bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo tolerance induction and associated cytokine production by subsets of murine CD4+ T cells.
Authors: Chu E B , Hobbs M V , Ernst D N , Weigle W O
Published: December 2022

Male BXSB mice develop lupus-like disease and die early in life (4 to 5 mo) whereas female mice do not. Others have demonstrated that CD4+ cells from male mice support B cell resistance to tolerance induction to human gamma-globulin (HGG). In this study, male and female mice tolerized at 2 mo of age with deaggregated HGG and subsequently immunized with HGG in comparison with mice immunized only were tested for anti-HGG Ab responses. CD4+ cells from draining lymph nodes of these mice were tested in culture for proliferation and production of cytokine mRNA and protein in response to HGG plus APC. Tolerized male but not female mice produced anti-HGG Abs of both the IgG1 and IgG2a isotypes. HGG-stimulated CD4+ cells from immunized male and female mice that were not tolerized produced IL-2, IL-4, IL-5, IFN-gamma, and TNF-beta mRNA as well as IL-2 and IL-4 protein, whereas tolerized, immunized mice of both sexes failed to proliferate or produce either IL-2 or IL-4 or express any cytokine mRNA in response to HGG in vitro. A resistance in tolerance induction in male mice, as determined by anti-HGG Abs, was also observed at 3 mo of age. Although a resistance to tolerance was also seen in terms of proliferation in the 3-mo-old males, production of IL-2 or IL-4 protein was still not observed. Thus, all T cell subsets identified by cytokine expression profiles were tolerized not only from females but also from males, of which the latter appeared to show some resistance to tolerance induction.
Transfection of TCR alpha-chains into suppressor and T helper cell hybridomas. Production of suppressor factors with predicted antigen specificity.
Authors: Kuchroo V K , Byrne M C , Greenfield E , Whitters M J , Nalefsky E A , Rao A , Collins M , Dorf M E
Published: December 2022

Conditioned medium from Ag-specific suppressor T cell hybridomas contains soluble factors (TsF) that modulate immune responses in an Ag-specific manner. We previously generated a series of TCR-alpha- and TCR-beta- expression variants from a 4-hydroxy-3-nitrophenyl acetyl (NP)-specific inducer suppressor T cell hybridoma and demonstrated that loss of TCR alpha-chain mRNA, but not TCR-beta chain mRNA, was accompanied by concomitant loss of suppressor bioactivity. Suppressor factor bioactivity was restored by expression of TCR alpha-chain cDNA, suggesting that the TCR alpha-chain plays a critical role in Ag-specific suppressor cell function. We have now transfected TCR alpha-chain from a Th cell clone specific for arsanylated peptides plus I-Ad into a TCR-alpha- derivative of an NP-specific inducer suppressor T cell hybridoma. The transfectants expressed a new hybrid TCR-alpha beta complex and produced soluble factors that suppressed azobenzenearsonate hapten (ABA) but not NP delayed-type hypersensitivity responses. These supernatants mediated suppression of the induction, but not the effector phase of the delayed-type hypersensitivity reaction. In reciprocal experiments we transfected a TCR alpha-chain from an NP-specific suppressor T cell hybridoma into a TCR-alpha- hybridoma derived from the ABA-specific Th cell hybridoma. The NP-specific TCR alpha-chain was expressed in the Th cell hybridoma, but the supernatant from this transfectant did not suppress DTH responses to either NP or ABA. However, the latter supernatants, when combined with cell lysates derived from a TCR-alpha- Ts hybridoma, specifically suppress NP DTH responses. These data are consistent with the interpretation that TCR alpha-chain imparts Ag specificity to the suppressor molecule and a second, yet undefined, component produced by the Ts hybridoma controls the immunoregulatory bioactivity.
Met-enkephalin-containing peptides encoded by proenkephalin A mRNA expressed in activated murine thymocytes inhibit thymocyte proliferation.
Authors: Linner K M , Quist H E , Sharp B M
Published: December 2022

Murine thymocytes activated with the mitogen Con A express proenkephalin A mRNA (PEA mRNA) and met-enkephalin and/or met-enkephalin-containing peptides ("enkephalins"). This Con A-induced expression of PEA mRNA is modulated by the delta opioid receptor agonist, deltorphin I, in a biphasic, dose-dependent manner. That is, 10(-13) M to 10(-11) M deltorphin enhanced PEA mRNA expression 3- to 3.5-fold over the level induced by Con A alone, and 10(-9) M to 10(-7) M deltorphin inhibited it 40 to 70%. delta opioid receptor antagonists recognizing the delta-2 (naltrindole (NTI) and naltriben (NTB)), but not the delta-1 (7-benzylidenenaltrexone (BNTX)), subtype of opioid receptor described in brain, reversed both the enhancing and inhibiting effects of deltorphin on Con A-induced PEA mRNA expression. In addition, the delta-2 receptor-specific antagonists, NTI and NTB, directly inhibited Con A-induced PEA mRNA expression. The function of the enkephalins expressed by thymocytes was examined by using 1) delta opioid receptor antagonists, 2) PEA mRNA-specific antisense cDNA, and 3) Ab to met-enkephalin, and measuring cell proliferation. All three reagents caused enhancement of Con A-induced proliferation, with effects ranging from two- to fourfold over the response to Con A alone. Again, the delta-2 subtype-specific antagonists, NTI and NTB, were functional and the delta-1 subtype-specific antagonist, BNTX, was not. The PEA mRNA-specific antisense cDNA blocked translation but not transcription of PEA mRNA. The data suggest that 1) endogenous enkephalins induced in thymocytes modulate their own expression through delta-2-like opioid receptors, and 2) these endogenous enkephalins function to inhibit the proliferation of activated thymocytes.
Effect of IL-12 on immune suppression and suppressor cell induction by ultraviolet radiation.
Authors: Schmitt D A , Owen-Schaub L , Ullrich S E
Published: December 2022

