bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of polymorphonuclear neutrophil CD16 and CD11b/CD18 expression by matrix proteins during hypoxia is VLA-5, VLA-6 dependent.
Authors: Simms H , D'Amico R
Published: December 2022

We investigated the effects of hypoxia on matrix protein regulation of polymorphonuclear neutrophil (PMN) CD16 and CD11b/CD18 expression. Adherence of PMN to fibronectin increased CD16 expression during hypoxia over levels seen during normoxia, while adherence of PMN to either fibronectin or laminin increased CD11b/CD18 expression during hypoxia over levels seen during normoxia. Kinetics assays demonstrated a t1/2 approximately 60 min and 30 min of hypoxia for maximal up-regulation of CD16 and CD11b/CD18, respectively. Incubation of fluid-phase PMN with anti-VLA-5 (alpha 5/beta 1) and anti-VLA-6 (alpha 6/beta 1) mAbs blocked the effect of fibronectin and laminin on CD16 and CD11b/CD18 expression. Cross-linking of both fluid-phase and adherent PMN VLA-5 and VLA-6 receptors resulted in a progressive increase in CD16 and CD11b/CD18 expression during hypoxia, but not normoxia. Increases in CD16 and CD11b/CD18 expression resulted in increased E anti-Fc gamma RIII and EC3bi rosetting. Inhibition of GPLC activity and IP3 production with U73122 and cyclopiazonic acid blocked the ability of fibronectin to increase CD16 expression and E anti-Fc gamma RIII rosetting. Inhibition of protein tyrosine kinase with genistein and herbimycin A blocked the ability of laminin to increase CD11b/CD18 expression and EC3bi rosetting. Depletion of intracellular Ca2+ abrogated the effects of fibronectin and laminin on CD16 and CD11b/CD18 expression, while restoration of intracellular Ca2+ restored the ability of fibronectin and laminin to increase CD16 and CD11b/CD18 expression. These results demonstrate that during hypoxia integrin signaling via alpha 5/beta 1 and alpha 6/beta 1 results in the increased expression of CD16 and CD11b/CD18. This increase in receptor expression results in biologically active receptors and is dependent upon intracellular Ca2+, GPLC, and protein tyrosine kinase activity.
Neuroantigen-specific Th2 cells are inefficient suppressors of experimental autoimmune encephalomyelitis induced by effector Th1 cells.
Authors: Khoruts A , Miller S D , Jenkins M K
Published: December 2022

We have identified a method of polarizing polyclonal populations of activated T helper cells toward either the Th1 or Th2 phenotype using different short-term in vitro culture conditions. Since the Ag used was an encephalitogenic peptide for SJL/J mice, the pathogenic potential of these cell populations was tested in an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE). Th1 cells reproducibly caused severe EAE, whereas highly polarized Th2 cells did not. Furthermore, highly polarized Th2 cells did not suppress EAE caused by Th1 cells. The anti-inflammatory cytokines made by Th2 cells may simply fail to inhibit tissue destruction mediated by differentiated Th1 cells at the effector phase of the disease. It is also possible that highly polarized Th2 cells may be inefficient at crossing the blood-brain barrier, which may limit their suppressive potential. In contrast, incompletely skewed T cell populations that produced high levels of both Th1 and Th2 cytokines were consistently only weakly encephalitogenic. Therefore, disease inhibition by Th2 cytokines may best be accomplished by intervention at earlier points preceding development of differentiated Th1 cells.
Elevated Th1- or Th0-like cytokine mRNA in peripheral circulation of patients with rheumatoid arthritis. Modulation by treatment with anti-ICAM-1 correlates with clinical benefit.
Authors: Schulze-Koops H , Lipsky P E , Kavanaugh A F , Davis L S
Published: December 2022

The current studies examined whether cytokine patterns indicative of an imbalance in Th1 and Th2 cells could be identified in PBMC of patients with active rheumatoid arthritis (RA). To investigate this possibility, a reproducible PCR technique to assess cytokine mRNA levels in PBMC was employed that minimized in vitro manipulation of the cells. Seven of 14 RA patients had increased mRNA levels for IL-2, 5/14 for IFN-gamma, 3/14 for IL-4, and 4/14 for the IL-2R alpha-chain, compared with normal donors. Whereas 4 patients had elevated mRNA for IL-2 and IFN-gamma, indicative of an increase in activated Th1 or Th0 cells, 1 of 14 patients expressed low levels of IL-2 and IFN-gamma and high levels of IL-4 mRNA. Seven RA patients were treated with a mAb to ICAM-1 (CD54). To determine whether changes in cytokine mRNA levels might be associated with and/or account for the anti-inflammatory effect of anti-ICAM-1 mAb therapy, changes in cytokine mRNA levels were assessed and correlated with clinical improvement. Anti-ICAM-1 mAb administration was followed by a prompt and transient increase of IFN-gamma mRNA. Elevation of IFN-gamma mRNA expression throughout the treatment period reflected a temporary increase in the number of circulating CD3+CD4+ T cells, suggestive of altered circulatory patterns of activated Th1-like cells and was related to clinical efficacy. The results indicate that elevated cytokine mRNA levels characteristic for Th1 cells can be detected in the PBMC in active RA and, furthermore, that anti-ICAM-1 mAb may be beneficial in RA by altering the recruitment of activated Th1-like cells into the synovium. This assumption further strengthens the hypothesis of a significant contribution of Th1-like cells to the pathogenesis of RA.
Localization of an Fc-binding reactivity to the constant region of human IgG4. Implications for the pathogenesis of rheumatoid arthritis.
Authors: Zack D J , Stempniak M , Wong A L , Weisbart R H
Published: December 2022

