bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen-independent processes in antigen-specific immunity. A role for alpha 4 integrin.
Authors: Ferguson T A , Kupper T S
Published: December 2022

It has been proposed that the migration of immune T cells out of blood vessels through connective tissue to the site of antigenic challenge is facilitated by the interaction of VLA integrins on lymphocytes with endothelial cell adhesion molecules and matrix proteins. Indeed, we have recently demonstrated that peptides derived from sequences in fibronectin abrogate the T cell-mediated contact hypersensitivity (CHS) reactions in vivo. These peptides blocked the interaction of the integrins VLA-4 and VLA-5 with fibronectin (FN), and our results suggested that by preventing the interaction of T cell integrins with FN, we successfully prevented the migration of T cells to sites of antigenic challenge. To further explore the role of integrins in T cell migration, we have used an antibody (R1-2) specific for the alpha-chain of alpha 4 integrins. Our data show that this antibody stains more than 90% of CD3+ T cells, and this percentage does not change after the mice have been immunized with a contact sensitizer. We also show that this antibody blocks the interaction of cells with immobilized FN and vascular cell adhesion molecule-1 on activated endothelial cells. Although R1-2 identifies alpha 4 integrin on a large number of T cells, it does not discriminate between activated and resting forms of this integrin, because very few T cells from immune or nonimmune mice actually bind FN or activated endothelium. In vivo, antibody R1-2 effectively blocks Ag-specific CHS, but has little effect on the non-Ag-specific cells that localize to the site of antigenic challenge. The population of cells that adoptively transfer CHS exits with the population of cells that bind to FN or to activated endothelial cells. Our data demonstrate that a small number of Ag-specific T cells use alpha 4 integrin to enter sites of inflammation and mediate effector immune responses. Ag-independent accumulation of T cells near sites of inflammation does not appear to be mediated by alpha 4 integrin.
An evaluation of mechanisms by which tolerance to organ-specific antigens is lost using a transgenic mouse model.
Authors: Martin L , Mora-Worms M , Lucas C , Reynolds C , Stewart T A
Published: December 2022

Patients with type I diabetes lose immunologic tolerance to beta cell Ag and produce anti-self (beta cell) antibodies. We have examined mechanisms by which this self tolerance is lost using transgenic mice in which islet cells express human CD4. These mice are immunologically tolerant to this human protein; the tolerance appears to be characterized by the presence of self reactive B cells and nonresponsive T cells. Autoantibodies can be induced by immunization with CD4 in CFA; this autoantibody response is accompanied by minor peri-islet inflammation but diabetes is not induced. Tolerance can also be broken by provision of an independent covalently linked T cell epitope. Although this mimic can lead to the appearance of an autoantibody response, the autoimmune response can not be maintained by the endogenous Ag. We have also demonstrated that an inflammatory lesion leading to death of the CD4-containing beta cells may not be sufficient to break tolerance. These results have implications for potential mechanisms relating to the appearance of autoantibodies in patients with type I diabetes.
Inhibition of tyrosine phosphorylation prevents IFN-gamma-induced HLA-DR molecule expression.
Authors: Ryu K , Koide Y , Yamashita Y , Yoshida T O
Published: December 2022

We have previously demonstrated that HLA-DR molecule expression induced by IFN-gamma is associated with phosphatidylinositide turnover, activation of protein kinase C, and elevation of intracellular calcium. Because phosphorylation of phospholipase C-gamma 1 on tyrosine residues is known to be involved in the activation of phosphatidylinositide turnover, we investigated the role of tyrosine protein kinase (TPK) in the signal transduction for IFN-gamma-inducible DR molecule expression on T98G cells. The effects of three specific TPK inhibitors, genistein, herbimycin A, and tyrphostin, suggest that TPK is involved in the signal transduction. These inhibitors inhibited the IFN-gamma-inducible DR molecule expression in a dose-dependent manner. Being consistent with this, immunoblotting with an anti-phosphotyrosine mAb revealed that IFN-gamma induces a rapid increase in protein tyrosine phosphorylation. Genistein not only abrogated the IFN-gamma-induced enhancement of tyrosine phosphorylation, but also inhibited the IFN-gamma-induced production of inositol-4-5-triphosphate and the elevation of intracellular calcium. However, these three TPK inhibitors failed to inhibit the DR molecule expression induced by PMA and A23187. These findings suggest that the tyrosine phosphorylation is an early and critical event that precedes phosphatidylinositide turnover leading to activation of protein kinase C and elevation of intracellular calcium concentration during IFN-gamma-inducible DR molecule expression.
The rabbit neutrophil N-formyl peptide receptor. cDNA cloning, expression, and structure/function implications.
Authors: Ye R D , Quehenberger O , Thomas K M , Navarro J , Cavanagh S L , Prossnitz E R , Cochrane C G
Published: December 2022

