity and epitope maintenance for glycosphingolipids with in vivo differentiation of SMC from IL-3-dependent BMMC progenitors. Thus, although mast cells derived after coculture of these progenitors for 21 days with fibroblasts assume multiple SMC-like properties in terms of their histochemical staining and their secretory granule proteoglycan and neutral protease constituents, they do not lose the ability to express complex glycosphingolipids. The finding that glycosphingolipid composition does not change coordinately with other secretory granule markers defines a new stage of mouse mast cell development between the BMMC and SMC and provides evidence that mast cell development is more complex than previously appreciated.
Responses of mouse lymphocytes to extracellular ATP. II. Extracellular ATP causes cell type-dependent lysis and DNA fragmentation.
Authors: Zanovello P , Bronte V , Rosato A , Pizzo P , Di Virgilio F
Published: December 2022

Extracellular ATP (ATPo) caused dose-dependent lysis of YAC-1 and P-815 mouse tumor cells. This event, assessed by 51Cr release, was accompanied by sustained depolarization of the plasma membrane potential and Ca2+ influx. Plasma membrane depolarization and Ca2+ influx occurred within a few seconds of ATPo addition to both cell types, whereas 51Cr was released without apparent lag in YAC-1 cells and after 2 h in P-815 cells. Furthermore, a rise in [Ca2+]i was required for ATPo-dependent lysis of YAC-1 but not P-815 cells. In P-815 cells, ATPo caused an early and [Ca2+]i-independent DNA fragmentation that occurred at lower nucleotide concentrations than those required to trigger 51Cr release. Instead in YAC-1 cells very low concentrations of ATPo caused early lysis (ED50 for lysis about 200 microM) accompanied by only barely detectable DNA fragmentation. Previous studies disclosed that lymphokine-activated killer cells are fully resistant to the membrane-perturbing effects of ATPo. We show that lymphokine-activated killer cells also do not undergo DNA fragmentation even in the presence of high ATPo concentrations. This study complements previous observations on the lytic effects of ATPo and shows that this nucleotide can also cause DNA fragmentation, one of the earliest target cell alterations observed during CTL-mediated lysis.
The identification of an Onchocerca-specific recombinant antigen containing a T cell epitope.
Authors: Colina K F , Perler F B , Matsumura I , Meda M , Nutman T B
Published: December 2022

Recombinant Onchocerca volvulus Ag have been derived from expression libraries and examined for their ability to stimulate PBMC from patients infected with O. volvulus. Ten clones producing recombinant Ag were selected and plaque purified; lysogens were produced and found to express beta-galactosidase fusion proteins ranging in molecular mass from 115 to 138 kDa. When ammonium sulfate-precipitated lysates of these recombinant phage clones were examined for their ability to stimulate PBMC from a patient with onchocerciasis, all 10 recombinants produced stimulation above that to nonrecombinant phage. When individual fusion proteins, affinity purified on anti-beta-galactosidase linked to agarose, were used to stimulate PBMC from patients with onchocerciasis, only one of the recombinant Ag induced PBMC proliferation (stimulation index greater than 4) above that to Ag from nonrecombinant phage. Characterization of the DNA coding for this Ag showed it to be 1.2 kb in length with a small (90 bp) open reading frame; furthermore, it appears to be Onchocerca specific (on genomic dot blots) and single copy. Using overlapping peptides encompassing the entire open reading frame, one T cell epitope has been localized.
Increased sensitivity to MHC-nonrestricted lysis of BL6 melanoma cells by transfection with class I H-2Kb gene.
Authors: Gorelik E , Jay G , Kwiatkowski B , Herberman R B
Published: December 2022