The majority of plasma cells in rheumatoid arthritis (RA) synovium produce rheumatoid factors (RF). IgG RF predominate in the immune complexes found in RA synovial fluid and have been implicated in the pathogenesis of RA. IgG4 RF are a major component of IgG RF produced in serum and synovium of RA patients, even though this subclass comprises only 4% of the serum IgG. We produced an IgG4 mAb, hRF-1, with RF reactivity from the synovial tissue of a patient with RA. mAb hRF-1 had binding specificity for mammalian IgG similar to Staphylococcus aureus protein A, which is characteristic of RF from patients with RA. To determine the molecular basis of this particular RF reactivity, the heavy and light chain genes of mAb hRF-1 were amplified by PCR, cloned, and ligated into the pSG5 plasmid for expression in COS-7 cells. Chain recombination experiments localized the Fc-binding reactivity to the hRF-1 heavy chain. Using a series of chimeric Ab sequences, the Fc-binding reactivity was mapped to the constant region of IgG4 rather than the variable region involved in classic RF reactivity. Multiple domains, including Hinge, CH2, and CH3 of the IgG4 constant region were required for Fc binding. Our studies demonstrate an example of RF-like Fc-binding reactivity that is conferred by the gamma-4 constant region rather than the classic Ag binding site and suggest that increased production of IgG4 may contribute to the pathogenesis of RA.
Defective TCR-mediated signaling in anergic T cells.
Authors: Migita K , Eguchi K , Kawabe Y , Tsukada T , Ichinose Y , Nagataki S , Ochi A
Published: December 2022

Extrathymic T cell tolerance to Ags can be achieved through clonal deletion by activation-induced programmed cell death as well as by functional unresponsiveness (anergy) of Ag reactive-T cells. Previous studies demonstrated that in vivo administration of staphylococcal enterotoxin B (SEB) induces SEB-specific T cell anergy. To investigate the molecular mechanisms of T cell anergy, we analyzed the TCR-mediated signal transduction in normal and anergic T cells. Anergic T cells exhibited impaired protein tyrosine phosphorylation on TCR-mediated stimulation. The altered tyrosine phosphorylation in anergic T cells may be caused by a defect in tyrosine phosphorylation of TCR zeta-chain and its subsequent association with ZAP-70 tyrosine kinase. Our data demonstrated that TCR zeta-chain phosphorylation and its sequential interaction with ZAP-70 are essential for the initiation of TCR-mediated signal transduction.
Differences in the avidity of TCR interactions with a superantigenic ligand affect negative selection but do not allow positive selection.
Authors: Chervonsky A V , Golovkina T V , Ross S R , Janeway C A
Published: December 2022

The products of the sag genes of the exogenous mouse mammary tumor virus (MMTV) genome and of endogenous Mtv integrants have been demonstrated to affect the T cell repertoire in mice by causing the deletion of T cells expressing receptors encoded by particular V beta gene segments. Since these deletions affect large populations of T cells with receptors of heterogeneous specificity, they serve as an important model for the study of T cell development in normal mice. Using several C3H/HeN-based strains that express different MMTV(C3H) transgenes, we demonstrate here that the stage of development at which T cell deletion occurs is determined by the level of ligand expression. Although at low levels of ligand expression in the thymus some signs of activation were observed in immature thymocytes, we were unable to detect a level of Sag expression that led to net positive selection. Moreover, we detected a level of Sag-transgene expression that did not cause negative selection in the thymus; no signs of positive selection were observed either. Inclusion of the env gene in the construct, earlier shown to markedly potentiate stimulation by Sag in mixed lymphocyte reactions, also markedly increased the ability of Sag to drive negative selection. These data are interpreted as showing that marked quantitative differences in expression of superantigens does not reveal a level at which only positive selection occurs. This, in turn, suggests that positive selection will occur on ligands distinct from those that drive clonal deletion.
Mapping the Ig superantigen-binding site of HIV-1 gp120.
Authors: Goodglick L , Zevit N , Neshat M S , Braun J
Published: December 2022

The envelope glycoprotein, gp120, of HIV-1 has recently been identified as a member of the new family of Ig superantigens (Ig-SAg). This classification is based on the selective binding of gp120 to an unusually high proportion of endogenous, nonimmune Ig, and the selective activation of nonimmune B cells by gp120 in vitro. Many, if not all of the nonimmune Ig that bind to gp120 are members of the VH3 Ig gene family. The aim of this study was to determine the epitope on gp120 that was responsible for its Ig-SAg binding activity. To do this, we utilized a panel of 30 peptides derived from gp160 in a competition-binding assay. For five Igs that were tested, as well as for polyclonal serum IgM, two overlapping peptides (each 20 amino acids in length) were identified that were potent inhibitors of gp120 binding. Similarly, the 10 amino acid overlap region of these two peptides had inhibitory activity. Thus, this decamer sequence represented the optimal Ig-SAg epitope or mimotope. The amino acid residue at position 1 of the decamer, and to a lesser extent at position 10, was critical for peptide binding. In addition to this decamer peptide, other peptides that shared modest sequence homology were also selectively inhibitory for specific Ig samples. These findings provide the first definition of an Ig-SAg ligand at the peptide level and will facilitate further structural and biologic characterization of this new class of pathogenic Ags.
Inhibition of expression of cyclin A in human B cells by an immunosuppressant mizoribine.
Authors: Hirohata S , Yanagida T
Published: December 2022