The rabbit neutrophil N-formyl peptide receptor (FPR) has been well studied for its ligand binding properties. Recent gene cloning experiments have established the existence of a subfamily of G protein-coupled receptors that share extensive sequence homology with the FPR, yet lack the capability of high affinity binding to FMLP. These findings prompted us to identify the structural requirement for formyl peptide ligand binding by delineation of the primary structure of the rabbit FPR. A rabbit neutrophil cDNA library was screened with a cloned human FPR cDNA probe and the insert of one positive isolate (B6) was sequenced. The 1268-bp cDNA insert encodes a peptide of 352 amino acids. Stably transfected L cell fibroblasts expressing the rabbit cDNA displayed specific binding of the ligand fMet-Leu-[3H]Phe with two affinities (Kd = 0.31 and 7.5 nM). Addition of the nonhydrolyzable guanosine triphosphate analogue, GTP gamma S, converted > or = 85% of the high affinity sites to the low affinity sites. FMLP induced mobilization of intracellular calcium in the transfected cells (EC50 = 0.5 nM), a response sensitive to pertussis toxin. FMLP stimulation desensitized the receptor such that subsequent stimulation with the same ligand produced a significantly reduced signal. These results indicate that the cloned rabbit receptor represents a high affinity FPR, and that FPR-mediated early signal transduction events can be fully reconstituted in transfected mammalian cells. The rabbit FPR sequence is 78% identical to that of the human FPR, and 68% identical to FPR2, a homologue of FPR with a low binding affinity (Kd > or = 400 nM) for FMLP. Analysis of the aligned sequences of these three proteins revealed that: 1) the amino termini and the second extracellular loops have the lowest sequence homology; 2) sequence in the intracellular domains that couple to G protein are highly conserved; and 3) the first and the third extracellular loops and their adjacent transmembrane domains of the FPR may contain residues essential for the high affinity binding of FMLP.
Immunogenicity of multiple antigen peptides containing B and non-repeat T cell epitopes of the circumsporozoite protein of Plasmodium falciparum.
Authors: Calvo-Calle J M , de Oliveira G A , Clavijo P , Maracic M , Tam J P , Lu Y A , Nardin E H , Nussenzweig R S , Cochrane A H
Published: December 2022

We have characterized the immune response of mice to multiple Ag peptide systems (MAP) containing the immunodominant B cell epitope (NANP)3 and one of three distinct Th epitopes, Th2R, Th3R, and CS.T3, of the C terminal region of the circumsporozoite protein of Plasmodium falciparum, a human malaria parasite. Mice of three different MHC haplotypes (H-2k, H-2d, and H-2a) were immunized with the various MAP constructs. Mice of all three strains produced antibodies, but their anti-sporozoite titers were considerably lower than their anti-peptide titers as detected by ELISA. These antibodies reacted at high titers not only with the repeat polymer (NANP)50, but also with MAP that contained only the respective Th sequence. The antibody binding site within each of the Th sequences was mapped, using truncated peptides, in an inhibition assay. A primary antibody response, induced by a single i.v. inoculation of sporozoites, was greatly enhanced by the injection of MAP.
Association of a V beta 2-specific superantigen with a tumorigenic milk-borne mouse mammary tumor virus.
Authors: Hodes R J , Novick M B , Palmer L D , Knepper J E
Published: December 2022

A number of endogenous mouse mammary tumor virus (MMTV) proviruses encode superantigens that have the property of stimulating mature T lymphocytes in a TCR V beta-specific fashion and of mediating V beta-specific clonal deletion in developing T cells. The tumorigenic milk-borne MMTV carried by C3H and GR mice also have superantigen properties in vivo, and it has been proposed that this superantigenic function is critical to the infectivity and/or tumorigenicity of the virus. To test the requirement for superantigen properties in tumorigenic MMTV, a highly tumorigenic strain of MMTV isolated from BALB/c mice (BALB/cV virus) was analyzed for its effect on TCR V beta expression. It was found that exposure of newborn mice to milk-borne virus results in marked deletion of V beta 2-expressing CD4+ peripheral T cells. This deletion is detected in mature TCRhigh thymocytes as well as in peripheral T cells from virus-exposed mice. Deletion is dependent on expression of a permissive MHC type in mice exposed to virus. Subcutaneous injection of adult mice with virus-containing milk induces a substantial increase in V beta 2+ CD4+ cells in draining lymph nodes within 4 days. Thus, tumorigenic BALB/cV is associated with V beta 2-specific superantigen activity capable of mediating both T cell expansion and clonal deletion in vivo. These findings are consistent with a critical role of superantigen-mediated T cell activation in MMTV infection and tumorigenesis.
Role of neurogenic inflammation in antigen-induced vascular extravasation in guinea pig trachea.
Authors: Bertrand C , Geppetti P , Baker J , Yamawaki I , Nadel J A
Published: December 2022

Conflicting results have been reported about the role of sensory nerves in the allergen-induced plasma extravasation in sensitized guinea pigs using capsaicin desensitization. To investigate the role of tachykinins released from sensory nerves in the anaphylactic reaction in guinea pigs in vivo, we used a selective inhibitor of neutral endopeptidase, phosphoramidon, and a selective neurokinin (NK)-1 receptor antagonist, CP-96,345. Male Hartley guinea pigs were sensitized to OVA by two i.p. injections (70 mg) at 1-wk intervals. Two wk later, the animals were anesthetized and OVA was administered for 2 min by aerosol through a tracheal cannula. Plasma extravasation was assessed by the photometric measurement of the extravasated Evans blue after formamide extraction. Administration of aerosolized OVA to sensitized guinea pigs increased dye extravasation in the trachea in a dose-dependent manner, an effect that was demonstrable 5 min after exposure to allergen and that reached a maximum 10 min after exposure. At 5 min after OVA (5%), phosphoramidon (2.5 mg/kg, i.v.) did not increase the amount of dye in the trachea significantly and CP-96,345 (4 mg/kg) did not decrease the extravasated dye. At 10 min after OVA, allergen-induced plasma extravasation was potentiated by phosphoramidon by 56%, and was inhibited by CP-96,345 (4 mg/kg) by 42%. In the presence of phosphoramidon, CP-96,345 reduced the OVA-evoked plasma extravasation at 10 min in a dose-related manner (0.1-4 mg/kg). CP-96,345 (4 mg/kg) did not affect plasma extravasation induced by platelet-activating factor (100 nmol/kg, i.v.). These results suggest that tachykinin release from sensory nerves after allergen challenge to airways in sensitized guinea pigs is not responsible for the early increase in plasma extravasation, but, tachykinin release appears to play an important role in the subsequent extravasation.
Stimulation of human neutrophils with formyl-methionyl-leucyl-phenylalanine induces tyrosine phosphorylation and activation of two distinct mitogen-activated protein-kinases.
Authors: Torres M , Hall F L , O'Neill K
Published: December 2022