An H-2Kb- negative clone of BL6 melanoma (BL6-8) was transfected with neor, H-2Kb, or H-2IAk genes. In an 18-h cytotoxicity assay clones with high levels of H-2Kb Ag expression were found more sensitive to lysis by spleen cells of syngenic and allogeneic mice than H-2Kb low clones. NK cells were involved in the lysis of H-2Kb+ BL6 melanoma clones, with spleen cell cytotoxicity of mice increased after poly I:C stimulation or decreased after pretreatment with anti-asialo GM1 serum or NK1.1 mAb. Anti-TNF Ab were also able to reduce the cytotoxicity of normal spleen cells and completely abolished the cytotoxicity of the NK-depleted spleen cells suggesting involvement of NC cells in lysis of H-2Kb+ BL6 melanoma clones. Increase in sensitivity of H-2Kb+ BL6 cells to natural cell-mediated cytotoxicity was associated with the appearance of NK recognizable determinants as assessed by the cold target inhibition assay. All BL6 clones, irrespective of sensitivity to natural cell-mediated cytotoxicity, showed high sensitivity to lysis by LGL-derived granules. In contrast, all H-2Kb low BL6 clones were resistant and all H-2Kb highly positive clones were sensitive to lysis by TNF-alpha. When an H-2Kb highly positive clone was selected in vitro for resistance to TNF, it concomitantly showed increased resistance to cytotoxicity by spleen cells, confirming the importance of TNF in spleen cell cytotoxicity against H-2Kb+ melanoma cells. Taken together, the data indicate that class I H-2Kb but not class II H-2IAk gene product could increase the sensitivity of BL6 cells to lysis by NK and natural cytotoxic cells as well as TNF. We hypothesize that these effects could be due to pleiotropic effects of H-2Kb gene products on various biologic properties of BL6 melanoma cells some of which may be more directly involved in regulation of tumor cell sensitivity to lysis by NK and/or natural cytotoxic cells.
Mouse IgA Fc receptor on CD3+ T cells. Molecular forms of IgA that bind to a 38-kDa Fc alpha R protein and development of an anti-Fc alpha R antisera.
Authors: Aicher W K , McGhee M L , McGhee J R , Eldridge J H , Beagley K W , Meyer T F , Kiyono H
Published: December 2022

Previous studies have shown that splenic T cells from mice that bear IgA myelomas, as well as certain T cell lines, express receptors for the Fc of IgA, and are termed Fc alpha R. In this study, we have isolated and characterized two CD3+ T cell lines derived by fusion of murine Peyer's patch (PP) CD4+ T cells with the BW 5147 lymphoma cell line. These cell lines, designated PPT4-6 and PPT4-16, were shown to bind monomeric or dimeric IgA, whereas the fusion partner did not bind either form of IgA. However, polymeric IgA (m.w. 600,000) bound equally well to all three cell lines. Similar results were also obtained with two known Fc alpha R+ T cell lines, ThHA1 nos. 9 and 10. Immunoprecipitation studies with IgA on PPT4-16 and ThHA1 no. 9 have shown that IgA binds to a 38-kDa protein. A rabbit antiserum was prepared to a 38-kDa fraction of Fc alpha R+ T cell membranes, and heterophilic antibody was removed from the antiserum by adsorption with mouse thymocytes, BW 5147 and R1.1 lymphoma. The antiserum bound to both PPT4-16 and ThHA1 no. 9 as well as to other Fc alpha R+ T cells, but did not bind to thymocytes or to the T lymphomas R1.1 or BW 5147. The antiserum appeared specific for the Fc alpha R, because it failed to block binding of anti-CD3 (145 2C11) or other surface molecule-specific antibodies. Further, competitive inhibition studies with IgA and anti-Fc alpha R (38 kDa) showed that preincubation of Fc alpha R+ T cells with the anti-38-kDa protein completely eliminated IgA binding, whereas IgA partially blocked the binding of the anti-Fc alpha R antibodies to the cell membrane. Immunoisolation with the anti-Fc alpha R antibody of radioiodinated cell membrane proteins from Fc alpha R+ T cells, but not from Fc alpha R- cells, gave a distinct band at 38 kDa. To further test the specificity of this antiserum, we have isolated T cells from spleens of IgA-myeloma bearing mice, and tested the phenotype and IgA binding. A subset consisting of 15 to 20% of CD3+, CD8+ T cells was found that bound monomeric or dimeric IgA. Further, the anti-Fc alpha R antiserum also recognized this CD8+ T cell subset, and preincubation of the cells with antibody resulted in their failure to bind IgA. Our results indicate that the Fc alpha R on T cell lines derived from PP is a 38-kDa protein.(ABSTRACT TRUNCATED AT 400 WORDS)
Characterization of the specificity of peptide binding to four DR haplotypes.
Authors: O'Sullivan D , Sidney J , Appella E , Walker L , Phillips L , Colón S M , Miles C , Chesnut R W , Sette A
Published: December 2022