Mizoribine has been used to prevent rejection of organ allografts in humans and in animal models. Recent clinical trials have demonstrated its efficacy in rheumatoid arthritis and lupus nephritis, in which abnormalities of B cell functions are also involved. We therefore examined the effects of mizoribine on the in vitro function of human B cells. IgM production was induced from highly purified B cells obtained from healthy donors by stimulation with Staphylococcus aureus Cowan I (SA) plus IL-2. Mizoribine suppressed the production of IgM at its pharmacologically attainable concentrations (1 to 3 micrograms/ml) in a dose-dependent manner. Mizoribine had to be present within the first 96 h after the initiation of cultures to exert its suppressive effects on B cell responses. Cell cycle analysis disclosed that mizoribine significantly decreased the numbers of B cells in S + G2 + M phases. Mizoribine did not decrease GTP levels in SA-stimulated B cells, whereas mizoribine led to a decrease in GTP levels in activated T cells, which was reversed by addition of GMP. Consistently, the suppressive effects of mizoribine on the IgM production of SA-stimulated B cells was not reversed by the addition of GMP as much as 40 microM, which overcame the inhibitory effects of mizoribine on the proliferation of anti-CD3-stimulated T cells. Although mizoribine did not suppress the expression of CD25 and cdc2 kinase, mizoribine markedly suppressed the expression of cyclin A in SA-activated B cells. These results indicate that mizoribine directly suppresses the function of human B cells without interfering with the initial phase of activation. Moreover, the data demonstrate that mizoribine interferes with the cell cycle progression of activated B cells by suppressing the expression of cyclin A by a mechanism distinct from guanine ribonucleotide depletion.
Listeriolysin is processed efficiently into an MHC class I-associated epitope in Listeria monocytogenes-infected cells.
Authors: Villanueva M S , Sijts A J , Pamer E G
Published: December 2022

Listeria monocytogenes is an intracellular pathogen that enters the cytoplasm of infected cells by secreting listeriolysin (LLO), a protein that destroys the phagosomal membrane. In infected mice, LLO is a major Ag detected by protective, MHC class I-restricted CTLs. Although the role of LLO in pathogenesis and host immunity is well established, its rate of intracellular synthesis has yet to be determined. Herein we show that cytosolic L. monocytogenes secrete LLO at a relatively low rate of approximately one molecule per bacterium per minute. Under extracellular labeling conditions, the rate of LLO secretion is approximately 50-fold higher. Intracellular LLO synthesis suffices, however, for the accumulation of 600 to 1000 H-2Kd-associated LLO 91-99 epitopes per cell. We calculate that between four and 11 LLO molecules are degraded for each LLO 91-99 epitope bound by H-2Kd. Our findings indicate that the antigenicity of LLO, with respect to MHC class I-restricted CTLs, cannot be attributed to high levels of intracellular secretion. Rather, LLO is a dominant Ag because it is rapidly degraded and very efficiently processed into an MHC class I-associated epitope.
Positive selection of thymocytes involves sustained interactions with the thymic microenvironment.
Authors: Wilkinson R W , Anderson G , Owen J J , Jenkinson E J
Published: December 2022

CD4+8+ cortical thymocytes are critically dependent upon interaction with the thymic epithelium to undergo positive selection and maturation into single-positive CD4+ or CD8+ cells. Here we investigate further the nature of this interaction and provide evidence that positive selection requires sustained, rather than "single hit," interaction with thymic stromal cells. We also show that calcineurin-mediated signaling in thymocytes is required for the initial stages of positive selection, but is not essential throughout the period of thymocyte dependence on stromal cell contact during positive selection. In addition, we show that double-positive thymocytes that have initiated positive selection (CD69+4+8+) and newly generated single-positive (CD69+4+) cells differ markedly in response to the same stimulus through the TCR. The former undergo deletion, whereas the latter proliferate, indicating that a critical change in response to TCR ligation occurs within the narrow developmental window between these two stages.
Nuclear factor-IL6 activates the human IL-4 promoter in T cells.
Authors: Davydov I V , Krammer P H , Li-Weber M
Published: December 2022

Positive regulatory element I (PRE-I) is a strong enhancer element essential for expression of the human IL-4 gene. To identify transcription factors binding to PRE-I, we screened a cDNA expression library from Jurkat T cells and isolated a cDNA encoding nuclear factor (NF)-IL6 (also known as C/EBP beta). NF-IL6 mRNA was found in human Jurkat T cells and in the mouse Th2 clone D10, but not in Th1 clone 29. rNF-IL6 expressed in bacteria was shown to specifically bind to PRE-I. PRE-I forms multiple DNA-protein complexes with nuclear extracts from Jurkat cells. Some of these complexes were demonstrated to contain NF-IL6 by using anti-C/EBP beta Abs. Overexpression of NF-IL6 enhanced expression of the chloramphenicol acetyl transferase reporter gene linked to the PRE-I-thymidine kinase or the human IL-4 promoter more than 10-fold in Jurkat cells. Promoter deletion studies revealed two additional NF-IL6 binding sites located at positions -44 to -36 (C/EBP proximal) and -87 to -79 (C/EBP medial), respectively. Our results demonstrate that NF-IL6 is involved in transcriptional activation of the human IL-4 promoter in T cells.
Molecular characterization of two murine eosinophil beta chemokine receptors.
Authors: Post T W , Bozic C R , Rothenberg M E , Luster A D , Gerard N , Gerard C
Published: December 2022