Stimulation of human polymorphonuclear neutrophils with the chemotactic peptide FMLP induces an increase in the phosphorylation of several proteins on tyrosine residues. Immunoblotting of whole cell lysates with antiphosphotyrosine antibodies demonstrated this increase to be predominant in two proteins with apparent m.w. of 40,000 and 42,000, respectively. We identify these two proteins as members of the mitogen-activated-protein (MAP) kinase family. These results are based on comigration of the tyrosine phosphorylated proteins with two proteins of same apparent m.w., recognized by a series of antibodies raised against sequences contained within members of this family. This was further confirmed by immunoprecipitation with either an antiphosphotyrosine antibody or a MAP kinase-specific antibody. To assess whether neutrophil stimulation with FMLP increased MAP kinase activity, the cells were stimulated with various doses of FMLP and the phosphotransferase activity in cell lysates was measured against two identified substrates of MAP kinase: myelin basic protein and T-669, the epidermal growth factor receptor Thr 669 synthetic peptide modeled after the major site of phosphorylation of the receptor. A dose- and time-dependent increase in MAP kinase activity was detected in the lysates from FMLP-stimulated PMN, which closely correlated with the dose and time course of tyrosine phosphorylation of the 40- and 42-kDa proteins. Additionally, the MAP kinases in crude lysates were enriched by DEAE-cellulose chromatography and further resolved on a MonoQ column by fast performance liquid chromatography. The respective eluates contained the two proteins, each recognized by anti-MAP kinase antibodies and, after stimulation, each was phosphorylated on tyrosine residues and expressed T-669 phosphotransferase activity. Treatment with genistein, a tyrosine kinase inhibitor, reduced the tyrosine phosphorylation of the 40- and 42-kDa proteins in a dose-dependent manner, decreased the activation of the MAP kinases, and inhibited the production of superoxide anion by FMLP-stimulated neutrophils. Therefore, this study demonstrates that human polymorphonuclear neutrophils contain two distinct members of the MAP kinase family, antigenically related to sea star p44mpk and rat p43erk1 and that both MAP kinase isoforms are implicated in the cascade of protein phosphorylation induced by neutrophil stimulation with FMLP. Furthermore, these data suggest that tyrosine phosphorylation and activation of these two enzymes may play a regulatory role in the signal transduction pathway leading to the respiratory burst induced by FMLP.
Antibodies to Trypanosoma cruzi express idiotypic patterns that can differentiate between patients with asymptomatic or severe Chagas' disease.
Authors: Reis D D , Gazzinelli R T , Gazzinelli G , Colley D G
Published: December 2022

Immunization of rabbits with pools of immunoaffinity-purified anti-Trypanosoma cruzi epimastigote antibodies derived from patients with different clinical forms of Chagas' disease induces antiidiotypic sera that can distinguish between anti-epimastigote antibodies from patients with asymptomatic (indeterminate (IND)) or severe (cardiac (CARD)) Chagas' disease. These idiotypically different anti-EPI antibodies from patients with the different clinical forms do not differ in their anti-epimastigote activities or isotypes. Analysis of immunoaffinity purified antibodies from individual chagasic patients by specific competitive ELISA generally confirms that Id-specific rabbit antisera can differentiate the clinical forms of the source of the antibodies. Based on these data, immunoaffinity-purified antibodies from patients share many Id with those from IND patients, although antibodies from IND patients express much lower levels of the distinctive Id characteristic of CARD patients. Reduction and alkylation of antibodies from IND patients reduces somewhat, but does not abolish, the ability of their Id to be recognized idiotypically, and to effectively inhibit in competitive ELISA. In contrast, reduction and alkylation of antibodies from CARD patients almost completely eliminates the ability of their predominant Id to be either recognized by, or inhibit, the appropriate systems. These data imply that the expression of the major Id that define CARD patients by these serologic anti-Id systems is largely dependent on the tertiary conformation of the Ig molecule. This agrees with our earlier studies on the respective differential abilities of CARD vs IND Id to stimulate anti-Id T cells by direct stimulation vs processing and presentation mechanisms.
Stimulation of protein tyrosine phosphorylation in human B cells after ligation of the beta 1 integrin VLA-4.
Authors: Freedman A S , Rhynhart K , Nojima Y , Svahn J , Eliseo L , Benjamin C D , Morimoto C , Vivier E
Published: December 2022

B lymphocytes express several adhesion molecules that are involved in cell-cell and cell-extracellular matrix interactions. The alpha 4 beta 1 integrin VLA-4, expressed on pre-B and mature/activated B cells, mediates adhesion of these cells to its two ligands, VCAM-1 and fibronectin. Recent evidence suggests that VLA-4 is involved in T lymphocyte activation; however, relatively little is known of the role of VLA-4 in B cell differentiation. To begin to assess the potential involvement of VLA-4 in B cell activation, we have examined the effect of ligation of VLA-4 on protein tyrosine phosphorylation in B cells. We found that cross-linking of VLA-4 by either mAb or natural ligands (i.e., VCAM-1 and the FN-40 cleavage fragment of fibronectin) induced the tyrosine phosphorylation of a 110-kDa protein in a human pre-B cell line (Nalm-6), an EBV-transformed B cell line (SB), and normal tonsillar B cells. These findings suggest that VLA-4 can activate a tyrosine kinase in B cells and B cell lines. These signals may be involved in the subsequent differentiation of pre-B and mature B cells within specific microenvironments where VLA-4 mediated adhesion is operational.
Exogenous antigens internalized through transferrin receptors activate CD4+ T cells.
Authors: McCoy K L , Noone M , Inman J K , Stutzman R
Published: December 2022

