bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective reduction of V alpha 14+ NK T cells associated with disease development in autoimmune-prone mice.
Authors: Mieza M A , Itoh T , Cui J Q , Makino Y , Kawano T , Tsuchida K , Koike T , Shirai T , Yagita H , Matsuzawa A , Koseki H , Taniguchi M
Published: December 2022

A novel peripheral T cell subset characterized by the expression of a NK marker and invariant TCR encoded by V alpha 14 J alpha 281 gene segments with a 1-base N-region was investigated in relation to autoimmune disease development. First, we observed that invariant V alpha 14+ NK T cells are specifically reduced with aging in C57BL/6 lpr/lpr or MRL lpr/lpr mice, whereas no change was observed in age-matched control C57BL/6 or MRL +/+ mice as determined by FACS analysis and RNase protection assay. This reduction precedes the disease development and could also be detected in other autoimmune disease-prone mice, such as C3H gld/gld and (NZB x NZW)F1 mice. These results suggest that the specific decrease in invariant V alpha 14+ NK T cells correlates strongly with the development of autoimmunity. Second, injection of MRL lpr/lpr mice with anti-V alpha 14 mAb resulted in the early onset and exacerbation of lymphosplenomegaly due to the accumulation of abnormal CD3+ B220+ CD4-CD8- T cells as well as an increase in the titers of anti-dsDNA autoantibodies. These results indicate that V alpha 14+ NK T cells regulate autoimmune responses and play a crucial role in controlling the development of autoimmune diseases.
Flow cytometric and functional analyses of central nervous system-infiltrating cells in SJL/J mice with Theiler's virus-induced demyelinating disease. Evidence for a CD4+ T cell-mediated pathology.
Authors: Pope J G , Karpus W J , VanderLugt C , Miller S D
Published: December 2022

Theiler's murine encephalomyelitis viruses (TMEVs) are endemic enteric pathogens of mice that cause immune-mediated, chronic, progressive, central nervous system (CNS) demyelinating disease in susceptible strains. Analysis of T cell phenotype and functional state from TMEV-infected SJL/J mice by flow cytometry reveals that 13.5 to 25% of the CD4+ T cells in the CNS express high affinity IL-2R, a marker of recent T cell activation, whereas splenic levels of CD4+IL-2R+ T cells generally range between 2 and 8.5%. In contrast, very few CD8+ T cells (<1-2%) from either site express IL-2R. From days 20 to 119 postinfection, the percentage of CD4+IL-2R+ T cells increases gradually in the CNS, but varies little in the spleen. CD4+ T cells isolated from the spinal cord of infected mice proliferate in vitro in response to viral Ag. Similar T cell phenotypes were found in experimental autoimmune encephalomyelitis, an established model of CD4+ T cell-mediated demyelination. In addition, most CD4+ and CD8+ T cells in CNS isolates from TMEV-infected mice are CD44+, indicating that prior activation may be required to traffic through and/or be retained in the CNS. Finally, TCR V beta region usage as well as IL-2R expression by individual V beta region subsets are heterogeneous in both the CNS and spleen. These results are consistent with a model in which a polyclonal population of TMEV-specific, CD4+ Th1 cells plays a major effector role in the demyelinating process.
TCR-independent activation of human CD4+ 45RO- T cells by anti-CD28 plus IL-2: Induction of clonal expansion and priming for a Th2 phenotype.
Authors: Brinkmann V , Kinzel B , Kristofic C
Published: December 2022

In this study we show that uncommitted human CD4+ CD45RA+ RO- CD25- CD71- HLA-DR- T cells can be primed for a Th2 phenotype before they encounter TCR signals and before they are exposed to IL-4. We found that >99% of uncommitted T cells proliferated upon costimulation by immobilized anti-CD3 plus anti-CD28 mAbs and differentiated into pure Th1 cells. In contrast, uncommitted T cells did not respond to stimulation by either anti-CD3 or anti-CD28, or by IL-2 alone. Interestingly, 5% of uncommitted T cells proliferated efficiently in response to stimulation by immobilized anti-CD28 plus IL-2 (in the absence of TCR/CD3 signals) and differentiated into pure Th2 "precursor" cells. Like murine CD4+ NK1.1+ T cells, human Th2 precursors promptly expressed mRNA for Th2 cytokines upon stimulation via the TCR/CD3 complex by anti-CD3 mAb or staphylococcal enterotoxin B, and secreted up to 50 ng of IL-4, IL-5, and IL-13 per 10(6) cells. Th2 "precursors" developed only in the complete absence of IL-4, as addition of 0.1 U (5 pg) of exogenous IL-4 suppressed their clonal expansion by >90%, whereas addition of neutralizing anti-IL-4 mAb had no effect. Together these results suggest that, in vivo, a significant fraction of uncommitted T cells may be primed for a Th2 phenotype independent of Ag and IL-4 if they are exposed to Th1 cell-derived IL-2 and simultaneously interact with accessory cells bearing the natural CD28 ligands B7-1 and B7-2. When stimulated by specific Ag, such primed Th2 precursor cells may provide a source of IL-4 to promote Th2 immunity.
Roles of proteasomes, transporter for antigen presentation (TAP), and beta 2-microglobulin in the processing of bacterial or particulate antigens via an alternate class I MHC processing pathway.
Authors: Song R , Harding C V
Published: December 2022