beta or C-C chemokines including RANTES, MCP-3, MIP-1 alpha, and eotaxin have been implicated in the pathogenesis of eosinophilic inflammation. Two human beta chemokine receptors have been cloned and characterized: the MIP-1 alpha/RANTES receptor or C-C chemokine receptor 1 (CCR-1) and the MCP-1 receptor or C-C chemokine receptor 2 (CCR-2). However, no murine beta chemokine receptors have thus far been reported. Molecular cloning from mouse genomic DNA and cDNA libraries yielded two murine beta chemokine receptors with 79% and 65% sequence identity with human CCR-1, and 50% and 55% with human CCR-2. COS cells transiently transfected with the murine homologue of human CCR-1 bind murine MIP-1 alpha and human RANTES with Kds of 3.4 nM and 4.2 nM and murine MIP-1 beta with an EC50 of 8.9 nM. The other murine beta chemokine receptor, which we have designated murine CCR-3, also binds murine MIP-1 alpha. The mRNAs for both receptors are expressed in eosinophils from IL-5 transgenic mice. The level of murine CCR-3 mRNA in these mouse eosinophils exceeds that of CCR-1 mRNA and approaches actin levels. Murine MIP-1 alpha was found to be a potent chemoattractant for murine eosinophils. Our findings suggest that the murine MIP-1 alpha ligand/receptor system is an important mediator of murine eosinophil trafficking.
Protein tyrosine phosphatase activity associates with the high affinity IgE receptor and dephosphorylates the receptor subunits, but not Lyn or Syk.
Authors: Swieter M , Berenstein E H , Siraganian R P
Published: December 2022

Protein tyrosine phosphorylation is an early event in the high affinity IgE receptor (Fc epsilon RI)-mediated signaling cascade leading to secretion in mast cells. Numerous proteins, including the beta- and gamma-subunits of Fc epsilon RI, become tyrosine phosphorylated after receptor aggregation. Dephosphorylation of these proteins may be important to reverse and limit transmembrane signaling. RBL-2H3 mast cell lysates were found to contain protein tyrosine phosphatase activity that dephosphorylated the tyrosine-phosphorylated beta- and gamma-subunits of Fc epsilon RI. The protein tyrosine phosphatase activity associated with Fc epsilon RI and was equally present with receptors from nonactivated and stimulated cells. Moreover, the phosphatase eluted from the immunoprecipitates and, when added back, dephosphorylated both tyrosine-phosphorylated beta- and gamma-subunits, but not tyrosine-phosphorylated Lyn or Syk. These results strongly suggest that the IgE receptor-associated protein tyrosine phosphatase may regulate the steady state level of tyrosylphosphate of the beta- and gamma-subunits and, therefore, may modulate the interaction of these subunits with other downstream molecules, such as Syk.
Rapid oscillations of actin polymerization/depolymerization in polymorphonuclear leukocytes stimulated by leukotriene B4 and platelet-activating factor.
Authors: Omann G M , Rengan R , Hoffman J F , Linderman J J
Published: December 2022

We previously showed that activation of polymorphonuclear leukocytes by leukotriene B4 (LTB4) and platelet-activating factor produces a rapidly oscillating actin polymerization/depolymerization response. In this study, we show that 1) oscillations are not due to the stimulated cyclic release of autocoids that could bind to cell surface receptors and activate subsequent cycles; 2) oscillations are not related to oscillations of ligand binding; and 3) the particular kinetic pattern is a property of the receptor, not of the binding constants of the ligand. The major conclusion of these studies is that the oscillations are a property of the intrinsic signaling pathways triggered by these chemoattractants. We also questioned whether increased actin nucleation activity was induced by LTB4 and found that, although LTB4 induced a transient actin nucleation response, there was not a direct correlation between oscillations of the actin polymerization/depolymerization and the actin nucleation activity. This suggests that processes other than actin nucleation, such as release of monomeric actin from monomer sequestering proteins and regulation of depolymerization, are likely to be involved.
The effect of pregnancy on polymorphonuclear leukocyte function.
Authors: Crouch S P , Crocker I P , Fletcher J
Published: December 2022

Pregnancy exerts suppressive effects on a number of chronic inflammatory conditions, particularly rheumatoid arthritis. We isolated peripheral blood polymorphonuclear leukocytes (PMN) from pregnant women at 30 to 34 wk (n = 34) and showed significant reductions in respiratory burst activity compared with nonpregnant controls (n = 34), as determined by lucigenin-enhanced chemiluminescence (LUCL). Responses to FMLP were reduced by 54% (p = 0.0046) and to zymosan-activated serum (ZAS) by 69% (p = 0.0043). Following LUCL responses to these agonists in women throughout the course of their pregnancy (n = 7) revealed significantly reduced responses by the second and third trimesters (p < 0.005). Intracellular H2O2 production in PMN at 30 to 34 wk gestation was significantly reduced (p = 0.0454) in response to FMLP, compared with the nonpregnant controls. Investigation of adhesion molecule expression revealed no differences in CD11b or CD18. However, loss of CD62L from the PMN surface in response to FMLP and ZAS was significantly reduced at 30 to 34 wk, as compared with controls (FMLP, p = 0.049; ZAS, p = 0.01; n = 34). There were no significant differences in cell surface formyl peptide receptor expression, although there were statistical differences in LUCL responses to all concentrations of FMLP used (p < 0.05). Incubating PMN with TNF, IL-8, and granulocyte-macrophage CSF increased formyl peptide receptor expression but revealed no differences between the two groups. Priming of pregnancy PMN with the same cytokines gave significantly reduced LUCL when cells were subsequently stimulated with FMLP (p < 0.05; n = 6). Our results show a reduction in PMN NADPH-oxidase activity during pregnancy and may offer a partial explanation for the remission of symptoms observed in rheumatoid arthritis.
Relative perforin- and Fas-mediated lysis in T1 and T2 CD8 effector populations.
Authors: Carter L L , Dutton R W
Published: December 2022