This study describes the establishment of a peptide-binding assay for purified, detergent-solubilized DR molecules. For each of the DR specificities and peptides studied, a unique pattern of interaction was observed. Excellent correlation was detected between the DR1-, 2-, 5-, and 52a-binding capacities and the known DR restrictions of a panel of synthetic peptides. This supports the immunologic relevance of the binding assay, and emphasizes the importance of determinant selection in defining the immune response of individuals. We have also examined the capacity of a panel of DR-restricted peptides to compete with one another for binding to DR1. The results obtained are compatible with a single peptide-binding site on DR molecules. The peptide-binding capacity of the four different DR types (DR1, DR2, DR5, and DR52a) has been further examined by testing a collection of 133 different peptides. This collection is unbiased with respect to previously known DR binding and restrictions, and includes peptides of eukaryotic, bacterial, and viral origins. It was found that: 1) approximately 15 to 35% of the peptides tested bound any given DR type; 2) DR-binding capacities appeared to correlate with each other, suggesting that different alleles of the DR isotype may recognize related structures on an Ag molecule; and 3) despite the statistical correlation between binding capacity of different DR types, approximately 50% of the peptides that were positive binders still were specific in that they could bind only one of the four DR molecules tested. Degenerate binding (i.e., binding to most or all the DR molecules tested) was detected in only a minority of the cases analyzed (approximately 25%).
Induction of apoptosis (programmed cell death) in human leukemic HL-60 cells by inhibition of RNA or protein synthesis.
Authors: Martin S J , Lennon S V , Bonham A M , Cotter T G
Published: December 2022

Apoptosis is regarded as a suicidal cell response since the dying cell appears to be an active participant. Previous studies have shown that apoptosis of various murine cell types, induced by a variety of stimuli, required RNA and/or protein synthesis. However, when human promyelocytic leukemia HL-60 cells were induced to undergo apoptosis by treatment with the calcium ionophore A23187 or microtubule-disrupting agents, in the presence of inhibitors of macromolecular synthesis, apoptosis of these cells was neither abrogated nor delayed. Furthermore, the presence of either cycloheximide, an inhibitor of protein synthesis, or actinomycin D, an RNA synthesis inhibitor, alone was found to induce large scale apoptosis of these cells. Apoptosis in these cells was characterized by cell and chromatin condensation followed by nuclear and DNA fragmentation. In common with many other studies, this DNA fragmentation was found to have an approximately 200-bp multiple pattern, which is consistent with the activation of an endogenous endonuclease which cleaves at internucleosomal sites. Calcium-dependent endonuclease activity of this type was also detected in the isolated nuclei of untreated HL-60 cells. The morphologic and biochemical changes characteristic of apoptosis were found to precede cell death, as measured by trypan blue uptake and were completely distinct from death caused by toxic stimuli such as azide, ethanol, or heat treatment. Similar experiments with six other human cell lines confirmed that this phenomenon was not peculiar to the HL-60 cell line. These results suggest that certain dividing cell populations do not require RNA or protein synthesis to undergo apoptosis and further, that continuous transcription and translation of some regulatory protein(s) may be required to maintain control over the apoptotic "machinery" of such cells.
The antiinflammatory effects of adenosine receptor agonists on the carrageenan-induced pleural inflammatory response in rats.
Authors: Schrier D J , Lesch M E , Wright C D , Gilbertsen R B
Published: December 2022

Adenosine and adenosine receptor agonists have a variety of inhibitory effects on the generation of inflammatory mediators by neutrophils and other cell types. In human neutrophils stimulated with the chemotactic peptide FMLP, adenosine agonists inhibit O2- generation and degranulation. Because these findings suggest that the agonists may have potential as antiinflammatory agents, several compounds were evaluated for effects on the exudative and cellular phases of carrageenan-induced pleural inflammation in rats. All of the agonists tested inhibited both parameters of the inflammatory response. Inhibition appeared to correlate better with binding to the A1 than to the A2 receptor and was reversible by a known adenosine receptor antagonist, 8-phenyltheophylline. In mechanistic studies, R-N-(1-methyl-2-phenylethyl)adenosine, a standard A1 selective agonist, reversed the drop in circulating neutrophil count that occurs after injection of carrageenan. These results suggest that the agonists may prevent cell emigration by inhibiting adhesion to the endothelium or diapedesis. In addition (R)-N-(1-methyl-2-phenylethyl)adenosine had weak inhibitory effects on superoxide production by FMLP-stimulated rat neutrophils. Control studies showed that the effects of the agonists were not the result of agonist-induced hypotension nor corticosterone production by the adrenal glands. These findings indicate that adenosine receptor agonists are effective new pharmacologic tools for the study of inflammatory processes.
Effect of protease inhibitors on human monocyte IgG Fc receptor II. Evidence that serine esterase activity is essential for Fc gamma RII-mediated binding.
Authors: van de Winkel J G , Jansze M , Capel P J
Published: December 2022

