bluuebunny/crossref_metadata_2025 huggingface dataset:
Antigen entry into early endosomes is insufficient for MHC class II processing.
Authors: Niebling W L , Pierce S K
Published: December 2022

Helper T-cell recognition of Ag requires that the Ag be processed and presented by class II-expressing Ag-presenting cells. Processing involves the introduction of Ag into acidic compartments where proteolysis occurs producing peptides that bind to the class II molecules. Although Ag can enter the processing pathway through fluid phase pinocytosis, Ag processing can be made over 1000-fold more efficient by binding the Ag to a variety of Ag-presenting cell surface structures. The increased efficiency in processing is presumably the result of the ability of such structures to deliver the bound Ag to compartments involved in processing. Here we report that Ag bound to the transferrin receptor (TfR), which cycles predominantly through early endosomal compartments, does not enter the processing pathway. We found that cytochrome c(c)covalently coupled to monovalent iron-saturated transferrin (Tf), (c-Tf), is not processed or presented significantly better than unconjugated c, indicating that the majority of cycling TfR does not enter compartments where processing proceeds. The conjugation of Tf to c does not affect its binding to the TfR, as the binding is both saturable and compatible with unmodified Tf. Moreover, c-Tf and unmodified Tf cycle equivalently with a t1/2 of internalization of 3 to 5 min and are released outside the cell with little detectable degradation. Significantly, we found that c-Tf is efficiently processed and presented when the TfR is cross-linked, altering its normal cycling. Indeed, c covalently coupled to polymerized Tf is presented at 1/100th the concentration of c alone. Cross-linking of c-Tf bound to the TfR using c-specific antibodies also results in efficient processing and presentation. Thus, the endosomal compartments through which Tf normally cycles are not sites of processing, whereas compartments into which cross-linked Tf is diverted allow efficient processing and presentation of Ag.
Comparative in vitro analysis of proliferation, Ig secretion, and Ig class switching by murine marginal zone and follicular B cells.
Authors: Snapper C M , Yamada H , Smoot D , Sneed R , Lees A , Mond J J
Published: December 2022

We have previously demonstrated that activation of murine B cells by dextran-conjugated anti-IgD antibodies may serve as a polyclonal, in vitro model system for studying immune responses to T cell-independent type 2 (TI-2) Ag, as exemplified by the bacterial polysaccharides. Because in vivo Ig responses to TI-2 Ag are mediated primarily by B cells resident in the splenic marginal zone, we wished to determine whether this reflected an intrinsic difference in the responsiveness of marginal zone B cells (MZB) compared with follicular B cells (FB) to this class of Ag. In this report we demonstrate that highly purified MZB, isolated by electronic cell sorting, exhibit a lower proliferative response in vitro in response to unconjugated anti-Ig antibody as well as to dextran- or Sepharose-conjugated anti-IgM or anti-IgD antibodies, whereas they proliferate equal to or better than FB when stimulated by other B cell mitogens including LPS, Salmonella typhimurium mitogen, or anti-CD3-activated CD4+ Th2 cell clone. Despite the different proliferative responses of MZB and FB induced by anti-Ig, Ag receptor cross-linkage stimulates comparable increases in intracellular free calcium concentrations in both of these B cell populations. Furthermore, MZB secrete Ig and undergo Ig isotype switching to a comparable degree, relative to FB, in response to both T cell-dependent and T cell-independent stimuli. This suggests that the compartmentalization of TI-2 responses to the splenic marginal zone rather than the follicular zone reflects something other than the intrinsic responsiveness of the B cells from these two sites.
The gamma subunit of the B cell antigen-receptor complex is a C-terminally truncated product of the B29 gene.
Authors: Friedrich R J , Campbell K S , Cambier J C
Published: December 2022

The predominant Ag-receptor complex of B cells consists of mIgM or mIgD noncovalently associated with glycosylated heterodimers of Ig-alpha and Ig-beta or Ig-alpha and Ig-gamma. Upon B cell stimulation the associated proteins are phosphorylated, giving rise to pp32/33 (alpha), pp37 (beta), and pp34 (previously designated gamma). Ig-alpha and Ig-beta contain extended cytoplasmic structure (61 and 48 amino acids, respectively) and associate with cytoplasmic effectors indicating that they are directly involved in signal transduction. Here we report analysis of the structural relationship of mIgM- and mIgD-associated Ig-beta and Ig-gamma chains from mice. N-terminal sequence, immunoblotting, and physicochemical analyses show that both Ig-beta and Ig-gamma are products of the B cell-specific B29 gene and demonstrate that the 37-kDa Ig-beta protein is the full length predicted product of the B29 gene. The Ig-associated protein that migrates in the 34-kDa range is actually two distinct species. The minor species is a phosphorylatable and underglycosylated form of full length Ig-beta, and the major species is a C-terminally truncated form of B29, which we now designate Ig-gamma. This conclusion is based on the observations that Ig-gamma is composed of a core protein which is 3 to 4 kDa smaller than deglycosylated Ig-beta, it is not phosphorylated, unlike Ig-beta, and it does not react with an antiserum raised against a peptide of the seven C-terminal amino acids of B29. Based on these findings we estimate that Ig-gamma is truncated by about 30 to 36 amino acid residues and hypothesize that the most 3' B29 exon, which encodes the 32 C-terminal residues, may not be expressed in Ig-gamma. All of the documented B29 products are found in association with both mIgM and mIgD. Interestingly, Ig-gamma is found in intermediate and low density splenic B cells, but is not detectable in resting B cells. This raises the possibility that it may confer some distinct signaling function on the Ag receptors of these cells.
Characterization of the alpha subunit of the IFN-alpha receptor. Evidence of N- and O-linked glycosylation and association with other surface proteins.
Authors: Platanias L C , Pfeffer L M , Cruciani R , Colamonici O R
Published: December 2022