IFN-gamma-secreting T1 and IL-5-secreting T2 subsets of CD8 effectors were generated in vitro using freshly isolated cells from wild-type and perforin knockout mice stimulated with allogeneic Ag-presenting cells. Both T1 and T2 effectors from wild-type mice exhibited perforin-mediated cytolysis. T1, but not T2, populations from perforin knockout mice displayed significant lysis by the Fas-mediated pathway. Th1 cells have recently been shown to be regulated by Fas and we speculate that Fas-mediated mechanisms are involved in the regulation of both Th1 and T1 populations of T cells.
Regulation of NK cell functions by TGF-beta 1.
Authors: Bellone G , Aste-Amezaga M , Trinchieri G , Rodeck U
Published: December 2022

The contribution of exogenous and endogenous TGF-beta 1 to human peripheral blood NK cell proliferation and activity in vitro was investigated. Exogenous bioactive TGF-beta 1 inhibited NK cell DNA synthesis and production of IFN-gamma, TNF-alpha, and granulocyte-macrophage CSF (GM-CSF) by NK cultures consisting of > 98% CD56+ cells. The cytotoxic activity of NK cells was also weakly inhibited by exogenous TGF-beta 1. All TGF-beta 1-induced inhibitory effects occurred in the absence and presence of the NK cell-activating cytokines IFN-alpha, IL-2, and IL-12. Unstimulated NK cell cultures expressed steady state TGF-beta 1 mRNA detected by Northern blot analysis and produced TGF-beta protein (1.6 ng/ml), as determined by ELISA. When NK cell proliferation was induced by IL-2, IL-12, IFN-alpha, or a combination of IL-2 and IL-12, expression of TGF-beta 1 mRNA and protein was moderately and consistently reduced by approximately 20%, as compared with unstimulated control cultures. Unstimulated and rapidly proliferating NK cell cultures secreted primarily latent TGF-beta into their culture medium, as determined by the Mv1Lu bioassay. These results indicate that, in vitro, endogenous NK cell-derived TGF-beta 1 has no negative autocrine effect upon activation of NK cells by various cytokines.
IL-10 induces the tyrosine phosphorylation of tyk2 and Jak1 and the differential assembly of STAT1 alpha and STAT3 complexes in human T cells and monocytes.
Authors: Finbloom D S , Winestock K D
Published: December 2022

IL-10 affects monocytes and T cells by driving the progression of immune responsiveness such that Th2 lymphocyte-mediated effects predominate. In this report, we show that in monocytes and T cells IL-10 stimulates tyrosine phosphorylation of the signal transducers and activators of transcription, STAT1 alpha and STAT3, in a differential manner such that the relative formation of homo- and heterodimers varies between the two cell types. Moreover, monocytes express a novel IL-10-stimulated STAT protein with an M(r) of 70 kDa that is recognized by the anti-STAT3 Ab but is not observed in T cells. IL-10 treatment of both T cells and monocytes results in the ligand-induced tyrosine phosphorylation of tyk2 and Jak1, but not Jak2 or Jak3. Selective modulation of immune responsiveness by IL-10 in cells such as monocytes and T cells may result in part from the differential activation of STAT protein pairs.
Structure of the gene for the human myeloid IgA Fc receptor (CD89).
Authors: de Wit T P , Morton H C , Capel P J , van de Winkel J G
Published: December 2022

A better understanding of IgA's role in immunity requires insight in IgAR complexity. We have now isolated, characterized, and sequenced the gene encoding the prototypic human myeloid IgA FcR (CD89). The gene consists of five exons and spans approximately 12 kilobase pairs. The leader peptide is encoded by two exons, the second of which is 36 bp long and specifies the predicted peptidase cleavage site. A similar, but shorter (21 bp) mini-exon has been found in the FcR for IgG (Fc gamma R) genes, and the FcR for IgE (Fc epsilon RI alpha) gene (human and rodent). The third and fourth exons code for two homologous Ig-like domains. The final exon encodes a short extracellular region, a hydrophobic transmembrane region, and a short cytoplasmic tail. The sequence of the 5'-flanking region was determined, and one major and several minor transcription initiation sites were mapped by primer extension studies. A putative TATA box was located at an appropriate location relative to the start site. Southern blot analyses of genomic DNA confirm the restriction map generated from cloned DNA. These data define the Fc alpha R gene as a distantly related member of the IgR gene family.
Functional dissection of the murine lck distal promoter.
Authors: Wildin R S , Wang H U , Forbush K A , Perlmutter R M
Published: December 2022

The lymphocyte-specific proto-oncogene lck is transcribed from two developmentally regulated, independently functioning promoters. The proximal promoter is used in thymocytes, but not in peripheral T lymphocytes. The distal promoter operates in all stages of T cell development, but predominates in more mature cells. Both promoters lack a TATAA element and they share little sequence similarity with each other. Using transgenic mice to locate in vivo functional cis-acting regions of the murine distal promoter, we defined a region from -1786 to -2913 that is essential for consistent insertion site-independent expression of a heterologous cDNA reporter. The transgene is lymphoid specific and expressed predominantly in T cells. One of four transgenic mice bearing a shortened distal promoter (-886 to +41) expressed the reporter in the expected developmental pattern, suggesting that important regulatory elements that require favorable flanking sequences for expression are present nearer the transcription start site. The DNA sequence from -4032 to +623 contains few consensus binding sites for previously described T lymphocyte-specific trans-acting factors, and their locations do not correlate well with the functional data. However, the locations of tissue-specific modifications of chromatin structure in the promoter region, manifest as sites of DNase hypersensitivity, correlated with these two functional regions in normal mice. The identification of lck distal promoter regulatory regions provides a useful control element for deliberate expression of transgenes in mature T lymphocytes. In addition, these regulatory regions should assist in defining T cell-specific trans-acting factors.
Plasminogen activator inhibitor type 2 prevents programmed cell death of human macrophages infected with Mycobacterium avium, serovar 4.
Authors: Gan H , Newman G W , Remold H G
Published: December 2022