We have shown previously that certain proteases can modulate the affinity of human Fc gamma RII for IgG. To study whether proteolytic events not only increase FcR affinity, but are essential for Fc gamma R functioning, we evaluated the effect of different protease inhibitors on binding mediated by two classes of human monocyte IgG FcR. These R, Fc gamma RI and Fc gamma RII, can be analyzed selectively in rosetting assays by employing E sensitized by either human IgG or mouse IgG1. Rosetting by both classes of R was inhibited profoundly by incubation of monocytes with different types of serine protease inhibitors such as diisopropylfluorophosphate, PMSF, or N alpha-tosyl-L-lysyl-chloromethylketone. The type II Fc gamma R was much more sensitive to inhibition than Fc gamma RI. We, therefore, studied these effects in more detail by using cell line K562, which expresses only Fc gamma RII. PMSF, diisopropylfluorophosphate, and N alpha-tosyl-L-lysyl-chloromethylketone were, again, inhibiting Fc gamma RII-mediated binding dose-dependently, whereas several inhibitors of metal, aspartic, or thiol proteases proved ineffective. Furthermore, Fc gamma RII-mediated rosetting on both cell types was profoundly inhibited by the addition of different small synthetic substrates of serine esterases. In an attempt to discriminate whether the proteolytic event is an intra- or extracellular process, macromolecular antiproteases such as soybean or ovomucoid trypsin inhibitor or alpha 1-antiprotease were tested. Fc gamma RII-mediated binding by K562 cells was not susceptible to macromolecular antiproteases, in contrast to monocytes. In the presence of drugs which interfere both with receptor recycling and intracellular traffic between endosomal compartments (e.g., primaquine or monensin), the effects of inhibitors were largely abrogated. This showed that endocytosis of inhibitors might be essential, indicating the proteolytic event to be intracellular. Our findings suggest that human monocyte Fc gamma RII-mediated functioning is dependent upon the action of one or more serine proteases.
Mapping of distinct serologic and T cell recognition epitopes on an HLA-DR beta-chain.
Authors: Gorski J , Radka S F , Masewicz S , Mickelson E M
Published: December 2022

A new DR beta-chain allele is defined that is identical to the previously described DR6b molecule except for the first hyperpolymorphic region, where the new allele displays the same polymorphisms found on DR8 and DR12 genes. Two distinct epitopes have been mapped on this new allele. The polymorphism in common with DRw8 and DRw12 is recognized by mAb GS313-9D11. However, alloreactive T cell clones specific for DR6b cells (Dw9) recognize this allele, whereas Dw8-specific T cell clones do not. The mAb determinant maps to the first beta-sheet and probably involves a polymorphic residue lying outside the helix. The binding of mAb 9D11 to this region does not interfere with TCR binding. Alloreactive T cell recognition is associated with polymorphisms located predominantly on the alpha-helical portion of the molecule.
Low dose ultraviolet B-irradiated Langerhans cells preferentially activate CD4+ cells of the T helper 2 subset.
Authors: Simon J C , Cruz P D , Bergstresser P R , Tigelaar R E
Published: December 2022

UVB radiation distorts the Ag-presenting function of epidermal Langerhans cells (LC); this has been shown for the presentation of soluble Ag to primed T cells in vitro and for the initiation of delayed-type hypersensitivity in vivo, such as contact hypersensitivity (CH). Previous work has also demonstrated UVB-induced suppression of CH to be mediated ultimately by T cells. Two subsets of CD4+ Th cells, Th1 and Th2, have been identified, based on their cytokine production and functional activities. In particular, Th1 mediate delayed-type hypersensitivity, whereas Th2 do not. To investigate whether the perturbation of LC function induced by UVB radiation leads to a differential activation of these subsets of CD4+ cells, we examined the capacity of unirradiated and irradiated (200 J/m2) APC from adult BALB/c mice to present keyhole limpet hemocyanin to Ag-specific, H2d-restricted Th1 and Th2 cell lines. Four sources of APC were utilized: epidermal cells (EC), flow microfluorometry-purified Ia+ EC (LC), flow microfluorometry-purified Ia- EC, and splenic adherent cells (SAC). Unirradiated EC, LC, and SAC, but not Ia-EC, presented keyhole limpet hemocyanin to both Th1 and Th2. Irradiated EC and LC lost their ability to stimulate Th1, but retained fully their capacity to stimulate Th2. On the other hand, irradiated SAC were unable to induce proliferation of either Th1 or Th2. These findings indicate that suppression of CH mediated by UVB-irradiated LC may result from an alteration of the ratio and/or activity of Th1 and Th2 cells normally generated during the induction of such responses.
Two mechanisms of transferrin receptor induction by anti-Ig in B cells.
Authors: Vora A , Stunz L L , Kemp J D , Ashman R F
Published: December 2022