The carcinogenic potential of UV radiation (the primary cause of nonmelanoma skin cancer) is associated with its ability to suppress cell-mediated immune responses. Previous studies have shown that this UV-induced immune suppression is caused by the secretion of immunosuppressive cytokines such as IL-10. Because the effects of IL-10 on the immune response are countered by IL-12, we injected irradiated mice with IL-12 to determine whether it could overcome UV-induced immune suppression. Administration of IL-12 blocked the suppression of delayed-in-time hypersensitivity reactions observed in UV-irradiated animals. Moreover, IL-12 prevented the induction of suppressor T cells, in that adoptive transfer of spleen cells from UV-irradiated mice treated with IL-12 had no effect on the immune response of the recipient mice, whereas transfer of spleen cells from UV-irradiated mice treated with the vehicle inhibited the immune response. In addition, IL-12 neutralized the activity of UV-induced suppressor T cells. Although the adoptive transfer of UV-induced suppressor T cells from irradiated mice suppressed the immune response of the recipient mice, treatment of the recipient mice with IL-12 following the adoptive transfer overcame the immune suppression. The results of these experiments demonstrate that IL-12 can overcome UV-induced immune suppression by preventing the induction of, as well as neutralizing the activity of pre-formed suppressor T cells.
Comparison of the binding of C3S and C3F to complement receptors types 1, 2, and 3.
Authors: Bartók I , Walport M J
Published: December 2022

The two common polymorphic variants of C3 are C3S/HAV4.1- and C3F/HAV4.1+. It was reported previously that erythrocytes coated with C3F rosetted more strongly with mononuclear cells than erythrocytes coated with C3S. We examined the binding of C3S/HAV4.1- and C3F/HAV4.1+ to complement receptors CR1, CR2, and CR3. The binding of 125I-labeled C3b dimers to erythrocyte CR1 was measured. Scatchard analysis showed a two-binding constant model with very similar binding constants for dimers prepared with C3S and C3F: for C3S Kd1 = 18.3 +/- 2 nM; Kd2 = 6.2 +/- 1 nM; for C3F Kd1 = 21.5 +/- 4 nM; Kd2 = 7.2 +/- 3 nM (mean +/- SEM). One-third of the binding sites were of the higher affinity. The rosetting of erythrocytes with different densities of iC3b, prepared from C3S or C3F, to CR2 on Raji cells was analyzed. The percentage of Raji cells rosetted was related to the coating dose of EC3bi: 400 molecules/cell = 9% rosettes; 5000 molecules/cell = 75%. The dose-response curves were very similar for C3S- and C3F-coated erythrocytes. CR3-dependent rosetting was studied in a similar manner by using neutrophils activated with f-met-leu-phe. CR3-dependent rosette formation with the indicator erythrocytes (600 to 6000 iC3b/cell) increased from 10% to 60% in a dose-dependent manner and was closely similar for C3S and C3F. Inhibition of CR2 and CR3 rosetting by fluid phase ligand was also studied. iC3b dimers (0.4 to 50 micrograms/ml) inhibited CR2-dependent rosetting in a dose-dependent manner but had no inhibitory effect on CR3-dependent rosetting. When the dimers were absorbed to fluorescent microspheres, they mediated phagocytic uptake of the microspheres in a CR3-dependent manner by neutrophils. CR3-dependent binding by activated neutrophils required surface-bound ligand.
Anti-inflammatory effects of an adenosine kinase inhibitor. Decreased neutrophil accumulation and vascular leakage.
Authors: Rosengren S , Bong G W , Firestein G S
Published: December 2022

Adenosine inhibits neutrophil function, but also causes cardiovascular side effects when administered systemically. To regulate local adenosine concentrations and minimize toxicity, a novel adenosine kinase inhibitor, GP-1-515, was tested in several acute inflammation models in rats. GP-1-515 inhibited carrageenan-induced rat paw swelling in a dose-dependent manner (maximum inhibition, 47 +/- 3%). In a rat skin lesion model, GP-1-515 significantly reduced cutaneous neutrophil infiltration following an intradermal injection of carrageenan or zymosan-activated plasma, or induction of a reverse passive Arthus reaction. This action appeared to be mediated by endogenous adenosine, inasmuch as a specific A2 adenosine receptor antagonist reversed the effect. GP-1-515 also decreased vascular leakage induced by carrageenan (which is partly neutrophil dependent) and by the neutrophil-independent mediators histamine and bradykinin. Inhibition of leakage was reversed by co-administration of adenosine receptor antagonist. Treatment with anti-inflammatory doses of GP-1-515 had no effect on heart rate or blood pressure. In conclusion, GP-1-515 significantly reduced both neutrophil infiltration and vascular leakage through the release of endogenous adenosine without evidence of cardiovascular side effects.
Aspartate at position 57 of nonobese diabetic I-Ag7 beta-chain diminishes the spontaneous incidence of insulin-dependent diabetes mellitus.
Authors: Quartey-Papafio R , Lund T , Chandler P , Picard J , Ozegbe P , Day S , Hutchings P R , O'Reilly L , Kioussis D , Simpson E
Published: December 2022