We studied the association of the alpha subunit of the (IFN-alpha-receptor) to other receptor components in the human H-929 and U-266 myeloma cell lines. Immunoprecipitation performed with the IFNaR3 mAb showed that two proteins with molecular masses of 205 and 145 kDa are co-precipitated with the alpha subunit. These complexes may not bind IFN-alpha as shown by studies using the heterobifunctional cross-linking reagent Denny-Jaffe and by partial cleavage of the homobifunctional cross-linker dithio succinimidyl propionate. These studies also provided evidence that at least two subunits with molecular mass of 130 kDa (alpha subunit) and 110 kDa (including 20 kDa corresponding to IFN-alpha) contribute to the formation of the IFN-alpha-receptor complex. To further characterize the alpha subunit of the IFN-alpha-receptor, immunoprecipitates using the mAb IFNaR3 were sequentially treated with N-glycanase, neuraminidase and O-glycanase. These studies showed that the alpha subunit is heavily glycosylated and has a protein precursor with a molecular mass of 68 kDa. Binding studies provided evidence for high and low affinity binding sites for IFN-alpha 2. Affinity cross-linking experiments under low and high affinity conditions suggest that the high affinity binding site of the IFN-alpha-receptor is formed by a complex containing the alpha subunit, whereas the 110-kDa subunit may bind IFN-alpha 2 under low affinity conditions.
Effects of cytokines on multiplication of Toxoplasma gondii in microglial cells.
Authors: Chao C C , Hu S , Gekker G , Novick W J , Remington J S , Peterson P K
Published: December 2022

Microglia may play a role in host defense against Toxoplasma gondii in the central nervous system. In our study, the antitoxoplasma activity of murine microglia was investigated. T. gondii multiplied readily in cultured microglial cells, and activation of these cells with IFN-gamma plus LPS suppressed (p < 0.01) intracellular growth of this parasite. This antitoxoplasma defense of activated microglia was significantly blocked by neutralizing antibodies against TNF-alpha and transforming growth factor-beta, suggesting that these cytokines play a role in the inhibitory process. Consistent with this hypothesis, activation of microglia with IFN-gamma plus TNF-alpha or transforming growth factor-beta inhibited, in a dose-dependent manner, T. gondii multiplication. Treatment with pentoxifylline or dexamethasone, drugs that suppress production of TNF-alpha, prevented (p < 0.01) the microglia from exerting an anti-T. gondii effect. These findings suggest that the host defense function of microglia against T. gondii is dependent primarily on the activating properties of IFN-gamma and TNF-alpha, and that agents that interfere with cytokine-mediated activation may promote the pathogenesis of this infection.
Functional antagonism between vitamin D3 and retinoic acid in the regulation of CD14 and CD23 expression during monocytic differentiation of U-937 cells.
Authors: Oberg F , Botling J , Nilsson K
Published: December 2022

1,25 alpha-Dihydroxicholecalciferol (VitD3) and retinoic acid (RA) are important regulators of the proliferation and differentiation of several cell types. This paper describes how the expression of the monocyte-macrophage Ag, CD14, and the low affinity Fc receptor for IgE, CD23, were inversely regulated during VitD3- and RA-induced monocytic differentiation of human U-937 monoblasts. PMA induced the expression of both CD14 and CD23 mRNA and protein. Exposure to VitD3 rapidly induced the de novo expression of CD14 mRNA and protein. The addition of cycloheximide completely blocked the VitD3 induction of CD14 mRNA expression, indicating that the induction was dependent on ongoing protein synthesis. While inducing CD14 expression, VitD3 concomitantly suppressed the basal, PMA-, and RA-inducible CD23 expression in a dose-dependent manner. In contrast, U-937 cells induced by RA strongly increased their expression of CD23 mRNA and protein, whereas they completely lacked detectable CD14 cell surface or mRNA expression. Furthermore, the VitD3- and the PMA-induced CD14 expression was inhibited as a temporal consequence of the RA-induced differentiation. The results suggest that there exists a functional antagonism between VitD3 and RA that may have important implications for the regulation of certain immune and inflammatory responses through their inverse effects on CD14 and CD23 gene expression.
Enhancement of antigen-induced bronchoconstriction after intravascular complement activation with cobra venom factor. Reversal by granulocyte depletion.
Authors: Regal J F , Fraser D G , Anderson D E , Solem L E
Published: December 2022

Our previous studies have demonstrated that activation of the C system with cobra venom factor (CVF) in a passively sensitized guinea pig results in an enhanced bronchoconstrictor response to Ag but not to other constrictor agents. Thus, our immediate goal was to determine the mechanism of the CVF-induced enhancement of the Ag-induced bronchoconstriction. Isolated airways from sensitized guinea pigs that had been treated with CVF responded normally to Ag. Because such a system lacks the normal circulating cell populations, we hypothesized that the CVF-induced enhancement of the Ag-induced bronchoconstriction was dependent on the presence of circulating white blood cells or platelets. Guinea pigs were depleted of circulating granulocytes, platelets, or both using specific antisera and the effect on the CVF-induced enhancement of the Ag-induced bronchoconstriction was determined. We found that CVF treatment did not result in an enhanced Ag-induced bronchoconstriction in guinea pigs depleted of either granulocytes or both granulocytes and platelets. However, the enhanced response was still apparent in guinea pigs depleted of just platelets. We investigated the effects of CVF itself and found that CVF treatment did not alter the number, or percentages, of different cell populations in the bronchoalveolar lavage, did not alter the protein or albumin content of the lavage fluid or the wet:dry ratio of the lung. In addition, CVF did not cause an increase in airway microvascular permeability as assessed by leakage of Evans blue. However, CVF did substantially increase granulocytes sequestered in the lung as measured by increased myeloperoxidase content. Thus, C activation by CVF results in an increase in neutrophils in the lung and an enhanced Ag-induced bronchoconstriction dependent on the presence of circulating granulocytes. These studies suggest that C activation and/or retention of granulocytes in the lung may be important in determining the severity of an Ag-induced bronchoconstriction.
Effect of docosahexaenoic acid on the generation of platelet-activating factor by eosinophilic leukemia cells, Eol-1.
Authors: Shikano M , Masuzawa Y , Yazawa K
Published: December 2022