By cross-linking surface Ig to the Fc gamma R, whole (IgG)-rabbit anti-Ig antibodies have been shown to substantially inhibit proliferation induced by LPS and F(ab')2 anti-Ig, and polyphosphoinositide hydrolysis induced by F(ab')2 anti-Ig. Surprisingly, however, whole anti-Ig was unable to inhibit induction of transferrin receptor (TfR) expression by LPS or F(ab')2 anti-Ig. Indeed, whole anti-Ig on its own induced TfR as early as 4 h. TfR induction by F(ab')2 anti-Ig and by LPS was accompanied by an early increase in TfR mRNA, and was prevented by inhibitors of protein and RNA synthesis and therefore can be ascribed to a transcriptional mechanism. In contrast, whole anti-Ig induced TfR even in the presence of protein and RNA synthesis inhibitors. Little or no TfR mRNA was detectable after 4 or 16 h of exposure to whole anti-Ig, whereas increased TfR mRNA was evident after 4 h of F(ab')2 anti-Ig or LPS. Antibody to the Fc gamma R (2.4G2) restores the ability of whole anti-Ig to generate increased TfR expression via the transcriptional route. We conclude that whole anti-Ig induces TfR mostly by using preexisting TfR molecules through a mechanism different from the transcriptional mechanism triggered by F(ab')2 anti-Ig and LPS.
Characterization of the heterodimeric complex of human IL-2 receptor alpha.beta chains reconstituted in a mouse fibroblast cell line, L929.
Authors: Minamoto S , Mori H , Hatakeyama M , Kono T , Doi T , Ide T , Uede T , Taniguchi T
Published: December 2022

The receptor for IL-2 has been known to exist in three forms on the basis of their affinities to IL-2: high, intermediate, and low affinity forms. Two IL-2R components have been identified as IL-2R alpha (p55, Tac Ag) and IL-2R beta (p70-75) chains, both bind IL-2 with low and intermediate affinities, respectively. Recently, we cloned human IL-2R beta chain cDNA and demonstrated that the cDNA product binds IL-2 with intermediate affinity and forms high affinity IL-2R with coexpressed IL-2R alpha chain in a human T cell line, Jurkat. In this study, we report the establishment of the mouse fibroblast transformants expressing either the IL-2R beta chain alone or both the IL-2R alpha and IL-2R beta chains. In contrast to lymphoid cells, significant IL-2 binding was not detected in the transformants expressing the IL-2R beta chain alone at IL-2 concentrations (50 pM to 10 nM) generally utilized. Nonetheless, the transformants expressing both IL-2R alpha and IL-2R beta chains displayed two forms of the IL-2R with high and low affinities to IL-2. However, neither IL-2 internalization nor signal transduction via the high affinity IL-2R complex were observed in the L929 transformants. Those findings suggest that the interaction of the IL-2R beta chain with the IL-2R alpha chain occurs in the absence of additional lymphoid specific component(s) to form high affinity IL-2R, but that this interaction is insufficient for IL-2 internalization and signal transduction just as observed in lymphoid cells. The experimental approach described here may allow further dissection of the molecular architecture of the IL-2R complex in the ligand binding, internalization, and signal transduction.
Fc gamma receptor-mediated functions in neutrophils are modulated by adenosine receptor occupancy. A1 receptors are stimulatory and A2 receptors are inhibitory.
Authors: Salmon J E , Cronstein B N
Published: December 2022