The role of endosomes in exogenous Ag processing was investigated by targeting Ag into the endosomal transport pathway via transferrin receptors. The Ag, pigeon cytochrome c and chicken OVA, were coupled to human ferric transferrin by a heteroligation technique. The conjugates were significantly more efficient than native Ag in stimulating Ag-specific CD4+ T cells, when the APC expressed transferrin receptors. The addition of ferric transferrin eliminated the enhanced response. Paraformaldehyde-fixed APC did not present the conjugates, indicating that the conjugates still required processing to activate T cells. An augmented level of T cell activation was not observed when the APC lacked transferrin receptors or when the conjugate contained the apoenzyme form of transferrin, which does not bind the receptor. The conjugate followed an intracellular pathway similar to that for transferrin, remaining in low density vesicles. Degraded conjugate appeared rapidly in culture supernatants, within 5 min, and peaked by 20 min; under these conditions a T cell response to the conjugate was elicited that was consistent with an early processing compartment. Our results suggest that antigenic peptide fragments can be generated in the early endosomes, without delivery of these Ag to the lysosomes. Thus, various Ag may have differential processing requirements, dictated by their molecular nature, that determine the site of Ag processing.
Mapping epitopes of human Fc gamma RII (CDw32) with monoclonal antibodies and recombinant receptors.
Authors: Ierino F L , Hulett M D , McKenzie I F , Hogarth P M
Published: December 2022

Fc gamma RII is a low affinity FcR for IgG with two Ig-like extracellular domains (D1 gamma and D2 gamma), a transmembrane domain, and a cytoplasmic domain. The production, characterization, and epitope analysis of four anti-human Fc gamma RII mAb (8.2, 8.7, 8.26, and 7.30) are detailed, and the mAb are compared with two defined CDw32 mAb, IV.3 and CIKM5. Reactivity of all mAb with Fc gamma RII was demonstrated by (a) specific binding to Fc gamma RII+ L cells (produced after transfection of L cells with human Fc gamma RIIa cDNA, HFc3.0), by using flow cytometry, (b) inhibition of the binding of SRBC sensitized with rabbit antibody (EA) to Fc gamma RII+ L cells, and (c) immunoprecipitation and SDS-PAGE, which detected a 42-kDa protein on K562 and U937 cells and a single 45-kDa protein on Fc gamma RII+ L cells. The mAb were able to detect different forms of Fc gamma RII, by flow cytometry, on Daudi cells (8.7 and 7.30) and U937 cells (8.2, IV.3, and CIKM5); 8.26 stained Daudi cells with intermediate fluorescence and U937 cells with the highest fluorescence, relative to the remaining mAb. Binding to transiently expressed isoforms of Fc gamma RII (a and b1) and four allelic variants of Fc gamma RIIa in COS-7 cells did not distinguish the mAb epitopes. Further mapping of the mAb epitopes was determined by (a) EA inhibition assays, (b) mAb blocking studies, and (c) the binding of the mAb to segments of human Fc gamma RIIa by using genetically engineered chimeric receptors. Chimeric receptors expressing either D1 gamma linked to domain 2 of Fc epsilon RI or domain 1 of Fc epsilon RI linked to D2 gamma were produced by exchanging homologous, but antigenically different, regions of Fc gamma RIIa and the high affinity receptor for IgE. Four clusters of mAb were identified, each mapping to discrete epitopes of Fc gamma RII. Cluster I (mAb 8.2 and CIKM5) defines a combinatorial epitope with determinants in D1 gamma and D2 gamma distant from the IgG Fc binding site, inasmuch as F(ab')2 fragments of 8.2 and CIKM5 do not inhibit the binding of EA to Fc gamma RII. The epitopes of clusters 2 (mAb 8.26), 3 (mAb IV.3), and 4 (mAb 8.7 and 7.30) are located entirely in D2 gamma and all involve the IgG Fc binding region, because F(ab')/F(ab')2 fragments of the mAb inhibit EA binding to Fc gamma RII. Thus, all mAb that inhibit the binding of EA map totally to D2 gamma; it is likely the IgG Fc binding region is also contained in D2 gamma.
Macrophage activation: lipoarabinomannan from avirulent and virulent strains of Mycobacterium tuberculosis differentially induces the early genes c-fos, KC, JE, and tumor necrosis factor-alpha.
Authors: Roach T I , Barton C H , Chatterjee D , Blackwell J M
Published: December 2022