Human eosinophilic leukemia (Eol-1) cells were examined for their ability to generate platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) (PAF) and the effect of supplementation of docosa-hexaenoic acid (C22:6n-3) (DHA) on the PAF synthesis was explored in relation to the fatty acid composition of phospholipids and the liberation of arachidonic acid (C20:4n-6 AA). Although undifferentiated cells did not produce PAF, the exposure of IFN-gamma differentiated Eol-1 to generate PAF in response to the Ca-ionophore. In addition, the IFN-gamma-treated cells acquired the ability to release free fatty acids, approximately 55% of which was found to be AA. When DHA was supplemented into the culture of Eol-1 for 24 h, PAF production decreased by 40 to 50% at concentrations of 3 to 10 microM. On the other hand, supplementation of 10 microM eicosapentaenoic acid (C20:5n-3) did not significantly decrease PAF production. With the supplementation of 10 microM DHA, DHA levels in phospholipid subclasses, including alkylacylglycerophosphocholine, were greatly increased with concurrent decreases in other unsaturated fatty acids. In these cells, the liberation of AA in response to an ionophore was decreased by 55%. Even when DHA was enriched in phospholipids, DHA release in response to ionophore stimulation was almost negligible, indicating that the DHA moiety of phospholipids is not susceptible to the action of phospholipase A2. Furthermore, DHA supplementation appeared to attenuate phospholipase A2 reaction by some unknown mechanism because the decrease in AA release was much more than that for the AA level in phospholipids. Acetyl-CoA:1-alkylGPC acetyltransferase activity of stimulated cell lysate was also reduced by DHA supplementation but the reduction was much less when compared with that of PAF synthesis or AA release. These results implicated that enrichment of DHA attenuates enzymic reactions for PAF synthesis, mainly the initial reaction catalyzed by AA-specific phospholipase, and thereby reduces PAF synthesis in Eol-1.
Effects of exogenous eicosapentaenoic acid on generation of leukotriene C4 and leukotriene C5 by calcium ionophore-activated human eosinophils in vitro.
Authors: Thien F C , Hallsworth M P , Soh C , Lee T H
Published: December 2022

Exogenous eicosapentaenoic acid (EPA) has been compared with exogenous arachidonic acid (AA) for its ability to modulate the oxidative metabolism of membrane-derived arachidonic acid by the 5-lipoxygenase pathway in ionophore-activated human eosinophils, and for its suitability as a parallel substrate in this pathway. Products were quantitated by specific RIA and tetraene and pentaene leukotrienes (LT) were separated by reverse-phase HPLC. Eosinophils were preincubated with control buffer, exogenous EPA or AA and stimulated optimally with 10 microM calcium ionophore (A23187) for 15 min. Mean generation of LTC4 in the absence of fatty acid was 6.0 = 1.1 ng/10(6) eosinophils (mean = SEM, n = 5). In the presence of EPA, the amount of LTC4 generated rose to peak at 16.5 +/- 1.9 ng/10(6) eosinophils at 10 micrograms/ml EPA and then fell to 8.3 +/- 3.1 ng/10(6) cells at 40 micrograms/ml EPA. The EPA derivative, LTC5 was first detectable at 5 micrograms/ml EPA with 4.8 +/- 1.2 ng/10(6) cells and gradually rose with increasing dose of EPA to be maximal at 40 micrograms/ml with 12.7 +/- 2.2 ng/10(6) cells. Identity of the LTC5 was confirmed by an identical retention time to synthetic LTC5 standard, immunoreactivity to a specific antibody against LTC4 and LTC5 and a typical UV absorbance spectrum. When eosinophils were preincubated with AA and similarly stimulated, LTC4 generation gradually increased from a baseline of 6.7 +/- 0.7 ng/10(6) cells in the absence of fatty acid to reach a maximum of 12.9 +/- 0.8 ng/10(6) cells at 40 micrograms/ml of AA. Total LTC generation was nearly twofold more with cells incubated with EPA than with cells incubated with AA (p < 0.05). Thus, EPA does not suppress LTC generation from eosinophils but stimulates it at lower doses and is a substrate for LTC5 generation.
Engraftment and activity of anti-CD3-activated human peripheral blood lymphocytes transferred into mice with severe combined immune deficiency.
Authors: Murphy W J , Conlon K C , Sayers T J , Wiltrout R H , Back T C , Ortaldo J R , Longo D L
Published: December 2022