Although Mycobacterium avium is usually nonpathogenic in healthy individuals, in vitro infection of macrophages (M phi) from the majority of healthy donors induces death of the cells 2 wk after infection; this effect is in contrast to noninfected M phi, which survive for months in culture. We demonstrate here that treatment of normal M phi with indomethacin further shortens the life of these cells to 48 h after infection with M. avium. Indomethacin treatment of the M phi also prevents M. avium-dependent accumulation of mRNA-encoding plasminogen activator inhibitor type-2 (PAI-2), an inhibitor of urokinase-type plasminogen activator. Occurrence of nuclear condensation and DNA fragmentation in M phi pretreated with indomethacin and infected with M. avium indicates that the early death of these cells is caused by apoptosis. In contrast, priming of M phi with GM-CSF significantly prolongs their survival after M. avium infection and enhances M. avium-induced accumulation of PAI-2 mRNA. Most importantly, addition of PAI-2 is sufficient to prevent apoptosis of M phi infected with M. avium in the presence of indomethacin. Finally, M phi not treated with indomethacin also die of apoptosis 7 to 10 days after M. avium infection and can be rescued by PAI-2. These studies indicate that production of PAI-2 by normal M phi as a consequence of M. avium infection inhibits programmed cell death, a mechanism that might serve to prevent the spread of the infection.
Integration of mycobacterial lipoarabinomannans into glycosylphosphatidylinositol-rich domains of lymphomonocytic cell plasma membranes.
Authors: Ilangumaran S , Arni S , Poincelet M , Theler J M , Brennan P J , Nasir-ud-Din  , Hoessli D C
Published: December 2022

Lipoarabinomannans (LAMs) are major Ags of the mycobacterial cell envelope where they apparently insert through a glycosylphosphatidylinositol (GPI) anchoring structure. LAMs induce host macrophages to secrete TNF-alpha, IL-1, and IL-6 and inhibit T cell proliferative responses. The mechanisms by which LAMs mediate these effects remain poorly understood. We show that LAMs were efficiently inserted into the plasma membranes of human and murine lymphomonocytic cells through their GPI anchor. Prior deacylation of LAMs abrogated this event. Phosphatidylinositol hexamannoside (PIM6), the GPI anchor of all LAMs, competitively inhibited LAM insertion. Deacylated PIM6 was not inhibitory. The hexamannoside glycan of PIM6 appears to be important for LAM insertion, since phosphatidylinositol from soybean, lacking the glycan core, was not as efficient an inhibitor. Interaction of LAM with target cells was influenced by the gel/fluid phase distribution of membrane lipids, suggesting a direct interaction of the LAM-GPI anchor with the membrane bilayer. The inserted LAMs were mobile in the plane of the membrane and interfered with Ab-mediated mobilization of the GPI-anchored Thy-1 molecules. Further, LAMs were preferentially incorporated into isolated plasma membrane vesicles enriched in Thy-1. Our results strongly suggest that 1) interaction of LAMs with host lymphomonocytic cells is mediated through a preferential integration of LAM-GPI anchor into specialized plasma membrane domains enriched in endogenous GPI-anchored molecules, and 2) both the acyl chains and the mannoside core glycan of the LAM-GPI anchor contribute to the specificity of integration.
TGF-beta is important in determining the in vivo patterns of susceptibility or resistance in mice infected with Candida albicans.
Authors: Spaccapelo R , Romani L , Tonnetti L , Cenci E , Mencacci A , Del Sero G , Tognellini R , Reed S G , Puccetti P , Bistoni F
Published: December 2022

Resistance and susceptibility of mice to systemic infection with the fungus Candida albicans are associated with the preferential expansion of Th1 and Th2 cells, respectively. In this study, endogenous production of TGF-beta was found to be increased soon after infection of healer mice with a live vaccine strain of the fungus, but down-regulated in nonhealer mice with virulent yeast challenge. Although not affecting the outcome of primary challenge, serologic ablation of TGF-beta in the former animals abrogated development of acquired resistance and resulted in impaired production of IL-12/IFN-gamma and higher expression of IL-4/IL-10 at the time of reinfection with virulent yeast. A CD4+ population expressing the memory phenotype, CD44highMEL-14low, which appeared to be expanded by yeast infection of nonhealer mice, was similarly increased in the healer mice by anti-TGF-beta treatment. In vitro rTGF-beta impaired the candidacidal function of IFN-gamma-activated macrophages. Yet in nonhealer mice infected with virulent C. albicans, administration of rTGF-beta delayed progression of the disease, which was concomitant with the detection of lower levels of IL-4. In addition to previous evidence for an obligatory role of IFN-gamma and IL-12 in Candida-driven Th1 cell differentiation in vivo, the present data establish TGF-beta as a third cytokine, the presence of which may be required for optimal Th1 development leading to long-lived anticandidal resistance.
T cells infiltrating non-Hodgkin's B cell lymphomas show altered tyrosine phosphorylation pattern even though T cell receptor/CD3-associated kinases are present.
Authors: Wang Q , Stanley J , Kudoh S , Myles J , Kolenko V , Yi T , Tubbs R , Bukowski R , Finke J
Published: December 2022