Lipoarabinomannan (LAM) is a major cell-wall associated glycolipid produced by Mycobacterium tuberculosis and Mycobacterium leprae. Previous work demonstrated that LAM from avirulent (H37Ra) and virulent (Erdman) strains of M. tuberculosis differ in structure at their non-reducing termini. In this study the effects of the H37Ra and Erdman LAM on the activation of murine bone marrow-derived macrophages has been investigated. Their abilities to elicit immediate early gene responses at mRNA (c-fos, JE, KC) and protein (TNF-alpha secretion) levels, and nitrite production, was examined. H37Ra LAM, but not Erdman LAM, elicited TNF-alpha secretion at 1000 ng/ml. Neither stimulated production of reactive nitrogen intermediates (RNI). Addition of 25 U/ml IFN-gamma enhanced TNF-alpha secretion in response to H37Ra LAM, reducing the threshold level of LAM required to 10 to 100 ng/ml. In contrast, Erdman LAM at concentrations up to 1000 ng/ml could not induce macrophage TNF-alpha secretion even in the presence of 25 U/ml IFN-gamma. H37Ra LAM also synergized with IFN-gamma to stimulate enhanced production of RNI, whereas IFN-gamma and Erdman LAM did not elicit RNI production. Examination of events before TNF-alpha and RNI production revealed that H37Ra LAM, like LPS, was able to induce increased levels of mRNA expression for c-fos, KC, and JE, with similar kinetics but reduced potency compared with LPS. Erdman LAM in concentrations up to 2500 ng/ml was unable to stimulate c-fos, KC, or JE expression. IFN-gamma at 25 U/ml was itself a potent stimulus of JE expression, and synergized with 1000 ng/ml H37Ra, and to a lesser extent, Erdman LAM for the induction of JE. In contrast, IFN-gamma inhibited H37Ra LAM stimulation of KC expression. The phenomenon of avoiding the stimulation of macrophage immediate early gene expression may be an important determinant of mycobacterial virulence.
Recombinant migration inhibitory factor induces nitric oxide synthase in murine macrophages.
Authors: Cunha F Q , Weiser W Y , David J R , Moss D W , Moncada S , Liew F Y
Published: December 2022

A recombinant form of migration inhibitory factor (MIF) obtained from COS-1 cells transfected with a cDNA library from a human T cell hybridoma is able to activate, in a dose-dependent manner, murine macrophages to express nitric oxide (NO) synthase and to produce high levels of NO in vitro. The time course of the induction of NO synthase is similar to that produced by the IFN-gamma. Enzyme activity peaks at 24 h and is undetectable by 72 h. MIF can synergize with IFN-gamma in the induction of NO synthesis, and the induction of NO synthase by both MIF and IFN-gamma is sensitive to inhibition by dexamethasone. However, unlike IFN-gamma-induced NO generation, MIF is sufficient for the induction of the enzyme, does not synergize with LPS, and is highly sensitive to inhibition by transforming growth factor.
Characterization of the human basophil response to cytokines, growth factors, and histamine releasing factors of the intercrine/chemokine family.
Authors: Kuna P , Reddigari S R , Schall T J , Rucinski D , Sadick M , Kaplan A P
Published: December 2022

We have tested the histamine releasing properties and priming abilities of a wide range of recombinant or purified cytokines and growth factors on the basophils of 20 subjects (10 atopic and 10 nonatopic). We found that monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), RANTES, human macrophage inflammatory protein-1 alpha and human inflammatory protein-1 beta, Connective tissue activating peptide III and Neutrophil Activating Peptide-2 (NAP-2) cause histamine release from basophils and are all members of the intercrine/chemokine family. MCAF/MCP-1 was as potent as anti-IgE or C5a and it is clearly the major contributor to histamine releasing factor activity. RANTES was the second major histamine releasing factor among the positive cytokines. Both MCAF/MCP-1 and RANTES are present in conditioned mononuclear cell media and can be separated using Mono Q anion exchange chromatography. We also demonstrated that RANTES has unusual chromatographic properties in spite of its isoelectric point of > 9.0 because it is largely found in peak-2 of the Mono Q column rather than peak-1 in which intercrines such as MCAF/MCP-1, IL-8, and connective tissue activating peptide III are found. All other cytokines and growth factors tested were negative, with the exception of IL-3, which caused histamine release in a subpopulation of subjects, and also primed basophils for release by anti-IgE. Other basophil primers for anti-IgE-dependent histamine release were IL-5, mast cell growth factor (c-kit ligand), and insulin-like growth factor II. Using specific neutralizing antibodies we have shown that MCAF/MCP-1, RANTES, and IL-3 contribute significantly to the activity found in mononuclear cell culture supernatants. Granulocyte-macrophage-CSF, IP-10, I-309, IL-7, IL-8, IL-9, IL-10, IL-11, IgE-binding factor, TNF-alpha, TGF-beta 1, fibroblast growth factor, epidermal growth factor, and endothelial cell growth factor were negative for direct histamine release and as primers of basophils. Our results indicate that cytokines belonging to the intercrine/chemokine family are major constituents of the activity known as "histamine releasing factor" found in MNC supernatants.
Glucocorticoid therapy alters hormonal and cytokine responses to endotoxin in man.
Authors: Barber A E , Coyle S M , Marano M A , Fischer E , Calvano S E , Fong Y , Moldawer L L , Lowry S F
Published: December 2022