Human PBL (huPBL) were activated with anti-CD3 mAb in vitro and then were transferred into mice with severe combined immune deficiency (SCID) to determine the effect of activation on engraftment and to determine if the engrafted human cells could provide antitumor effects in mice. Some mice were also treated with human rIL-2 after huPBL transfer. Mice were analyzed 6 to 8 wk after cell transfer and the number of human cells in the peripheral lymphoid organs was determined. Mice receiving anti-CD3-activated huPBL demonstrated a significant increase in the incidence of human T cell engraftment in the periphery as assessed by flow cytometry. Human cells were also detected in the murine thymus after anti-CD3 stimulation indicating that human T cells can migrate to the murine thymus provided that they are activated before the transfer. The transfer of anti-CD3-activated huPBL also resulted in an xenogeneic graft-vs-host reaction manifested primarily by proliferation of murine splenic hemopoietic cells. When SCID mice received the human colon carcinoma HT29, the concurrent transfer of anti-CD3-activated human cells resulted in a significant increase in survival. However, the human cells displayed low cytolytic activity toward human tumor targets when they were recovered from the lymphoid organs of the SCID recipients. Supernatants from the anti-CD3-activated cells were able to inhibit the growth of HT29 in vitro, partly because of the presence of IFN-gamma, suggesting that the human T cells are producing cytokines in vivo that have antitumor effects. Thus, the use of anti-CD3-activated huPBL in SCID mice may be of value for optimizing human cell engraftment in the human/mouse lymphoid chimeras and may be used to evaluate potential anti-neoplastic therapies that employ human effector cells.
Human B cell proliferation and Ig secretion induced by recombinant CD40 ligand are modulated by soluble cytokines.
Authors: Armitage R J , Macduff B M , Spriggs M K , Fanslow W C
Published: December 2022

Recombinant human CD40 ligand (hCD40L) was expressed on the surface of CV1/EBNA cells and examined for its ability to induce proliferation and Ig secretion from human B cells in the presence or absence of soluble cytokines. hCD40L was directly mitogenic in a dose-dependent fashion for purified tonsil B cells with maximal proliferation occurring at days 5 to 7. Proliferation induced by CD40L was significantly enhanced in the presence of IL-2, IL-4, or IL-10 and strongly suppressed by transforming growth factor-beta. Although IL-5, TNF-alpha, and IFN-gamma had no stimulatory effect in the presence of hCD40L alone, if IL-4 was also present in cultures, these cytokines enhanced the proliferative response above that seen with IL-4 alone. Interestingly, in the absence of IL-4, IFN gamma had an inhibitory effect on hCD40L-induced proliferation. Although CD40L alone did not enhance Ig secretion, addition of IL-2 or IL-10 to the cultures significantly elevated the levels of IgM, IgG1, and IgA that were observed. Addition of IL-4 to the cultures did not enhance secretion of these isotypes but had a weak inhibitory effect. However, CD40L-mediated induction of IgG4 and IgE was dependent on the presence of IL-4. Of the cytokines examined, only IL-10 enhanced IgE secretion under these conditions. Although transforming growth factor-beta only partially inhibited secretion of IgM, IgG1, and IgA, it was strongly suppressive for IgG4 and IgE production. Our data demonstrate that proliferation and Ig secretion induced in the presence of CD40L can be modulated in a positive and negative fashion by soluble cytokines. IL-2 and IL-10 specifically enhance IgM, IgG1, and IgA production although IL-4, despite costimulating B cell proliferation, does not augment secretion of these isotypes but provided an essential cosignal with CD40L for the production of IgG4 and IgE.
Photoaffinity labeling of the T cell receptor on living cytotoxic T lymphocytes.
Authors: Romero P , Maryanski J L , Luescher I F
Published: December 2022

Using a direct binding assay based on photoaffinity labeling, we have studied the interaction of an antigenic peptide with MHC class I molecules and the TCR on living cells. Two photoreactive derivatives of the H-2Kd (Kd) restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) were used. The first derivative contained an N-terminal photoreactive iodo, 4-azido salicyloyl (IASA) group and biotin on the TCR contact residue Lys259 [IASA-YIPSAEK(biotin)I]. As previously described, this derivative selectively bound to and labeled the Kd molecule. The second photoreactive compound, the isomeric biotin-YIPSAEK(IASA)I, also efficiently bound to the Kd molecule, but failed to label this protein. A CTL clone derived from a mouse immunized with this derivative recognized this conjugate but not the parental P. berghei circumsporozoite peptide or the [IASA-YIPSAEK-(biotin)I] derivative in an Kd-restricted manner. Incubation of the cloned CTL cells with biotin-YIPSAEK(IASA)I, but not its isomer, followed by UV irradiation resulted in photoaffinity labeling of the TCR-alpha chain that was dependent on the conjugate binding to the Kd molecule. The TCR labeling was partially inhibited by anti-LFA 1 and anti-ICAM1 mAb, but was increased by addition of beta 2m or soluble KdQ10. The exquisite labeling selectivity of the two photoprobes opens a new, direct approach to the molecular analysis of antigen presentation and recognition by living CTL.
lpr T cells are necessary for autoantibody production in lpr mice.
Authors: Sobel E S , Cohen P L , Eisenberg R A
Published: December 2022

Mice homozygous for the gene Ipr develop a spectrum of autoantibodies closely resembling that of human SLE. Previous work has shown that the lpr defect must be expressed in the T cells that hyperproliferate and in the B cells that produce autoantibodies. Although autoantibody production in lpr mice requires T cells, it is not known whether these need to be lpr T cells. To ask whether normal (+/+) T cells can help lpr B cells produce autoantibodies, we have constructed chimeras containing mixtures of lpr-derived and normal-derived lymphoid cells, and have selectively eliminated the lpr-derived T cells by in vivo treatment with monoclonal anti-Thy-1 of the appropriate allotype. A mixture of T cell-depleted bone marrow from congenic strains of normal and lpr mice differentially marked by Ig H chain allotype and Thy-1 alleles was transferred into lethally irradiated lpr mice. The mice received weekly injections of either anti-Thy-1.2 to deplete specifically lpr T cells or an isotype-matched irrelevant control mAb. Absence of lpr-derived T cells in the experimental group was documented by immunofluorescence. In mice treated with control antibody, autoantibodies of Ipr origin were present in high titers, as determined by allotype-specific ELISA. In contrast, mice depleted of lpr-derived T cells had greatly reduced titers of antichromatin and rheumatoid factor. These mice also had increased levels of serum total IgM and IgG2a of +/+ origin. Parallel experiments were performed using a combination of two lpr marrow sources, also differentially marked by Ig H chain allotype and Thy-1 expression. Mice depleted of Thy-1.2-bearing T cells produced autoantibodies of both allotypes due to the presence of Thy-1.1-bearing T cells of Ipr origin. These data indicate that autoantibody production in lpr mice requires expression of the lpr gene in those T cells that provide help.
Increased IL-6 production and IL-6-mediated Ig secretion in murine host-vs-graft disease.
Authors: Vandenabeele P , Abramowicz D , Berus D , Van der Heyden J , Grooten J , Donckier V , Hooghe-Peters E L , Goldman M , Fiers W
Published: December 2022