MHC class II genes have been shown to influence the development of the autoimmune disease insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse. In human IDDM it has been suggested that the presence of an aspartate at position 57 of the DQ beta-chain might be important in determining resistance to development of IDDM. The involvement of MHC class II genes in IDDM was investigated through the introduction of MHC encoding transgenes. We show that introduction of a mutated I-Ag7 Ab gene which encodes an aspartate at position 57 reduces the incidence of IDDM but does not prevent insulitis, sialadenitis, or the development of insulin and nuclear autoantibodies.
Inhibition of antigen-specific T cell activation by staphylococcal enterotoxins.
Authors: Dowd J E , Jenkins R N , Karp D R
Published: December 2022

The staphylococcal enterotoxins SEA, SEB, SEC2, and TSST-1 bind to MHC class II molecules and stimulate polyclonal T cell populations on the basis of the expression of responsive TCR V beta domains. CL-1 is a human T cell clone that is specific for a peptide derived from influenza hemagglutinin (HA 307-319) presented in the context of HLA-DR1. CL-1 expresses the TCR V beta 13.1 domain, and does not respond to SEA, SEB, or TSST-1. This T cell was used to test the effect of nonstimulatory staphylococcal enterotoxins on a response to antigenic peptide. These toxins inhibit peptide-specific activation of CL-1 in a concentration-dependent manner. These toxins also inhibit the response of an HLA-DR1-specific alloreactive T cell clone. This inhibition seems to be a result of impaired access of TCR to the MHC/peptide complex rather than negative signaling by toxin via class II interaction or induction of T cell anergy. SEA, but neither SEB nor TSST-1 impedes avidin access to a biotin group attached to the amino terminus of HA 307-319. SEA partially impairs access of avidin to HA peptide biotinylated at residue 313, but is unable to inhibit avidin access to biotin at residue 318. This demonstrates that SEA binds to HLA-DR molecules that have also bound the antigenic peptide and suggests a topology for the interaction of SEA with class II, whereby the toxin interferes with peptide/MHC-TCR contact.
Specific suppression by prostaglandin E2 of activation-induced apoptosis of human CD4+CD8+ T lymphoblasts.
Authors: Goetzl E J , An S , Zeng L
Published: December 2022

Receptors (Rs) for prostaglandin E2 (PGE2) of the EP2 subtype are expressed at high levels in rodent and human thymuses, with preferential localization on immature thymocytes. Human cultured lymphoblasts of the Tsup-1 line express CD4 and CD8 but only a low level of CD3, typical of immature thymocytes, and bear EP2-type Rs for PGE2 that were identified by binding of [3H]PGE2 and Ab to recombinant EP2Rs, and by cAMP responses to PGE2. PGE2 protected Tsup-1 cells from apoptosis initiated by diverse stimuli, including mitogenic lectins and anti-Fas or anti-CD3 plus anti-CD28 Abs, but not ionomycin, as assessed by suppression of both fragmentation of [3H]thymidine-labeled DNA and appearance of free 3'-OH ends of cleaved DNA. An EP2R-selective synthetic agonist also significantly suppressed lectin-induced apoptosis of Tsup-1 cells. Phosphodiesterase inhibition synergistically enhanced PGE2-induced increases in cAMP and decreases in apoptosis in parallel, which suggests that the EP2R-specific protective effect of PGE2 is mediated predominantly by cAMP suppression of apoptosis. Dibutyrylcyclic AMP alone protected Tsup-1 cells against lectin-induced apoptosis, but the maximal effect was less than that for PGE2. The thromboxane A2 mimetic U46619 initiated apoptosis of Tsup-1 cells that was suppressed significantly by PGE2. Immune negative selection of immature thymocytes thus may be regulated by opposing effects of endogenous eicosanoids that include destruction by thromboxane A2 and protection by PGE2.
Acquisition of peptides by MHC class II polypeptides in the absence of the invariant chain.
Authors: Hitzel C , van Endert P , Koch N
Published: December 2022

Association of invariant chain with class II molecules has been suggested to inhibit binding of peptides that are available while the class II complex is present in the endoplasmic reticulum (ER) and subsequently transported to endosomes. We tested HLA-DR-transfected rat2 fibroblast cells lacking expression of invariant chain for their ability to form SDS stable class II dimers indicative of peptide binding in the class II cavity. No SDS-resistant class II dimers originating from short pulse-labeled immunoprecipitates can be identified. Prolonged ER retention of DR polypeptides by Brefeldin A treatment does not induce any stable class II dimers. In pulse-chase experiments, heat labile class II dimers are readily detectable after a 60-min chase, increasing in amounts by 4 h of chase. In vitro incubation of rat2DR cell lysate with DR3-binding peptides converts pulse-labeled class II molecules to SDS-resistant dimers. This indicates the ability of ER-resident DR dimers to bind peptides. Inhibition studies were conducted to define the intracellular site where stable class II complexes are formed in rat2DR cells. The lysosomotropic reagent chloroquine abrogates the appearance of SDS-resistant DR complexes in invariant chain-free rat2DR cells, which is consistent with the impact of chloroquine on peptide loading in other APCs. Leupeptin treatment strongly reduces the amount of heat-labile class II molecules but does not impair peptide loading when DR3-specific peptides were added to viable cells or to cell lysates. This result suggests that leupeptin inhibits intracellular degradation of polypeptides and thereby depletes the endocytic pathway of peptides available for class II binding.
Expression of VCAM-1 (CD106) by a subset of TCR gamma delta-bearing lymphocyte clones. Involvement of a metalloprotease in the specific hydrolytic release of the soluble isoform.
Authors: Leca G , Mansur S E , Bensussan A
Published: December 2022