Adenosine, an endogenously released purine, modulates the functions of many cells through surface A1 and A2 receptors. We examined the hypothesis that adenosine receptor ligation regulates Fc gamma R-triggered inflammatory response by polymorphonuclear leukocytes (PMN), a response which is critical to the pathogenesis of immune complex diseases. The effects of adenosine analogs on Fc gamma R-mediated phagocytosis and superoxide anion (O2-) generation in human neutrophils were investigated. 5'(N-ethyl)carboxamidoadenosine (NECA), the most potent A2 receptor agonist, inhibited Fc gamma R-mediated phagocytosis and O2- generation, whereas N6-cyclopentyladenosine (CPA), a highly selective A1 receptor agonist, enhanced these functions. The effects of the adenosine analogs were markedly accentuated in neutrophils adherent to biologic surfaces. Both the inhibition by NECA and the enhancement by CPA of PMN Fc gamma R functions were blocked by the adenosine receptor antagonist 8-p-sulfophenyltheophylline, which suggests that occupancy of surface adenosine receptors mediated the actions of these analogs. Because A1 receptors on PMN are linked to pertussis toxin-sensitive G proteins, our evidence that pertussis toxin blocked the effects on Fc gamma R function brought about by CPA but not by NECA further supports the hypothesis that CPA acts via an A1 receptor. Our data indicate that adenosine A1 and A2 receptors modulate neutrophil Fc gamma R function in opposing ways, allowing for a concentration-dependent, adenosine-regulated feed-back loop. At low concentrations there is enhancement of neutrophil Fc gamma R function via PMN A1 receptors, whereas at higher concentrations (those which may occur at sites of damaged tissues), there is inhibition via A2 receptors. Our observation that adenosine analogs had more potent effects on adherent neutrophils emphasizes the potential importance of adenosine as a modulator of Fc gamma R-triggered inflammation in vivo.
Treatment with monoclonal anti-CD3 antibody protects against lethal Sendai virus infection by induction of natural killer cells.
Authors: Kast W M , Bluestone J A , Heemskerk M H , Spaargaren J , Voordouw A C , Ellenhorn J D , Melief C J
Published: December 2022

C57BL/6 mice are protected from a lethal pneumonia caused by Sendai virus when treated with low doses of mAb directed to the CD3 Ag. The protective mechanism is not due to an accelerated Sendai virus-specific Th cell, CTL, or antibody response but to a strong NK cell response via the in vivo induction of lymphokines. Antibodies directed against the NK1.1 and asialo GM1 marker totally reversed the protective effect of anti-CD3 treatment. In vivo treatment with rIL-2 also induced NK activity and induced antiviral protection. Treatment with anti-CD3 protects when given in a narrow time window (1 day before until 1 day after Sendai virus inoculation), indicating that NK activity is protective in the early phase of virus infection.
Synthetic oligopeptides bearing a common or subtypic determinant of hepatitis B surface antigen.
Authors: Ohnuma H , Takai E , Machida A , Tsuda F , Okamoto H , Tanaka T , Naito M , Munekata E , Miki K , Miyakawa Y
Published: December 2022

Two determinants of hepatitis B surface Ag (HBsAg), identified by mAb raised against polypeptide components, were characterized immunochemically. One was expressed on HBsAg irrespective of the four major subtypes, i.e., adw, adr, ayw, and ayr, whereas the other was subtypic but not identical to any of d, y, w, and r determinants. The common determinant was generated by a synthetic pentadecapeptide with a sequence of Thr-Thr-Ser-Thr-Gly-Pro-Cys-Lys-Thr-Cys-Thr-Ile-Pro-Ala-Gln representing amino acids 115-129 of the S gene product, and detected invariably in 366 HBsAg samples in sera from asymptomatic carriers in Japan. The activity of the S gene product, as well as the peptide (115-129), to bind with the mAb was not affected by alkylation alone, but was completely lost after reductive alkylation. The antigenic activity was lost when the S gene product was severed between Lys122 and Thr123 by trypsin. A microconformation maintained by the -Cys121-Cys124 bond, therefore, would be required for the common determinant. The other mAb identified an epitope of HBsAg that was mimicked by a synthetic tetradecapeptide with a sequence of Thr-Cys-Thr-Ile-Pro-Ala-Gln-Gly-Thr-Ser-Met-Phe-Pro-Ser, representing amino acids 123-136 of the S gene product. Among 16 HBsAg samples with known S gene sequences, 5 with Ile126 possessed this subtypic determinant, but the remaining 11 with Thr126 did not. The 5 hepatitis B virus genomes encoding the subtypic determinant differed less than 5.6% from each other in the entire nucleotide sequence, but by 8.0% or more from any of the other 11 genomes without the capacity to encode it.
Human T cell leukemia virus type I prevents cell surface expression of the T cell receptor through down-regulation of the CD3-gamma, -delta, -epsilon, and -zeta genes.
Authors: de Waal Malefyt R , Yssel H , Spits H , de Vries J E , Sancho J , Terhorst C , Alarcon B
Published: December 2022