Previous experimental data have demonstrated that steroid pretreatment regulates endotoxin-elicited cytokine production. The timing of such hypercortisolemia also appears to be a major determinant of both the systemic and the cytokine response to infectious stimuli. Our study was undertaken to further study the in vivo influence of glucocorticoid infusion concurrent with and before endotoxin exposure in man. A total of 23 normal human subjects were given endotoxin (LPS) alone or pretreated with hydrocortisone infusion for 6 h immediately before and concomitant to LPS administration; or rendered hypercortisolemic for a 6-h period waiting 6, 12, or 144 h before LPS administration. LPS administration was followed by significant elevations in temperature (1.9 +/- 0.3 degrees C), pulse (29 +/- 6 bpm), and resting energy expenditure (26.2 +/- 0.4 kcal/kg/day) as well as epinephrine (236 +/- 59 pg/ml), cortisol (296 +/- 29), and C-reactive protein (4.2 +/- .03 mg/dl) as compared with base-line values. Levels of TNF and IL-6 that were not detectable before LPS administration, peaked, respectively, at 90 and 120 min after LPS (155 +/- 4 pg/ml, 12 +/- 1 U/ml). Glucocorticoids when given immediately before and concomitant with LPS significantly attenuated the temperature (0.8 +/- 0.01 degrees C) and pulse rate response (10 +/- 3 bpm) seen after LPS alone as well as suppressing peak levels of epinephrine (78 +/- 14 pg/ml) and C-reactive protein (undetected). TNF remained undetectable in this group although the IL-6 response (14 +/- 1 U/ml) was unchanged. With a 6-h interval between hydrocortisone infusion and LPS challenge, changes in temperature, pulse, resting energy expenditure, and hormone levels were similar to those seen after LPS alone whereas TNF remained undetectable and IL-6 levels were similar to subjects receiving LPS alone. Subjects receiving LPS after 12 or 144 h after hydrocortisone infusion displayed hemodynamic and hormonal responses similar to the LPS alone group, yet mounted significantly greater circulating levels of both IL-6 (117 +/- 14 U/ml, 160 +/- 51 U/ml at 12 and 144 h) and TNF (609 +/- 173, 671 +/- 132 pg/ml at 12 and 144 h) to those observed after LPS alone. We conclude that antecedent periods of hypercortisolemia participate in regulation of the hemodynamic, hormonal, and cytokine responses to endotoxin and that a complex temporal relationship between hypercortisolemia and LPS induced cytokine and systemic responses exists.
Requirements for CD28-dependent T cell-mediated cytotoxicity.
Authors: Azuma M , Cayabyab M , Phillips J H , Lanier L L
Published: December 2022

Small, resting human peripheral blood T cells are able to mediate anti-CD3 redirected lysis against murine P815 cells transfected with human B7, a ligand of CD28. We demonstrate that cytotoxicity is mediated by preexisting cytotoxic effectors within the small, resting "memory" T cell population and by the de novo generation of additional CTL within both the "memory" and "virgin" T subsets. This conclusion is based on analysis of the kinetics of the response and the effects of metabolic inhibitors on the generation of CTL function. Memory CD45RO+ T cells demonstrated cytotoxicity within 4 h of coculture with anti-CD3 mAb and B7+ P815 cells and cytolysis was only partially prevented by inhibitors of protein synthesis. By contrast, virgin CD45RO- T cells demonstrated anti-CD3-induced lysis against B7+ P815 targets only after 6 or 8 h of coculture and cytotoxicity was completely prevented by inhibiting protein synthesis. Induction of cytotoxicity was B7 dependent in that parental P815 cells and P815 cells transfected with CD72 and vascular cell adhesion molecule-1, ligands for T cell-associated membrane receptors CD5 and very late activation antigen-4, respectively, did not initiate cytotoxicity. Our studies also revealed cooperation between the CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 pathways in the generation of CTL from small, resting T cells. However, after CTL generation, the CD28-B7 interaction was not required for cytotoxic effector cell function. These observations may have important physiologic implications because this would permit activated CTL to lyse targets in vivo that do not express B7, after the CTL were generated by APC that do express B7 or possibly other costimulatory molecules.
Structure and diversity of rat T cell receptor alpha-chain genes.
Authors: Shirwan H , Ohanjanian M , Burcham G , Makowka L , Cramer D V
Published: December 2022

We have sequenced 22 TCR-alpha clones isolated from two cDNA libraries derived from rat thymocytes and heart allograft-infiltrating lymphocytes. Sixteen different V alpha and 17 J alpha genes were characterized in these clones. The V alpha genes could be divided into nine subfamilies, each containing from one to three members. Nucleotide sequence comparisons to mouse alpha genes revealed the existence of mouse homologues for all the J alpha s and 8 of 9 V alpha subfamilies. One of the rat V alpha subfamilies appeared to have no known mouse counterpart. Southern blot analysis of germ-line DNA with cDNA probes specific for six different subfamilies demonstrated the existence of two to nine V alpha genes per subfamily. A minimum number of 35 V alpha genes was estimated for the nine rat subfamilies characterized. These analyses also detected two different RFLP patterns among five different inbred rat strains, including strain-specific loss of hybridization bands for some V alpha, indicating that deletion events may have occurred in the rat alpha gene locus. The characterization of rat TCR V alpha genes should facilitate a better understanding of T cell role in pathologic conditions such as autoimmune diseases and graft rejection.
In vitro replication dynamics of human culture-derived macrophages in a long term serum-free system.
Authors: Bennett S , Por S B , Cooley M A , Breit S N
Published: December 2022

Human peripheral blood monocytes maintained in a long term serum-free system were found to undergo extensive replication. Newly replicated culture-derived macrophages initially appeared as colonies of small cells on the adherent monolayer. After the appearance of these colonies, large numbers of nonadherent macrophages were observed. Using PKH26, a fluorescent tracking dye, the increase in cell number was attributed to a replicating pool of cells. Half of the monocytes present in the original monolayer were able to undergo at least one cycle of replication and of these, approximately 16% underwent three or more cycles of replication. Macrophages in the nonadherent state contained a larger proportion of cells that had undergone division compared with adherent cells. However, it appeared that only adherent macrophages were capable of replication, suggesting movement between the adherent and nonadherent states. Culture-derived macrophages were also predisposed to multinucleated giant cell formation; and in the nonadherent state, their capacity to form these cells increased. At the end of the study period, approximately 25% of the cells maintained in a nonadherent state had two or more nuclei, and 3% had 10 or more nuclei. By comparison, the adherent cells, over the same period, had 10% of cells with two or more nuclei and none had 10 or more nuclei. These multinucleated cells were found to arise through cell fusion.
Transforming growth factor-beta potentiates vitamin D3-induced terminal monocytic differentiation of human leukemic cell lines.
Authors: Testa U , Masciulli R , Tritarelli E , Pustorino R , Mariani G , Martucci R , Barberi T , Camagna A , Valtieri M , Peschle C
Published: December 2022