The cytokine-inducible vascular cell adhesion molecule 1/CD106 is widely distributed in endothelial, epithelial, macrophage, and dentritic cells. We previously have reported a mAb termed sTA that recognizes the CD106 molecule on various TCR gamma delta T cell clones that do not proliferate in response to an anti-CD3 stimulation. In the present report, further biochemical analysis reveals two intracellular precursors (82 and 98 kDa) of the membrane-bound 110-kDa form of CD106. In addition, we detect a 100-kDa soluble form in the culture supernatant of the specific cloned lymphocytes. Phorbol ester raises the amount of the soluble CD106 in the supernatant while simultaneously inducing the disappearance of the membrane-bound form. We show that the membrane-anchored form of CD106 is converted to soluble form by a regulated proteolytic cleavage process involving a metalloprotease. EDTA and 1,10-phenanthroline, two potent inhibitors of metalloproteases, specifically inhibit the conversion of the membrane anchored to the soluble form of the CD106 molecule. In fact, these results implicate a Zn(2+)-activated metalloprotease in the regulation of CD106 expression in a subset of T cells and, therefore, represent a novel pathway of T cell functions.
Human p59fyn(T) regulates OKT3-induced calcium influx by a mechanism distinct from PIP2 hydrolysis in Jurkat T cells.
Authors: Rigley K , Slocombe P , Proudfoot K , Wahid S , Mandair K , Bebbington C
Published: December 2022

The earliest biochemical event after cross-linking of TCR is the tyrosine phosphorylation of a variety of substrates. At least three nonreceptor tyrosine kinases have been implicated in this signaling cascade: p59fyn(T), p56lck, and ZAP-70. Recently, PLC gamma 1 has been shown to be tyrosine phosphorylated in T cells after receptor activation. This increase in tyrosine phosphorylation correlates with the increased activity of the enzyme. The substrate for PLC gamma 1, phosphatidylinositol 4,5-bisphosphate (PIP2), is hydrolyzed to the protein kinase C activator diacylglycerol and inositol 1,4,5-triphosphate (IP3), which promotes calcium release from the endoplasmic reticulum. These results lend support to the notion that calcium mobilization after TCR cross-linking is mediated by increased levels of IP3. In this study we have cloned and transfected a human p59fyn(T) cDNA in the anti-sense configuration into the human T cell line, Jurkat, resulting in decreased expression of the protein. We find that cell lines expressing significantly reduced levels of p59fyn(T) exhibit significantly lower calcium influx following OKT3 activation. However, the level of IP3 production was unchanged and IP1 and IP2 levels were elevated. These data indicate that p59fyn(T) can regulate calcium influx by a mechanism distinct from PIP2 hydrolysis.
Analysis of an initial step of T cell adhesion to endothelial monolayers under flow conditions.
Authors: Yago T , Tsukuda M , Yamazaki H , Nishi T , Amano T , Minami M
Published: December 2022

We analyzed an initial step of T cell adhesion to endothelial cells under flow conditions. An initial step in T cell-endothelial cell interactions was characterized either by a rolling phase, or by an immediate arrest without rolling. Anti-E- and P-selectin Abs inhibited T cell rolling on endothelial cells under flow conditions. The rolling velocity of T cells on endothelial cells increased after treatment of endothelial cells with anti-E- or P-selectin Abs. Also, rolling of T cells was observed on E-selectin-transfected CHO cells but not on ICAM-1-transfected CHO cells. Thus, rolling of T cells under flow condition was dependent on E-selectin as well as P-selectin expressed on endothelial cells. In contrast, neither anti-E- nor P-selectin Abs inhibited the immediate arrest of T cells on endothelial cells significantly and anti-ICAM-1 Abs also failed to inhibit the immediate arrest of T cells. Therefore, other adhesion molecules may play an important role in the immediate arrest of T cells under flow conditions. The number of CD45RA- T cells rolling on E-selectin-transfected CHO cells was much higher than that of CD45RA+ T cells.
Analysis of homology-directed recombination in VDJ junctions from cytoplasmic Ig- pre-B cells of newborn mice.
Authors: Chukwuocha R U , Nadel B , Feeney A J
Published: December 2022