BALB/c mice neonatally injected with semiallogenic (A/J x BALB/c)F1 splenocytes develop a host-vs-graft (HVG) reaction between host T cells and donor B cells, resulting in hypergammaglobulinemia, splenomegaly, and increased serum levels of various autoantibodies. This syndrome is associated with a polyclonal activation of the donor-derived B cells. High serum levels of IL-6 were found in 4-wk-old mice undergoing HVG disease (mean +/- SEM, 132 +/- 93 as compared with 12 +/- 2 in control mice, p < 0.05). Also supernatants of spleen cell cultures from HVG mice contained increased levels of IL-6. In situ hybridization and cell depletion experiments demonstrated that host macrophages were responsible for this pathologic IL-6 secretion. The spontaneous in vitro production of autoreactive antibodies by donor B cells from HVG mice was further enhanced by adding human rIL-6, whereas addition of human rIL-1 beta, human rIL-2, murine rIL-4, murine rIL-5, or combinations of these cytokines had no effect. Finally, addition of blocking anti-IL-6 and anti-IL-6 receptor mAb markedly reduced hyper IgG1 production in cultures of spleen cells from HVG mice. These data suggest that an increased production of IL-6 by persistently stimulated host macrophages is involved in the activation of donor B cells leading to HVG disease.
Enhanced IL-1 beta and tumor necrosis factor-alpha release and messenger RNA expression in macrophages from idiopathic pulmonary fibrosis or after asbestos exposure.
Authors: Zhang Y , Lee T C , Guillemin B , Yu M C , Rom W N
Published: December 2022

Idiopathic pulmonary fibrosis (IPF) and asbestosis are fibrotic interstitial lung diseases characterized by alveolar wall fibrosis with accumulation of extracellular matrix, interstitial remodeling, and increased numbers of activated alveolar macrophages. Animal models and in vitro studies have shown that macrophage cytokines, namely IL-1 beta and TNF-alpha, play significant roles in the development of fibrosis. We found significant increases for TNF-alpha release in both diseases (p < 0.01) and a significant increase for IL-1 beta release in asbestosis compared to normal controls (p < 0.01). Also, the mRNA expression of these cytokines was increased in alveolar macrophages from patients with IPF or asbestosis compared with normals. The level of TNF-alpha release in macrophage supernatants correlated with the number of neutrophils per milliliter bronchoalveolar lavage fluid returned. Chrysotile, crocidolite, amosite asbestos, and silica stimulated IL-1 beta and TNF-alpha release and up-regulated their respective mRNA in macrophages or monocytes. To evaluate the role of IL-1 beta and TNF-alpha in the accumulation of extracellular matrix, we studied collagen types I and III and fibronectin gene expression in human diploid lung fibroblasts after short term (2 h) serum-free exposure to recombinant cytokines. Both cytokines up-regulated these genes 1.5- to 3.6-fold. These cytokines have the potential to influence the remodeling and fibrosis observed in the lower respiratory tract in IPF and asbestosis.
Surface transport and internalization of the membrane IgM H chain in the absence of the Mb-1 and B29 proteins.
Authors: Cherayil B J , MacDonald K , Waneck G L , Pillai S
Published: December 2022

The micron IgH chain is one component of the membrane IgM complex, the endocytic and signal transducing receptor for Ag on the surface of B lymphocytes. It is transported to the cell surface in association with three other B cell-specific proteins, an L chain and the products of the mb-1 and B29 genes. The roles played by these various proteins in mediating the functions of the complex are unclear. To analyze micron function in the absence of other lymphoid-specific proteins, we first attempted to express micron on the surface of nonlymphoid cells. Deletion of the CH1 domain was sufficient to allow surface expression of this protein in transfected COS cells as well as in mouse L cells. To determine whether this extracellularly truncated micron was capable of endocytosis, we used an assay to detect the internalization of anti-mu antibody bound to the surface of transfected cells expressing the protein. Under both cross-linking and non cross-linking conditions, the CH1-deleted micron protein was internalized in endocytic vesicles. We conclude from these observations that a) the CH1 domain of micron contains a retention signal, the elimination of which allows surface transport of this protein, and b) micron by itself is capable of at least one of the functions of the membrane IgM complex.
Organization of the murine Cd22 locus. Mapping to chromosome 7 and characterization of two alleles.
Authors: Law C L , Torres R M , Sundberg H A , Parkhouse R M , Brannan C I , Copeland N G , Jenkins N A , Clark E A
Published: December 2022