Although tumor infiltrating lymphocytes (T-TIL) from B cell non-Hodgkins lymphoma patients contain tumor-reactive T cells, they display poor proliferation and IFN-gamma production when stimulated through the TCR-CD3. To determine if there was altered signaling linked to TCR-CD3 ligation, tyrosine phosphorylation was examined in T-TIL because it represents an early and critical event in T cell activation. After stimulation with anti-CD3 Ab, Western blotting with anti-phosphotyrosine showed reduced phosphorylation in T-TIL when compared with peripheral blood-derived T cells from normal individuals. The altered phosphorylation was not due to the reduced expression of signaling elements linked to the TCR-CD3 complex. T-TIL expressed normal levels of CD3 epsilon, TCR zeta chain, and the three tyrosine kinases, p56lck (Lck), p59fyn, and ZAP-70. However, in T-TIL, anti-Lck Ab reacted with a 60-kDa protein, which appears to be the phosphorylated form of Lck. Binding of anti-Lck Ab to the 60-kDa protein was blocked by Lck peptide. In addition, anti-Lck Ab immunoprecipitated a phosphorylated 60-kDa protein from gamma-32P-labeled T-TIL that was not seen in normal resting T cells. In vitro kinase assay studies also demonstrated that TCR-CD3 engagement increased the kinase activity of Lck in normal T cells but not in T-TIL. These results suggest that although T-TIL from B cell non-Hodgkins lymphoma patients contain the signal transduction molecules associated with TCR-CD3 activation pathway, they are impaired in tyrosine phosphorylation and Lck activity, which may contribute to the functional defects of these cells.
Inhibition of IL-10 expression by IFN-gamma up-regulates transcription of TNF-alpha in human monocytes.
Authors: Donnelly R P , Freeman S L , Hayes M P
Published: December 2022

Stimulation of human monocytes with LPS induces expression of multiple cytokines, including TNF-alpha, IL-1 beta, IL-6, and IL-10, IL-10 expression is delayed relative to that of TNF-alpha, IL-1 beta, and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF-alpha, IL-1 beta, and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. The Th1-type lymphokine, IFN-gamma, markedly up-regulates TNF-alpha production in monocytes. However, the precise mechanism by which IFN-gamma mediates this effect is unknown. We examined the effects of IFN-gamma on IL-10 expression in LPS-stimulated monocytes, and the relationship between IL-10 and TNF-alpha production in these cells. LPS stimulation induced rapid, ordered expression of multiple cytokines. Steady-state mRNA levels for TNF-alpha increased rapidly, reached maximal levels by 2 to 3 h poststimulation, and then declined sharply. IL-1 beta and IL-6 mRNA levels also increased markedly following stimulation with LPS, but decreased more slowly than did TNF-alpha. Down-regulation of mRNA for TNF-alpha, IL-1 beta, and IL-6 coincided with a delayed and more gradual increase in IL-10 mRNA levels. Furthermore, neutralization of IL-10 with anti-IL-10 Abs prolonged TNF-alpha mRNA expression, and significantly increased net TNF-alpha production. IFN-gamma suppressed expression of IL-10 mRNA and protein in a dose-dependent manner. Moreover, inhibition of IL-10 production correlated with a marked increase in both the magnitude and duration of TNF-alpha expression. Thus, potentiation of TNF-alpha production by IFN-gamma in monocytes is coupled to inhibition of endogenous IL-10 expression.
Up-regulation of lung vascular ICAM-1 in rats is complement dependent.
Authors: Vaporciyan A A , Mulligan M S , Warren J S , Barton P A , Miyasaka M , Ward P A
Published: December 2022

Intrapulmonary deposition of IgG immune complexes in rats causes acute inflammatory lung injury that is neutrophil, complement, cytokines (IL-1, TNF-alpha), and intercellular adhesion molecule (ICAM-1) dependent. In the current studies involving the same model of lung injury, complement depletion or complement blockade resulted in substantial reductions in up-regulation of pulmonary vascular ICAM-1, accompanied by reduced lung injury and neutrophil accumulation. Complement depletion neither reduced the amount of immune complex deposited in lung nor the TNF-alpha content in bronchoalveolar lavage fluids. In the same model of inflammatory lung injury, neutrophil depletion (which is highly protective) did not affect up-regulation of lung vascular ICAM-1. Up-regulation of lung vascular ICAM-1 by intratracheally administered TNF-alpha was also prevented by complement depletion. These studies indicate an unexpected in vivo relationship between complement and up-regulation of lung vascular ICAM-1.
HIV infection suppresses type 1 lymphokine and IL-12 responses to Toxoplasma gondii but fails to inhibit the synthesis of other parasite-induced monokines.
Authors: Gazzinelli R T , Bala S , Stevens R , Baseler M , Wahl L , Kovacs J , Sher A
Published: December 2022