We previously showed that most VD and DJ gene combinations from newborn surface Ig- pre-B cells had one to three predominant junctions, all of which occurred at the sites of short sequence homologies between the two coding ends. Because the majority of sequences that are present in pre-B cells are in-frame, however, the possibility existed that the frequency of occurrence of predominant IgH junctions was skewed by proliferation of pre-B cells with productive rearrangements. In this study, we analyzed cytoplasmic Ig- pre-B cells, because these cells should not yet be subject to such selection. Two-thirds of the rearrangements from this population in the adult were out-of-frame, suggesting that these rearrangements are unbiased. In newborn cIg- pre-B cells, DJ junctions still showed the same predominant sequences as sIg- pre-B cells, but there was less use of predominant junctions in VD junctions for three of four different VH genes analyzed. For those three VH genes, an average of 30% of the sequences were in-frame. When only the in-frame rearrangements from these cIg- newborn cells were analyzed, frequencies of predominant VD junctions were comparable to those in sIg- pre-B cells. For sequences using the VHS107/V11 gene, however, 67% of the junctions were created at the site of the same dinucleotide in the V gene, and as a result, 73% of the sequences were in-frame. Thus homology-directed recombination does not initially produce as much junctional homogeneity as anticipated in all VD combinations, although it is a frequently used mechanism in the early fetal/neonatal gene rearrangements.
Molecular cloning, characterization, and chromosomal localization of a human lymphoid tyrosine kinase related to murine Blk.
Authors: Islam K B , Rabbani H , Larsson C , Sanders R , Smith C I
Published: December 2022

Triggering of Ag receptors on lymphocytes induces rapid phosphorylation of several receptor-associated protein tyrosine kinases (PTKs), implicating their role in controlling cellular growth and differentiation. In this study, we report the cloning of a human cDNA encoding a nonreceptor PTK with a calculated M(r) of about 58 kDa. The kinase has an overall amino acid identity of approximately 87% with the murine Blk. However, in the unique domain there is only 58% homology and an insertion of six amino acids in the N-terminal region. The nature of this insertion suggests a functional role in membrane attachment. Northern blot analysis showed expression in all stages of B cell development and in T cell lines. The message was not observed in the nonlymphoid tissues examined. In contrast, expression of murine blk in plasma cells and T lymphocytes has not been reported. Importantly, transcripts were seen in human embryonic liver as early as 7.5 wk of gestation before the rearrangement of Ig H chain locus. Furthermore, transcripts were detected in human thymocytes and not in mature T cells. Southern blot analysis revealed polymorphism of this gene in a Caucasian population but not in a Gambian population, indicating a recent origin of this polymorphism. The gene was localized to chromosome 8p22-23. The homology at the protein level suggests that this kinase may be the human homologue of murine Blk. Expression of BLK in immature T cells suggests that BLK may play an important role in thymopoiesis.
Docosahexaenoic acid, a major constituent of fetal serum and fish oil diets, inhibits IFN gamma-induced Ia-expression by murine macrophages in vitro.
Authors: Khair-el-Din T A , Sicher S C , Vazquez M A , Wright W J , Lu C Y
Published: December 2022

Decreased Ia expression by macrophages may account for the increased susceptibility of fetuses and neonates to infection. We chose to investigate the role of docosahexaenoic acid (DHA), an omega-3 fatty acid, on Ia expression in vitro, because neonatal serum concentrations of DHA (100-150 microM) are approximately 50 times higher than in the adult. In addition, DHA is a major component of fish-oil diets that ameliorate some autoimmune diseases and prevent renal allograft rejection. DHA inhibited IFN-gamma-induced Ia expression with a half-maximal inhibitory concentration of 25 microM. The inhibition was not caused by nonspecific damage, because oxidative metabolism via the mitochondrial electron-transport chain was not inhibited. There were strict biochemical requirements for inhibition of Ia expression. Polyenoic fatty acids with 22 carbons were more inhibitory than those with 20 carbons. Among 22-carbon fatty acids, those with more double bonds, and, in particular, with a double bond in the omega-3 position, were more inhibitory. Although DHA is known to inhibit cyclooxygenase and thus the production of eicosanoids, indomethacin did not inhibit Ia expression. This indicated that inhibition of cyclooxygenase was not responsible for inhibition of Ia expression. We divided induction of Ia expression by IFN-gamma into four phases, with IFN-gamma being present only during the second phase. DHA was most inhibitory when given before or with the IFN-gamma. This indicated that DHA inhibited early steps in IFN-gamma-induced Ia expression. Consistent with this idea, we found that DHA inhibited the increase in mRNA transcripts for Ia beta b, as assayed by Northern blotting. In summary, we found that DHA, a major component of fetal and neonatal sera as well as fish-oil diets, inhibited IFN-gamma-induced macrophage Ia expression in vitro by preventing increases in Ia mRNA transcripts.
Human granulocytes contain an opiate alkaloid-selective receptor mediating inhibition of cytokine-induced activation and chemotaxis.
Authors: Makman M H , Bilfinger T V , Stefano G B
Published: December 2022

Human peripheral blood granulocytes previously were found to contain opioid delta 2-receptors mediating stimulation by opioid peptides of chemotaxis. Studies presented in this work indicate that granulocytes also contain opiate alkaloid-selective, opioid peptide-insensitive receptors mediating inhibition by morphine and other opiates of cytokine-induced activation and chemotaxis. Binding studies with [3H]morphine and [3H]diprenorphine ([3H]DPN) indicated the presence of receptor sites, at considerable density with affinities and selectivity for opiates comparable with those of the mu 3-receptor of human peripheral blood monocytes (macrophages). The influence of the guanosine 5'-triphosphate (GTP) analogue GppNHp on binding indicated that the granulocyte receptor was linked to a G protein. Morphine but not opioid peptides interfered with activation and/or chemotaxis of the granulocytes induced by TNF-alpha, IL-1 alpha, IL-8, and FMLP (chemotactic peptide). These effects of morphine were blocked by the antagonist naloxone. Levorphanol inhibited TNF-alpha-induced activation, and also potentiated the inhibition by morphine. Furthermore, in binding assays, levorphanol enhanced the affinity of the receptor for morphine. Dextrorphan had no effect on activation or chemotaxis, and it also had no effect on binding, indicative of stereoselectivity for the effect of levorphanol. It is concluded that human granulocytes contain opiate alkaloid-selective mu 3-receptors that mediate inhibitory effects of morphine on cellular activation by cytokines.
Endothelial cell associated platelet-activating factor (PAF), a costimulatory intermediate in TNF-alpha-induced H2O2 release by adherent neutrophil leukocytes.
Authors: von Asmuth E J , Buurman W A
Published: December 2022