Murine CD22 (mCD22) is a B cell-associated adhesion protein with seven extracellular Ig-like domains that has 62% amino acid identity to its human homologue. Southern analysis on genomic DNA isolated from tissues and cell lines from several mouse strains using mCD22 cDNA demonstrated that the Cd22 locus encoding mCD22 is a single copy gene of < or = 30 kb. Digestion of genomic DNA preparations with four restriction endonucleases revealed the presence of restriction fragment length polymorphisms (RFLP) in BALB/c, C57BL/6, and C3H strains vs DBA/2J, NZB, and NZC strains, suggesting the presence of two or more Cd22 alleles. Using a mCD22 cDNA clone derived from the BALB/c strain, we isolated genomic clones from a DBA/2J genomic library that contained all the exons necessary to encode the full length mCD22 cDNA. Fifteen exons, including exon 3 that encodes the translation start codon, were identified. Each extracellular Ig-like domain of mCD22 is encoded by a single exon. A comparison between the nucleotide sequences of the BALB/c CD22 cDNA and the exons of the DBA/2J CD22 genomic clones revealed an 18-nucleotide deletion in exon 4 (encoding the most distal Ig-like domain 1 of mCD22) of the DBA/2J genomic sequence in addition to a number of substitutions, insertions, and deletions in other exons. These nucleotide differences were also present in a cDNA clone isolated from total RNA of LPS-activated DBA/2J splenocytes by reverse transcription-polymerase chain reaction. The Cd22 locus was mapped to the proximal region of chromosome 7, a region sytenic to human chromosome 19q, close to the previously reported loci, Lyb-8 and Mag (a homologue of Cd22). An antibody (CY34) against the Lyb-8.2 B cell marker reacted with a BHK transfectant expressing the full length mCD22 cDNA, thus demonstrating that Lyb-8 and Cd22 loci are identical. Furthermore, a rat anti-mCD22 mAb, NIM-R6, bound to sIgM+ DBA/2J B cells, confirming the expression of a CD22 protein by the Cd22a/Lyb-8a allele.
Altered protein tyrosine phosphorylation in anergic Th1 cells.
Authors: Cho E A , Riley M P , Sillman A L , Quill H
Published: December 2022

Clonal anergy as a mechanism for tolerance in T lymphocytes can be studied using an in vitro culture system, in which cloned CD4+ Th1-type murine T cells are rendered anergic for IL-2 transcription. The long-lasting molecular changes in anergic cells that prevent the response to Ag restimulation are not yet known. To determine whether the TCR might be uncoupled from normal intracellular signaling pathways, we investigated the response of anergic T cells to Ag, to anti-CD3 antibodies, or to anti-CD4 antibody restimulation in terms of early protein tyrosine phosphorylation events. Tyrosine phosphorylation of the CD3 zeta chain was apparently normal. In contrast, defects in the induction of tyrosine phosphorylation of three major T cell protein substrates were demonstrated. Altered phosphorylation correlated with functional nonresponsiveness for proliferation and reversal of anergy by growth in exogenous IL-2 resulted in reversal of the phosphorylation defects as well as in recovery of Ag responsiveness. These results suggest that specific defects in tyrosine phosphorylation pathways required for the induction of IL-2 synthesis may help to explain nonresponsiveness to Ag in tolerant T cells.
Traces of bacterial lipopolysaccharide suppress IFN-gamma-induced nitric oxide synthase gene expression in primary mouse macrophages.
Authors: Bogdan C , Vodovotz Y , Paik J , Xie Q W , Nathan C
Published: December 2022

A nitric oxide synthase (iNOS) inducible by cytokines and microbial products contributes to the cytotoxic and antimicrobial activity of mouse macrophages. Bacterial LPS interacts synergistically with IFN-gamma to induce iNOS when both stimuli are added together. In contrast, we show here that pre-exposure of peritoneal macrophages to low concentrations of LPS suppresses the induction of iNOS when IFN-gamma is added subsequently. Suppression required pretreatment with LPS for at least 8 h and was optimal with LPS concentrations in the range of 50 to 200 pg/ml. Suppression was exerted by smooth and rough forms of LPS from Escherichia coli and by lipid A from Salmonella minnesota, but not by a biologically inactive lipid A from Rhodobacter sphaeorides. Suppression of nitrite accumulation and iNOS enzyme activity by prior exposure of macrophages to LPS could be explained by their markedly decreased content of iNOS protein, as revealed by immunoblot with monospecific anti-iNOS IgG. Messenger RNA for iNOS was affected in a biphasic manner by pretreatment with LPS. Five hours after addition of IFN-gamma, iNOS mRNA levels were unaltered or even enhanced by pretreatment with LPS, but by 24 to 48 h, expression of iNOS mRNA was inhibited strongly enough to account for the reduced levels of iNOS protein. Suppression by LPS did not appear to be mediated by endogenous prostaglandins, transforming growth factor-beta, or TNF-alpha, even though pretreatment with exogenous TNF-alpha was also suppressive. These findings suggest that preactivation of pathways normally contributing to synergistic induction of iNOS may deplete macrophages of factors needed for its expression. Regulation of iNOS in vivo may depend on the relative tempo with which the inflammatory and immune responses evolve.
Characterization of influenza virus-induced leukocyte adherence to human umbilical vein endothelial cell monolayers.
Authors: Colden-Stanfield M , Ratcliffe D , Cramer E B , Gallin E K
Published: December 2022