We have investigated the effects of 1,25-dihydroxyvitamin D3 (D3) and/or transforming growth factor (TGF)-beta on one monocytic (U-937) and two human promyelocytic (HL-60 and AML-193) leukemic cell lines. D3 addition induces a partial monocytic maturation of the cell lines, whereas TGF-beta treatment is largely ineffective. Combined treatment with TGF-beta and D3 causes terminal monocytic maturation, as evaluated both by assessment of a large spectrum of membrane Ag and by functional assays. Furthermore, sequential addition of the two inducers showed that pretreatment with TGF-beta 1 followed by incubation with D3, but not vice versa, induces monocytic maturation as effectively as simultaneous treatment with both agents. In liquid culture the proliferative activity of these cell lines is slightly decreased by D3 and virtually unaffected by TGF-beta, whereas combined treatment with D3 and TGF-beta induces a markedly potentiated inhibitory effect. Furthermore, TGF-beta/D3 treatment (but not D3 alone) elicits the expression of membrane CD14, FcRI, FcRII, CD11a, CD11b, CD11c, ICAM-1, and PECAM-1 Ag at a level comparable to that observed on normal human monocytes. It is noteworthy that several of these Ag play an important role in monocyte physiology (e.g., CD14 Ag mediates the binding of bacterial LPS to monocytes). Treatment with both TGF-beta and D3 (but not D3 alone) induces superoxide anions and H2O2 production similar to that of circulating monocytes. In semisolid culture, D3 and TGF-beta alone cause, respectively, a marked and slight loss of cloning efficiency of the cell lines, whereas their combined addition synergistically results in a complete loss of the cloning capacity. These findings suggest a physiologic role for TGF-beta in monocyte maturation. Furthermore, they may pave the way to the design of clinical protocols combining D3 and TGF-beta in the differentiation therapy of acute promyelocytic/myelomonocytic leukemia.
HIV-gp 160-induced T cell-dependent B cell differentiation. Role of T cell-B cell activation molecule and IL-6.
Authors: Chirmule N , Kalyanaraman V S , Lederman S , Oyaizu N , Yagura H , Yellin M J , Chess L , Pahwa S
Published: December 2022

The HIV envelope glycoprotein gp160 has been previously demonstrated to induce differentiation of normal B lymphocytes into Ig-secreting cells; the response is T cell-dependent, and T cells pretreated with gp160 can support B cell differentiation. This study investigates the cell surface molecules and cytokines that play a role in the gp160-induced T-B cell interaction. Utilizing CD4+CD45RO+ cloned T cells as the source of helper cells, we observed that physical contact with B cells is essential for the gp160-induced B cell response; no IgG-secretion occurred if T cells were separated from the B cells by culturing them in Transwell chambers. The expression of T cell-B cell activation molecule, a novel surface molecule associated with T cell activation, was moderately increased by gp160, and antibody to T cell-B cell activation molecule abrogated the gp160-mediated Th cell function. Cell surface molecules LFA-1, ICAM-1, HLA-DR, CD28, and B7 were also involved in the T-B cell interaction since mAb to any of these molecules inhibited the gp160-induced B cell differentiation response. gp160 also induced IL-6R and CD23 molecule expression on B cells when added to cultures of T plus B cells; there was CD23 expression only in cells that formed conjugates with T cells. Paraforamaldehyde-fixed, gp160-pretreated T cells failed to elicit IgG responses in B cells, but did induce CD23 and IL-6R up-regulation on B cells. Addition of exogenous IL-6, but not IL-2 or IL-4, restored the IgG secretion. These findings indicate that the T cell dependence for gp160-induced B cell differentiation responses involves two steps: one requires contact-dependent interaction of several cell surface molecules, and the second requires IL-6 secretion.
Expression of CSF-1, c-fms, and MCP-1 in the central nervous system of rats with experimental allergic encephalomyelitis.
Authors: Hulkower K , Brosnan C F , Aquino D A , Cammer W , Kulshrestha S , Guida M P , Rapoport D A , Berman J W
Published: December 2022

We have examined the expression of factors associated with the growth, differentiation, and chemotaxis of cells of the monocyte/macrophage series in the central nervous system of Lewis rats sensitized to develop experimental allergic encephalomyelitis. CSF-1 mRNA increased significantly over that found in control animals (sensitized with OVA in CFA or CFA alone). The elevation in the levels of this growth factor commenced immediately before the onset of early clinical signs and peaked immediately before maximal clinical incidence of disease. Expression of CSF-1 message declined to base-line values with resolution of the disease process. CSF-1 protein was also detected in the central nervous system at the height of clinical disease. Expression of the receptor for CSF-1, the proto-oncogene c-fms, also paralleled the early disease process. Elevated levels of c-fms mRNA were detected immediately before the onset and peaked at the height of clinical signs of disease. In contrast to CSF-1 levels, elevated c-fms message expression persisted after resolution of the acute phase of experimental allergic encephalomyelitis. Levels of macrophage chemotactic factor-1 message were also elevated immediately before the onset of clinical signs, peaked with the height of clinical disease, and declined with resolution of the disease. Unlike CSF-1 or c-fms, no endogenous macrophage chemotactic factor-1 message was detected in control animals. Macrophage chemotactic factor-1 protein was demonstrated by Western blot in the central nervous system at the height of clinical disease. The results support the conclusion that expression of factors that specifically target cells of the monocyte/macrophage series are an important component of the disease process in experimental allergic encephalomyelitis.
A role for calcineurin in degranulation of murine cytotoxic T lymphocytes.
Authors: Dutz J P , Fruman D A , Burakoff S J , Bierer B E
Published: December 2022