TNF is a strong secretagogue for surface-contacting neutrophils. During inflammation, endothelium offers the first substrate for neutrophil adherence and for modulation of the toxic response of neutrophils to soluble agonists such as TNF. In this in vitro study, evidence is presented that endothelium participates actively in TNF-induced neutrophil respiratory burst activity by expressing platelet-activating factor (PAF) in response to initial neutrophil H2O2 release. Three findings are shown that favor such a mechanism. First, PAF receptor antagonists reduced H2O2 release by TNF-activated neutrophils placed on endothelium approximately by 50%, whereas H2O2 responses by neutrophils placed on serum-coated polystyrene remained intact. Second, preincubation of HUVEC with known PAF-inducing agents PMA, H2O2, and thrombin, followed by fixation, enhanced neutrophil H2O2 release in response to TNF. H2O2 release by these neutrophils was sensitive to the presence of PAF receptor antagonists, whereas H2O2-release from neutrophils placed on fixed nonactivated endothelial cells was not. Finally, replacing endothelium by monolayers of human renal cortical epithelial cells and human fibroblasts, cells that are known to produce less PAF than endothelial cells, reduced the effect of PAF receptor antagonists. P-selectin expression and IL-8 release, two other ways by which endothelial cells might influence H2O2-release by TNF preincubated neutrophils, were examined in parallel, and were found not to influence TNF-induced neutrophil H2O2-release. We conclude that during neutrophil-endothelial interaction in inflammation, endothelium modulates the toxic response of neutrophils to TNF. Endothelial cell-associated PAF, but not endothelial cell IL-8 release and P-selectin expression, is likely to participate in TNF-induced neutrophil respiratory burst activity.
CDR3-independent gamma delta V delta 1+ T cell expansion in the peripheral blood of HIV-infected persons.
Authors: Boullier S , Cochet M , Poccia F , Gougeon M L
Published: December 2022

A majority of circulating gamma delta T cells in humans express the V delta 2 variable segment associated with the V gamma 9 segment. A minor subset uses the V delta 1 gene mainly paired with a V gamma-chain from group I. Although little is known about the function and the Ags recognized by V delta 1 T cells, their expansion has been described in several diseases. Significant alterations of gamma delta subset distribution have been observed in PBMC from HIV-infected persons. In addition to their significant increase, gamma delta T cells showed an alteration in their subset representation because most of them expressed the V delta 1 receptor and, concomitantly, the V delta 2+ subset was under-represented. To gain insight into the mechanisms involved in this selective expansion, we characterized the V delta 1-J delta 1 junctional diversity in PBMC from healthy donors and HIV-infected individuals at different stages of the disease. We confirmed that the V delta 1 repertoire is restricted in most of the healthy donors. In HIV-infected subjects, we found that the increase of V delta 1 T cells is independent to a particular V gamma-chain expression, and the characterization of the TCR-delta diversity demonstrated a similar restricted V delta 1-J delta 1 rearrangement pattern, not significantly different from the pattern of healthy donors. Moreover, no amino acid junctional motif could be identified either in control or in HIV-infected donors. This report demonstrates that the V delta 1 selective expansion in the course of HIV infection is not the consequence of the emergence of some specifically CDR3-dependent expanded V delta 1 T cell clones. Interestingly, this subset showed an increased ability to be expanded in vitro in the presence of IL-2 alone and, although they did not harbor ex-vivo the phenotype of fully activated cells, they did express the activation marker CD38, a marker for disease progression. Altogether this report indicates that, although the patients' V delta 1 T cells seem to be in a pre-activated state, their selective expansion in the course of HIV infection is not the consequence of a peripheral CDR3-dependent antigenic selection.
T cell activation and anergy to islet cell antigen in type I diabetes.
Authors: Miyazaki I , Cheung R K , Gaedigk R , Hui M F , Van der Meulen J , Rajotte R V , Dosch H M
Published: December 2022