Infection and transformation by human T cell leukemia virus type I (HTLV-I) up-regulates expression of several inducible genes including those coding for cytokines involved in the proliferation of normal and leukemic T cells. We demonstrate that HTLV-I can also shut off expression of the CD3-gamma, delta, epsilon, and zeta genes that code for the constant elements of the TCR for Ag. In addition, the T cell-specific CD3-epsilon enhancer was found to be inactive in a HTLV-I-infected T cell clone. This HTLV-I-infected T cell clone (827-p19-II) that could be cultured in the absence of IL-2 lacked the CD3 proteins but did express the TCR-alpha and -beta proteins intracellularly. In the absence of the CD3-gamma, delta, epsilon, and zeta polypeptide chains the disulfide bridged TCR-alpha/beta heterodimer was not formed and the Ag receptor did not appear at the cell surface. These results allowed two major conclusions: first, HTLV-I infection has an effect on the T cell specific regulatory elements that coordinately regulate CD3-gamma, delta, epsilon, and zeta expression and second, the CD3-gamma, delta, epsilon, and zeta proteins are necessary for formation and routing the variable TCR-alpha/beta (or -gamma/delta) heterodimer to the human T cell surface.
Germline V genes encode viable motheaten mouse autoantibodies against thymocytes and red blood cells.
Authors: Kasturi K N , Mayer R , Bona C A , Scott V E , Sidman C L
Published: December 2022

Autoantibodies against thymocytes and RBC may contribute to the pathophysiology of homozygous viable motheaten (mev) autoimmune disease. Whether the production of these autoantibodies in mev mouse results from polyclonal nonspecific B cell activation or specific Ag-driven stimulation is not known. To understand the mechanisms involved in the induction of antithymocyte autoantibody response in mev mouse, we have studied the fine antigenic specificity, structure, and origin of three antithymocyte autoantibodies derived from mev splenic B cell hybridomas. Western blot analysis showed that these mAb bind to polypeptides of 33 and 105 kDa present in RBC and thymocytes, respectively. Additional specificities for the epitopes present in other polypeptides distinguished these three autoantibodies. Northern hybridization and flow microfluorimetry analysis indicated that these hybridomas are derived from the Ly1+ B cell subset. These autoreactive Ly-1 B cell hybridomas, chosen on the basis of their specificity, expressed L chain V genes from a single VK family (VK9) and VH genes from J606 and S107 families. Hybridomas UN34.11 and UN42.5 expressed the VK9 gene identical to that used by peritoneal Ly1+ B cells from various mouse strains and malignant B lymphoma cells secreting anti-mouse RBC treated with proteolytic enzyme bromelin and anti-SRBC antibodies. The third hybridoma, S2-14.2, used a VK9 gene identical to that expressed by MOPC41. None of the VK genes encoding these autoantibodies showed any somatic mutations. In the case of VH genes, the two hybridomas UN42.5 and S2-14.2 derived from two separate fusions, used identical VH genes from the J606 family. The third hybridoma UN34.11 used unmutated V11 germline VH gene, a member of the S107 family. Southern hybridizations, using oligonucleotide probes specific for CDR1 and CDR2, showed that the VH genes encoding the J606 autoantibodies were derived from a germline gene found in the 6.7-kb fragment of EcoRI-digested germline DNA. This germline VH gene is distinct from VH22.1 germline gene that codes for antigalactan antibodies. Sequence analysis of this gene showed perfect homology with the rearranged VH genes confirming the lack of somatic mutations. Thus, our data demonstrate that antithymocyte antibody response occurring in mev mouse is polyclonal and it involves Ly-1 B cells expressing unmutated germline VH and VK genes. These results indicate that antigen driven stimulation may not play an important role in the induction of anti-thymocyte antibody response in mev mouse.
Granulated metrial gland cells of pregnant mouse uterus are natural killer-like cells that contain perforin and serine esterases.
Authors: Parr E L , Young L H , Parr M B , Young J D
Published: December 2022