The immunosuppressive drugs cyclosporin A (CsA) and FK506 bind to distinct families of intracellular proteins, cyclophilins, and FK506 binding proteins (FKBP) respectively, termed immunophilins. Immuno-suppressant-immunophilin complexes bind to and inhibit the activity of calcineurin, a calcium-dependent serine/threonine phosphatase. CsA is known to inhibit degranulation in CTL as assessed by N benzyloxylcarbonyl-L-lysine thiobenzyl ester-esterase release assays. We have investigated whether calcineurin phosphatase activity is involved in this degranulation. Both CsA and FK506 are shown to inhibit N benzyloxylcarbonyl-L-lysine thiobenzyl esteresterase release in murine CTL clones induced either by cognate target or by PMA and the calcium ionophore A23187. Inhibition is concentration dependent and is observed at drug concentrations that specifically inhibit cellular calcineurin. The FK506-binding immunophilin FKBP12, as well as calcineurin, are shown to be present in these cells by immunoblotting analysis. Rapamycin, a macrolide antibiotic thought to compete with FK506 for binding to common FKBP receptor sites, antagonizes the effects of FK506 on both degranulation and calcineurin activity. Neither the degranulation nor the effect of the immunosuppressants is affected by the protein synthesis inhibitor cycloheximide. These observations suggest a role for calcineurin in CTL degranulation. Thus, in addition to its previously described role in lymphokine gene activation, calcineurin also appears to be involved in T cell activation processes which do not require protein synthesis.
Signal transduction pathways involved in T cell receptor-induced regulation of CD2 avidity for CD58.
Authors: Hahn W C , Burakoff S J , Bierer B E
Published: December 2022

Through physiologic interactions with its ligands CD58 (lymphocyte function-associated Ag-3, LFA-3) and CD59, the T cell glycoprotein CD2 plays a role in T cell signaling and promotes lymphocyte adhesion. We have recently demonstrated that the interaction of CD2 with CD58 is dynamic: TCR stimulation or treatment with the phorbol ester PMA rapidly up-regulates CD2 ligand avidity, and this regulation requires the carboxyl-terminal asparagine residue of the CD2 cytoplasmic domain. Here we have analyzed the regulation of CD2 avidity for CD58, as assessed by the binding of CD2+ cells to purified CD58 and by the formation of rosettes between CD2+ cells and SRBC. In murine T cell hybridomas transfected with human CD2, we show that, unlike CD2-mediated IL-2 production, cell surface expression of the TCR-CD3 structure is not required for up-regulation of CD2 ligand avidity. TCR-initiated up-regulation of CD2 avidity requires the activity of both protein tyrosine kinases and protein kinase C. Agents which elevate intracellular levels of cAMP also up-regulate CD2 ligand avidity and act either distal to or independently of protein kinase C and protein tyrosine kinases. Cell lines expressing single amino acid substitutions of the carboxyl-terminal asparagine of CD2 are incapable of avidity regulation by TCR signaling, PMA treatment, or elevation of intracellular cAMP levels, demonstrating that each of these stimuli utilizes a common structural element for regulating CD2 avidity. The response to both cAMP and phorbol ester treatment distinguishes the regulation of CD2 avidity from that of a second major adhesion pathway, LFA-1 (CD11a/CD18)/ICAM-1 (CD54) and from that of the TCR coreceptor CD8. These observations identify the signaling events involved in the regulation of CD2 avidity and help to define the signal transduction processes that participate in "inside-out" signaling.
Elevated proliferating cell nuclear antigen levels in immature thymocytes. Dissociation from cell cycle progression.
Authors: Turka L A , Gratiot-Deans J , Keim D , Bandukwala R , Green J , Strahler J , Hanash S M
Published: December 2022

Entry into and progression through the cell cycle is associated with a tightly regulated program of gene expression in mature T cells. One such gene product, proliferating cell nuclear Ag (PCNA), is the auxiliary protein of DNA polymerase delta, and is induced during late G1 and early S phase after stimulation of resting (G0) cells. Blockade of PCNA production has been found to inhibit cell division suggesting that PCNA plays an important role in cell proliferation. The extent to which PCNA and other proliferation-related gene products are similarly regulated in thymocytes has been largely undetermined. Here, we report that immature double positive (CD4+CD8+) thymocytes express high levels of PCNA protein and mRNA relative to mature single positive (CD4+CD8- or CD4-CD8+) thymocytes or peripheral blood T cells. Elevation of PCNA expression among double positive thymocytes is not the result of increased numbers of cycling cells in this subpopulation, being specifically observed in double positive thymocytes with normal diploid DNA content and resting (G0) levels of RNA. Unlike mature thymocytes and T cells, mitogenic stimulation did not induce an increase in PCNA expression in double positive thymocytes. These data indicate that immature thymocytes express high levels of PCNA in the absence of cell cycle progression, thus providing evidence for differential regulation of proliferation related pathways during lymphoid development. Altered expression patterns of these genes in immature vs mature thymocytes may contribute to the process of thymic selection.