Early exposure to cow milk proteins was linked to the development of type I diabetes by consistent epidemiology, and by feeding and tolerization studies in diabetes-prone rodents. Dietary BSA was suggested as the culprit because patients and relevant rodents have elevated anti-BSA Abs that precipitate the recently cloned protein, p69, from beta cell lysates. A total of 68 of 78 children with recent onset diabetes had BSA-reactive T cells at the time of diagnosis. Here we 1) map the fine specificity of these T cells, 2) delineate a homologous peptide sequence near the N-terminus of p69, and 3) demonstrate T cell recognition of this p69 sequence (T cell epitope p69, Tep69) by patient T cells. The Tep69 sequence is conserved in p69 of patients and diabetes-prone rodents. Whereas BSA triggers T cell proliferation, recombinant p69 and a synthetic Tep69 peptide induce early stages of T cell activation (IL-2R transcription) but insufficient IL-2 production and thus anergy. Exogenous IL-2 overrides anergy and allows proliferative expansion of p69-responsive T cells. In mixing experiments, p69 and Tep69 peptide prevented proliferative responses to BSA even at 100-fold smaller concentrations. These findings imply that high-affinity self-peptide triggers anergy, whereas low-affinity mimicry Ag triggers proliferative expansion of these T cells. This implies a disease model in which mimicry Ag would rescue autoreactive cells from ablation by self-Ag.
pp60c-src expression is induced by activation of normal human T lymphocytes.
Authors: Branch D R , Mills G B
Published: December 2022

We have re-examined whether pp60c-src, the normal cellular homologue of the transforming protein of Rous sarcoma virus, is present in human T cells. By in vitro immune-complex kinase assay or Western blotting with the anti-pp60c-src mAbs 327 or GD11, pp60c-src was found to be present in lysates of T cell lines, including the Jurkat T cell line. The 327 and GD11 mAbs have been reported to be specific for pp60c-src and not to cross-react with other src family members or other kinases. Furthermore, the size of the pp60c-src bands present on Western blotting and in vitro kinase assay were clearly different from those of p56lck or p59fyn. In addition, pp60c-src is detected in the HTLV-I-derived T cell lines S1T and C8, which lack expression of p56lck and p59fyn. RNase protection assays confirmed that pp60c-src mRNA is present in Jurkat T cells. We also found pp60c-src protein to be constitutively present in freshly isolated thymocytes. In contrast, pp60c-src was absent, or present at extremely low levels, in normal, resting peripheral blood T lymphocytes, which is in agreement with previous findings. However, after stimulation of resting T cells with the mitogenic lectin PHA or with Ab to the TCR complex, pp60c-src expression is induced in both CD4+ and CD8+ T cell subsets, with peak expression detectable 12 to 24 h after T cell activation. The levels of pp60c-src are low in all T cells except Jurkat, where levels of pp60c-src are comparable to levels found in a glioblastoma cell line (T98G). Nevertheless, significant levels of pp60c-src kinase activity are readily detectable in thymocytes and activated normal T cells as well as in T cell lines. The finding that pp60c-src is inducible following activation through the TCR suggests that pp60c-src may play a specific role in the normal T cell activation pathway.
The hinge deletion allelic variant of porcine IgA results from a mutation at the splice acceptor site in the first C alpha intron.
Authors: Brown W R , Kacskovics I , Amendt B A , Blackmore N B , Rothschild M , Shinde R , Butler J E
Published: December 2022

Recently published genomic and cDNA sequences for porcine IgA suggested that the splice acceptor site in the C alpha 1-C alpha 2 intron was an AA rather than an AG dinucleotide. This possibility was tested in an in vitro HeLa cell splicing system using an RNA substrate corresponding to the genomic DNA with the putative AA splice site. Data indicated that splicing occurred at a cryptic AG site 12 nucleotides into the C alpha 2 domain rather than at the AA site. The possibility that swine B cells could use either site was tested by preparing the cDNAs from 13 different samples representing nine animals and amplifying the segment from the first C alpha 1 nucleotide to nucleotide 532 in C alpha 2 (genomic DNA numbering system). Analysis on a 6% polyacrylamide sequencing gel revealed two polynucleotide products in most samples that differed by the expected 12 nucleotides, suggesting that swine could use both splice sites. Sequence analysis confirmed that the shorter form was spliced at the downstream site and the larger form at the apparent upstream AA site. However, when the genomic DNA from an animal expressing only the longer polynucleotide was cloned and sequenced, the upstream splice acceptor site was AG not AA. Thus the data suggested that porcine IgA occurred in two allelic forms, designated IgAa and IgAb, which differ by an apparent G to A mutation in the last nucleotide of intron 1 resulting in a short-hinged (two amino acids, IgAb) variant, in which the downstream cryptic splice site is used, as well as a "normal-hinged" (six amino acids, IgAa) variant. Evidence that IgAa and IgAb are allelic was confirmed by genotypic analyses of progeny from matings of IgAa/IgAb heterozygotes. Evidence that both transcripts are functional was confirmed by showing that serum IgA levels were similar in animals homozygous for each variant.
Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.
Authors: Blum-Tirouvanziam U , Servis C , Habluetzel A , Valmori D , Men Y , Esposito F , Del Nero L , Holmes N , Fasel N , Corradin G
Published: December 2022

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.
Gamma delta T cells function in cell-mediated immunity to acute blood-stage Plasmodium chabaudi adami malaria.
Authors: van der Heyde H C , Elloso M M , Chang W L , Kaplan M , Manning D D , Weidanz W P
Published: December 2022