The mouse uterus during pregnancy contains a large population of lymphoid cells termed granulated metrial gland (GMG) cells. Our observations suggest that these cells are highly activated cytolytic lymphocytes related to NK or lymphokine-activated killer cells. Immunostaining demonstrated asialo GM1 and Thy-1 on GMG cells, both of which are expressed by NK cells. Decidua basalis tissue and isolated GMG cells contained three proteins that are characteristic of activated cytolytic lymphocyte granules: perforin, serine esterase 1, and serine esterase 2. These mediators were demonstrated in GMG cells by Western blot analysis using polyclonal antisera and by Northern blot analysis using specific cDNA probes for their mRNA. The proteins were not detected in normal spleen or liver or in asialo GM1+ cells isolated from those organs, consistent with the absence of these mediators from resting cytolytic cells. The amount of perforin in GMG cells was similar to that present in cloned, IL-2-stimulated, CTL shown previously to contain a large amount of this protein. A large population of NK cells bearing the surface marker LGL-1 was demonstrated at the implantation site by labeling with monoclonal antibody 4D11, but T cells were not detected. Many LGL-1+ cells at the implantation site expressed the GMG cell markers asialo GM1, Thy-1, and perforin. Staining intensities were inversely correlated, with LGL-1-bright cells showing little or no staining of GMG cell markers and LGL-1-faint cells showing more obvious staining of GMG cell markers. This suggests that LGL-1+ NK cells may differentiate in situ to GMG cells, losing LGL-1 and gaining a high concentration of GMG cell markers in the process. Activated cytolytic cells related to NK or lymphokine-activated killer cells may function in the pregnant rodent uterus to intercept and kill aberrant placental or embryonic cells that might otherwise enter the female and proliferate.
Molecular analysis of a germ line-encoded idiotypic marker of pathogenic human lupus autoantibodies.
Authors: Young F , Tucker L , Rubinstein D , Guillaume T , André-Schwartz J , Barrett K J , Schwartz R S , Logtenberg T
Published: December 2022

Id-16/6 is an idiotypic marker found in both IgM and IgG antibodies, as well as in the tissue lesions of patients with SLE. The prototypic Id-16/6+ mAb is 18/2, whose VH3-derived H chain is encoded by an unmutated germ-line gene. We found that the H chains of VH3-derived Id-16/6+ antibodies contain the major determinants of Id-16/6. Moreover, B cell clones from which those antibodies were harvested produce RNA that hybridized under conditions of high stringency to oligonucleotide probes corresponding to the CDR of the VH segment of 18/2. Western blots of Id-16/6+ mAbs with anti-Id confirmed the association of the Id with H chains. Id-16/6 can identify a subgroup of VH3-derived antibodies we have termed the 18/2 CDR family. However, Id-16/6 can also be expressed in some antibodies unrelated to the 18/2 CDR family. No characteristic Ag-binding specificity was found among the members of the 18/2 CDR family. The principal phenotypic feature shared by all known members of the family is Id-16/6.
Delineation of the mechanism of inhibition of human T cell activation by PGE2.
Authors: Minakuchi R , Wacholtz M C , Davis L S , Lipsky P E
Published: December 2022

The capacity of PGE2 to inhibit human T cell responses was examined by investigating its effect on mitogen-induced IL-2 production and proliferation of highly purified CD4+ T cells. PGE2 inhibited both PHA and anti-CD3 induced proliferation and IL-2 production by an action directly on the responding T cell. Inhibition of IL-2 production reflected decreased accumulation of mRNA for IL-2. A variety of other cAMP elevating agents exerted similar inhibitory effects. Inhibition of proliferation could be overcome by supplemental IL-2, PMA, or the anti-CD28 mAb 9.3. Although PMA and 9.3 markedly increased the amount of IL-2 produced by mitogen-stimulated T cells, the percentage inhibition of IL-2 secretion caused by PGE2 and other cAMP elevating agents remained comparable in these costimulated cultures. Rescue of T cell DNA synthesis by these agents appeared to reflect the finding that, although PGE2 markedly inhibited IL-2 production, the absolute amount of IL-2 produced was increased sufficiently to sustain mitogen-induced proliferation. As anticipated, PGE2, forskolin, and cholera toxin increased T cell cAMP levels. The quantity of cellular cAMP generated in response to PGE2, cholera toxin, and forskolin could be inhibited by PMA or 2',5'-dideoxyadenosine. Using these reagents, the inhibitory effects of PGE2 were found to reflect intracellular cAMP levels, but only within a very narrow range. The results indicate that by elevating cAMP levels, PGE2 inhibits human T cell IL-2 production at a point that is common to both the CD3 and CD28 signaling pathways.