The adherence of undifferentiated 51Cr-labeled HL-60 (0.5 x 10(6) HL-60 cells/well) cells was monitored on influenza virus-infected HUVEC monolayers. Whereas only 3.0 +/- 1.6% (n = 36) of HL-60 cells adhered to uninfected HUVEC, adherence was increased to 41.7 +/- 2.2% (n = 6), 79.7 +/- 1.2% (n = 6), 83.9 +/- 0.7% (n = 6), and 84.4 +/- 0.5% (n = 6) on HUVEC infected for 7 h at a MOI of 1, 3, 6, and 9, respectively. In comparison, HL-60 cell adherence increased to 35% when HUVEC monolayers were stimulated with LPS (0.2-20 micrograms) for 4 h. Increased adherence to infected HUVEC occurred at 5 h postinfection, peaked at 7 h, and was maintained at 24 h postinfection. Active virus and metabolically active endothelial cells were required to mediate the virus-induced adherence. E-selectin and ICAM-1 Ag were upregulated 78.3- and 4.1-fold, respectively, by LPS (0.02-20 micrograms, 4 h) whereas virus infection (7 h) only increased these proteins 2.6- and 1.4-fold with a MOI > or = 16. Although the time courses of expression for both adhesion molecules after LPS treatment of virus infection were similar, the difference in the magnitude of upregulation suggests that virus-induced adherence is not a result of upregulation of E-selectin and ICAM-1. In contrast, surface expression of HA is involved in HL-60 cell adherence to virus-infected HUVEC because (1) the time course and magnitude of HA AG expression paralleled the time course and magnitude of HL-60 cell adherence after virus infection of HUVEC; (2) HL-60 cell aggregates were absent on infected HUVEC monolayers in the presence of anti-HA; (3) HL-60 cells competed with RBC for infected endothelial cells stained for cellular HA Ag and (4) anti-HA abolished the virus-induced adherence. Furthermore, it appears that HL-60 cells are binding directly to HA because HL-60 cell adherence to a cell-free surface was increased if virus was prebound and neuraminidase treatment of HL-60 cells prevented the HL-60 cell adherence to influenza virus-infected endothelial monolayers.
Human IgE receptor alpha-chain IgG chimera blocks passive cutaneous anaphylaxis reaction in vivo.
Authors: Haak-Frendscho M , Ridgway J , Shields R , Robbins K , Gorman C , Jardieu P
Published: December 2022

Cross-linking of the high affinity IgE receptor (Fc epsilon RI) expressed on mast cells and basophils is essential for triggering anaphylaxis in vivo. Previously, other investigators have tried to produce competitive inhibitors using IgE peptide analogues and anti-IgE antibodies with limited success. To create a novel specific inhibitor of IgE that can block binding of IgE to Fc epsilon RI without the capacity to stimulate degranulation, we made an Fc epsilon RI-IgG immunoadhesin. The Fc epsilon RI-IgG was constructed by gene fusion of the extracellular portion of the human alpha-chain of Fc epsilon RI, which contains the high affinity binding site for IgE, with a truncated human IgG1 H chain C region. The Fc epsilon RI-IgG recognizes both human and murine IgE. Coincubation of Fc epsilon RI-IgG with murine IgE prevented sensitization of RBL-2H3 cells and the subsequent histamine release in response to anti-IgE. Similarly, when the Fc epsilon RI-IgG was preincubated with equimolar concentrations of either hyperimmune mouse sera or purified mouse IgE, it completely blocked the passive cutaneous anaphylaxis reaction in rats. Furthermore, i.v. administration of Fc epsilon RI-IgG following intracutaneous injection of serum from DNP-immunized mice was able to block the passive cutaneous anaphylaxis reaction in a time-dependent fashion. These results demonstrate that Fc epsilon RI-IgG is a potent inhibitor of IgE binding to intracutaneous mast cells in vivo and may prove clinically useful for the treatment of IgE-mediated disease.
Stem cell factor-induced signal transduction in rat mast cells. Activation of phospholipase D but not phosphoinositide-specific phospholipase C in c-kit receptor stimulation.
Authors: Koike T , Hirai K , Morita Y , Nozawa Y
Published: December 2022

Stem cell factor (SCF), a hematopoietic growth factor for primitive hematopoietic stem cells, is also known as mast cell growth factor. SCF induced serotonin release from rat peritoneal mast cells, connective tissue-type mast cells. The treatment of rat peritoneal mast cells with SCF failed to produce inositol 1,4,5-trisphosphate, indicating the absence of involvement of phosphoinositide-specific phospholipase C pathway. 1,2-Diacylglycerol (1,2-DG) and phosphatidic acid, however, were increased after stimulation by SCF. Phosphatidylethanol formation catalyzed by phospholipase D (PLD) was observed, together with the release of choline but not phosphocholine. Propranolol, an inhibitor of phosphatidate phosphohydrolase, blocked the production of 1,2-DG. These results indicate that the phosphatidylcholine-specific PLD pathway is the main pathway for the production of 1,2-DG in SCF-stimulated rat peritoneal mast cells. Furthermore, treatment of cells with protein tyrosine kinase inhibitor, genistein, inhibited 1,2-DG formation and serotonin release dose-dependently. Taken together, SCF induces the activation of PLD through the protein tyrosine kinase pathway without activation of phosphoinositide-specific phospholipase C.
CD4+ T cell clones obtained from Plasmodium falciparum sporozoite-immunized volunteers recognize polymorphic sequences of the circumsporozoite protein.
Authors: Moreno A , Clavijo P , Edelman R , Davis J , Sztein M , Sinigaglia F , Nardin E
Published: December 2022

CD4+ T cell clones were derived from three volunteers who were protected against malaria after immunization with Plasmodium falciparum sporozoites. T cells specific for an epitope, Pf Th/Tc, contained in amino acids 326 to 345 of the circumsporozoite (CS) protein of P. falciparum (NF54) were derived from all three volunteers. DR1-, -4-, -7-, and -9-restricted T cell clones were found to recognize overlapping, but distinct, epitopes within a 20-mer peptide representing the amino acid 326 to 345 sequence. The Pf Th/Tc epitope contains part of the highly conserved region II as well as part of a polymorphic domain of the P. falciparum CS protein. All of the overlapping epitopes within peptide 326-345 contained at least three amino acids of the amino terminus of the conserved region II, in addition to a variable number of amino acids in the polymorphic region. The DR4-, -7-, and -9-restricted but not the DR1-restricted T cell clones recognized variant peptides representing this polymorphic region of the CS protein of P. falciparum isolates from Africa, Asia, and South America.
Regulation of CD4 and CD8 expression on mouse T cells. Active removal from the cell surface by two mechanisms.
Authors: Anderson S J , Coleclough C
Published: December 2022