To determine whether gamma delta T cells are essential for the resolution of acute Plasmodium chabaudi adami (P. c. adami) malaria, we depleted gamma delta T cells from C57BL/6 mice with hamster monoclonal anti-TCR gamma delta Ab treatment. During the period in which control mice that had received normal hamster IgG completely resolved infections, gamma delta T cell-depleted mice were unable to suppress their infections. Because the number of splenic CD4+ alpha beta T cells in these anti-TCR-gamma delta-treated mice with nonresolving malaria was similar to control mice, it appears that CD4+ alpha beta T cells alone cannot mediate early resolution even though they are known to play a critical role in immunity to blood-stage malaria. Mice treated with anti-CD4 mAb also failed to resolve P. c. adami malaria. Depletion of CD4+ alpha beta T cells from the spleens of infected mice resulted in minimal expansion of the splenic CD4- gamma delta T cell subset compared with infected control mice. Together, these findings indicate that activation of the gamma delta T cell subset, which requires the presence of CD4+ alpha beta T cells, is essential for resolution of acute P. c. adami malaria. To determine whether gamma delta T cells require either Abs or B cells to achieve their protective activity, B cell-deficient JHD mice were treated with the same depleting anti-TCR-gamma delta Abs. Whereas control JHD mice injected with hamster IgG resolved acute P. c. adami malaria, JHD mice depleted of gamma delta T cells failed to do so. We conclude that gamma delta T cells suppress P. c. adami parasitemia by mechanisms of immunity independent of Ab and B cells.
Taxol and colchicine increase LPS-induced pro-IL-1 beta production, but do not increase IL-1 beta secretion. A role for microtubules in the regulation of IL-1 beta production.
Authors: O'Brien J M , Wewers M D , Moore S A , Allen J N
Published: December 2022

IL-1 beta is a proinflammatory cytokine secreted chiefly by monocytes and macrophages. Currently, much of its mechanism of processing and secretion is poorly understood, but there is increasing evidence that the microtubule system may be involved. For example, it is known that taxol and colchicine, two drugs that affect microtubule structure and function, increase LPS-induced IL-1 beta release. However, it is not known whether these drugs affect the synthesis of the 31-kDa precursor (pro-IL-1 beta) or the processing and release of mature IL-1 beta. To test this, an assay was used that allowed for the temporal separation of IL-1 beta synthesis and release. The addition of taxol or colchicine to the secretory phase of the assay resulted in no significant change in IL-1 beta release. However, when these drugs were added to the stimulus for IL-1 beta production, there was a significant increase in both IL-1 beta release and total IL-1 beta production, as measured by ELISA. These findings indicate that taxol and colchicine increase LPS-induced IL-1 beta release by an increase in the production of the precursor molecule. Thus, it is unlikely that microtubules are involved in the IL-1 beta secretory machinery in any significant manner, but they do play a role in the regulation of pro-IL-1 beta production.
Lack of effect of recombinant platelet-derived growth factor on human neutrophil function.
Authors: Qu J , Condliffe A M , Lawson M , Plevin R J , Riemersma R A , Barclay G R , McClelland D B , Chilvers E R
Published: December 2022

Platelet-derived growth factor (PDGF) has been reported to induce chemotaxis, degranulation, and superoxide anion generation, and to increase the expression of CD11b/CD18 in human neutrophils; hence, it has been proposed as an important regulator of neutrophil function. Most of the studies on PDGF, however, have been complicated by the use of nonrecombinant PDGF or the use of mixed leukocyte cell preparations. Assessment of the effects of recombinant human PDGF-AB or -BB which display agonist activity against both PDGF receptor subtypes failed to demonstrate any effect of this peptide on neutrophil shape change, respiratory burst activity, CD11/CD18, or CD62-L expression, inositol 1,4,5-trisphosphate accumulation, or phosphorylation of mitogen-activated protein kinase. This apparent lack of effect of PDGF was consistent with our findings that neutrophils display no specific 125I-PDGF-AB or -BB binding and lack detectable mRNA for PDGF alpha-receptor and beta-receptors. These data indicate that human neutrophils do not possess functional PDGF receptors and question previous reports of a functional effect of this peptide in these cells.
Proteoglycan form of colony-stimulating factor-1 (proteoglycan-100). Stimulation of activity by glycosaminoglycan removal and proteolytic processing.
Authors: Partenheimer A , Schwarz K , Wrocklage C , Kölsch E , Kresse H
Published: December 2022

A proteoglycan had been isolated from the conditioned media of a human osteosarcoma cell line and had tentatively been named proteoglycan-100 (PG-100) because of the size of its core glycoprotein. Amino acid sequencing of the purified proteoglycan and cDNA analysis were consistent with the assumption that PG-100 is identical with the proteoglycan form of CSF-1 (or macrophage colony-stimulating factor). PG-100 induced mouse macrophage differentiation. Proliferation of macrophages was stimulated in a dose-dependent manner. On a molar basis, however, about 100- to 300-fold higher doses of PG-100 than of recombinant human (rh)CSF-1 were required for the half-maximal growth-stimulating effect. Upon enzymatic removal of the glycosaminoglycan chain, the purified core protein exhibited higher activity, but was still about 20-fold less active than rhCSF-1. Incubation of the purified proteoglycan for 48 h at 37 degrees C led to the formation of a glycosaminoglycan-free 50-kDa fragment either by autoproteolysis or by the action of a protease not yet identified. The purified fragment exhibited almost the same biologic activity as rhCSF-1. The glycosaminoglycan chain of the growth factor was not only shown to inhibit CSF-1 activity but also to increase the stability of the core protein when the CSF-1-producing osteosarcoma cells were maintained in a collagen lattice. These findings provide a link between a soluble, highly active cytokine and its extracellular matrix storage form of comparatively low activity.