Individuals infected with Toxoplasma gondii normally develop resistance to the parasite, resulting in an asymptomatic chronic infection. In AIDS patients, this resistance is lost leading to reactivation of infection and development of encephalitis. To characterize the cytokine response of T. gondii-infected individuals, PBMC were cultured in vitro in the presence or absence of crude tachyzoite Ags (STAg). When stimulated with STAg, PBMC from T. gondii-infected donors, but not controls, produced high levels of Type 1 lymphokines (IL-2 and IFN-gamma) as well as the monokine IL-12, in the absence of detectable Type 2 lymphokines (IL-4 and IL-5). In contrast, cells of individuals from both groups produced high levels of IL-1, IL-6, and TNF-alpha when exposed to the same Ag preparation. By using highly purified elutriated cells, we demonstrated that monocytes are a major source of these monokines. The above findings were further expanded by analyzing the cytokine responses induced by STAg in PBMC from patients co-infected with T. gondii and HIV. Our results demonstrate that parasite-specific IL-2 and IFN-gamma responses are greatly impaired even before AIDS development, as is IL-12 synthesis by PBMC from HIV-infected individuals stimulated with STAg. In contrast, the release of IL-6 and TNF-alpha triggered by STAg is either not affected or augmented during HIV infection.
Exquisite peptide specificity of oral tolerance in experimental autoimmune encephalomyelitis.
Authors: Javed N H , Gienapp I E , Cox K L , Whitacre C C
Published: December 2022

Experimental autoimmune encephalomyelitis (EAE), induced in Lewis rats by injection of myelin basic protein (MBP) and adjuvant, is a T cell-mediated autoimmune disease. Earlier studies from our laboratory have shown that oral administration of guinea pig MBP before encephalitogenic challenge induces T cell anergy and results in the suppression of clinical signs and CNS histopathologic changes of EAE. In contrast, oral administration of rat MBP did not confer a similar degree of protection. This study was undertaken to determine the tolerogenicity of the synthetic peptide 68-88 derived from guinea pig (GP) MBP and rat MBP. These peptides differ by a single amino acid at position 80. Lewis rats fed GP 68-88 were protected from EAE induced with GP 68-88 or rat 68-88. In contrast, feeding rats 68-88 did not protect the animals from challenge with either peptide. Measurement of the frequency of peptide-reactive Th1 cells showed results consistent with the clinical picture. The in vitro proliferative response was significantly suppressed following oral administration of either whole GP MBP, the GP peptide, or the rat peptide, irrespective of clinical status. These results extend our earlier observation at the whole molecule level that GP but not rat MBP confers oral tolerance. These findings suggest that small structural differences at the amino acid level can produce dramatic differences in clinical outcome, with important implications for the design of multiple sclerosis clinical trials.
Altered expression of monocyte IgA Fc receptors is associated with defective endocytosis in patients with alcoholic cirrhosis. Potential role for IFN-gamma.
Authors: Silvain C , Patry C , Launay P , Lehuen A , Monteiro R C
Published: December 2022

Expression, saturation, and endocytosis of IgA Fc receptors (Fc alpha R) were analyzed in blood phagocytic cells of patients with alcoholic liver cirrhosis (ALC). Surface Fc alpha R expression was decreased in monocytes but not in neutrophils, as evaluated by IgA binding and anti-Fc alpha R mAb. The Fc alpha R of ALC patients were saturated by IgA1 and IgA2. ALC Fc alpha R had a higher M(r) (60 to 90 kDa) than those of controls (55 to 75 kDa) with a similar 32-kDa protein core after N-glycanase treatment, suggesting the expression of Fc alpha R molecules with altered carbohydrate moieties. Treatment of U937 cells with IFN-gamma induced a decrease of surface Fc alpha R expression in a dose-dependent manner, with a similar M(r) as observed for ALC patient Fc alpha R (60 to 90 kDa). Fc alpha R endocytosis was induced by anti-Fc alpha R or IgA. Neutrophils internalized Fc alpha R molecules faster than did monocytes. Endocytosed Fc alpha R co-localized with cathepsin D, suggesting an endolysosomal compartment pathway. In ALC monocytes, Fc alpha R endocytosis was defective, with nearly 50 to 60% of receptors detected on the cell surface even after 90 min at 37 degrees C. Similarly, delayed Fc alpha R endocytosis was observed on IFN-gamma-treated U937 cells as compared with PMA-activated cells. Defective internalization of surface-bound IgA with reflux of IgA to cell surface was also observed on ALC monocytes, but not on normal cells preincubated with patients' plasma, ruling out direct effects of IgA. The inverse correlation between monocyte Fc alpha R levels and serum IgA levels associated with defective endocytosis suggest that altered Fc alpha R expression might contribute to receptor saturation and generation of increased plasma levels of IgA and IgA-immune complexes in ALC patients.
Analysis of the CD4 coreceptor and activation-induced costimulatory molecules in antigen-mediated mature T lymphocyte death.
Authors: Boehme S A , Zheng L , Lenardo M J
Published: December 2022

We have compared the signaling requirements for activation and lymphokine production in mature T lymphocytes to those required for TCR-driven programmed cell death (PCD). Both processes require TCR engagement and ligation of the CD4 coreceptor in the case of a T cell clone that recognizes Ag in the context of an MHC class II molecule. By contrast, stimulation through the CD28/B7 pathway does not appear to positively or negatively influence TCR-induced PCD, although it was required for IL-2 production in both resting and proliferating T cells. T cells that had been activated and induced to proliferate with IL-2 were found to express high levels of IL-2 mRNA upon TCR rechallenge, without a requirement for accessory cells. This was due to a strong up-regulation of the B7-1 molecule, but not the B7-2 molecule, on the T cell surface. These T cells that strongly costimulate each other are highly susceptible to TCR-induced death providing independent evidence that costimulatory signals are not protective. Thus, these results provide evidence that in mature T cells there exists a difference in the requirement for CD28 to achieve activation and IL-2 production compared with TCR-mediated PCD.