Phorbol esters are known to induce a loss of CD4 from the surface of mouse and human T cells, presumably through activation of protein kinase C. Here we describe additional, calcium-dependent processes that remove CD4 and CD8 from the surface of T cells and thymocytes, and that differ from the protein kinase C-mediated effect in that they require the expression of new gene products. Whereas PMA causes the disappearance of almost all CD4 from the surface of mouse CD4+8+ thymocytes, it induces only a partial (approximately 60%) and transient (10 to 12 h) loss of CD4 from the surface of mouse peripheral T cells, with no effect on CD8 expression. When T cells are exposed to a combination of PMA and the calcium ionophore, ionomycin (Cal), surface CD4 virtually disappears for a period of at least 24 h, and CD8 expression is also diminished. This additional, calcium-dependent effect, on both CD4 and CD8 expression, is abrogated by either cycloheximide or actinomycin D, and so depends on new RNA and protein synthesis. There appear, therefore, to be two distinct mechanisms for the removal of CD4 and CD8 from mouse peripheral T cells: one induced by PMA alone, the second by the combination of PMA and Cal. The second mechanism, but not the first, depends on the expression of new gene products. In contrast to mouse peripheral T cells, mouse thymocytes and human peripheral blood T cells respond to PMA alone with virtually a complete loss of surface CD4, and partial loss of CD8 expression. The addition of Cal has no synergistic effect on CD4 expression in either of these populations, but augments the loss of CD8 in mouse thymocytes.
Evidence that peritoneal exudate cells cultured with eye-derived fluids are the proximate antigen-presenting cells in immune deviation of the ocular type.
Authors: Hara Y , Okamoto S , Rouse B , Streilein J W
Published: December 2022

Immune privilege in the anterior chamber of the eye results in part from a selective deficit in delayed hypersensitivity that is elicited by antigenic materials placed in this unique tissue site. This distinctive systemic immune response to intraocular Ag (termed anterior chamber-associated immune deviation, ACAID) is fashioned by indigenous, intraocular bone marrow-derived cells that capture Ag within the anterior chamber and carry an Ag-specific ACAID-inducing signal via the blood directly to the spleen. An identical form of immune deviation can be evoked by the i.v. injection of peritoneal exudate cells (PEC) pulsed in vitro with soluble Ag in the presence of transforming growth factor-beta (TGF-beta). To determine whether eye-derived cells present Ag directly to responding splenic T cells or merely serve as vehicles to deliver Ag to the spleen, we have conducted MHC restriction experiments with PEC donors and recipients selected to differ at loci dictating MHC and/or minor histocompatibility Ag. When PEC were pulsed in vitro with soluble Ag (BSA) in the presence of culture fluid containing TGF-beta and injected into recipients with which they shared either class I or class II MHC molecules, BSA-specific ACAID was induced. By contrast, PEC pulsed with BSA and TGF-beta-containing culture fluid failed to induce ACAID when the cells were injected into recipients who were completely histoincompatible or were identical with the injected cells only at non-MHC loci. Using MHC class I-deficient transgenic mice, it was determined that intraocular injection of BSA failed to induce ACAID, and that class I-deficient PEC were incapable of inducing either BSA-specific ACAID or splenic regulatory T cells that suppress BSA-specific delayed hypersensitivity. We conclude that cells that carry an ACAID-inducing signal to the spleen are: 1) restricted in their ability to induce ACAID by MHC-encoded molecules, 2) these cells are the proximate APC ACAID, and 3) class I MHC molecules play a central role in presenting exogenous protein Ag to splenic T cells in ACAID.
Early mobilization of Ca2+ is not required for glucocorticoid-induced apoptosis in thymocytes.
Authors: Iseki R , Kudo Y , Iwata M
Published: December 2022

Contradictory results have been reported on the question of the role of Ca2+ in glucocorticoid-induced apoptosis in thymocytes. To resolve this problem, we investigated the effect of dexamethasone, a synthetic glucocorticoid, on intracellular Ca2+ concentration ([Ca2+]i), by microscopic fluorometry that enables us to monitor real-time [Ca2+]i of cells loaded with fura-2, a fluorescent Ca2+ indicator, on a single cell basis. The results indicated that dexamethasone does not induce an increase in [Ca2+]i above control level both in murine and rat thymocytes at least for 1 h after the start of the culture. We also investigated whether the depletion of extracellular Ca2+ with EGTA or buffering intracellular Ca2+ with quin-2/AM inhibited glucocorticoid-induced apoptosis as reported on rat thymocytes. Dexamethasone-induced apoptosis in both murine and rat thymocytes, however, was not inhibited by EGTA. High concentrations (25 microM and over) of quin-2/AM inhibited DNA fragmentation, but failed to inhibit cytolysis. Calmodulin inhibitors, trifluoperazine and calmidazolium, also inhibited DNA fragmentation as reported, although they markedly enhanced cytolysis. Therefore, glucocorticoid-induced death is not inhibited by quin-2/AM or calmodulin inhibitors. Furthermore, we have previously found that a proper combination of the calcium ionophore, ionomycin, and the protein kinase activator, PMA, inhibits corticosterone-induced apoptosis. These results suggest that an early increase in [Ca2+]i is neither induced by glucocorticoids nor responsible for glucocorticoid-induced apoptosis in thymocytes.