Latex-OVA and bacteria expressing an OVA fusion protein were processed by macrophages via an alternate class I MHC (MHC-I) processing pathway to present OVA(257-264):Kb. This pathway was resistant to dipeptide aldehyde proteasome inhibitors and brefeldin A, unlike the cytosolic MHC-I pathway. TAP1-/- macrophages exhibited decreases in cell surface peptide-receptive MHC-I and binding of extracellular peptide during transient incubations. This may explain an apparent influence of TAP on alternate MHC-I processing. Alternate MHC-I processing by TAP1-/- cells was enhanced by preincubation at 26 degrees C or with beta 2-microglobulin to increase peptide-receptive MHC-I. Thus, peptides may bind to MHC-I within post-Golgi vacuolar organelles accessible to exogenous beta 2-microglobulin or on the cell surface (following peptide regurgitation).
Hematopoietic cell protein-tyrosine phosphatase-deficient motheaten mice exhibit T cell apoptosis defect.
Authors: Su X , Zhou T , Yang P A , Wang Z , Mountz J D
Published: December 2022

We previously demonstrated that hematopoietic cell protein-tyrosine phosphatase is one of the molecules that can transduce Fas-mediated apoptosis signals in lymphoid cells. The present study analyzed the effect of defective Fas signaling on the T cell phenotype and apoptosis function in hematopoietic cell protein-tyrosine phosphatase-deficient motheaten mice. Viable motheaten (me(v)/me(v)) mice exhibited increased T cell proliferation and defective activation-induced apoptosis of Fas+ T cells in the lymph node, which was not ascribed to defective Fas ligand function. Furthermore, the Fas-mediated apoptosis defect in activated T cells from me(v)/me(v) mice was confirmed by their resistance to anti-Fas-induced apoptosis. No protein tyrosine dephosphorylation signal was delivered after anti-Fas cross-linking in the lymph node cells of me(v)/me(v) mice as revealed by 32Pi labeling of protein phosphatase substrates. The defective activation-induced apoptosis of Fas+ T cells in me(v)/me(v) mice led to lymphadenopathy with an accumulation of CD4- CD8- B220+ CD3+ T cells. Pneumonitis in me(v)/me(v) mice was associated with infiltration of cycling T cells detected by bromodeoxyuridine uptake in vivo. Thus, T cells from me(v)/me(v) mice are resistant to Fas-mediated apoptosis which results in lymphoproliferative disease and tissue infiltration.
EBV induces proliferation of immature human thymocytes in an IL-2-mediated response.
Authors: Todd S C , Tsoukas C D
Published: December 2022

The receptor for EBV, CD21 is expressed on a population of immature human thymocytes and facilitates infection of these cells by EBV. Thymocytes infected by EBV become responsive to exogenous rIL-2- or CD2-mediated stimulation in vitro. To address whether such costimulation may be provided by thymic presenting cells and to study the cellular effects of EBV infection, the present work utilizes thymocyte cultures containing autologous thymic presenting cells. In the presence of these presenting cells, EBV induces proliferation of thymocytes. EBV infection promotes the formation of adhesions between two populations of cells in an APC responder fashion, and separation of these two populations abrogates the proliferative response to EBV. The response is mediated by IL-2 because Ab blocking of the IL-2R inhibits proliferation as does cyclosporin A. EBV promotes an expansion in the number of CD4+8+ thymocytes, and the proliferating population is vulnerable to TCR/CD3-generated signals, indicating that the responding cells are phenotypically and functionally immature. Finally, addition of exogenous IL-2 to EBV-exposed thymocytes promotes a second wave of proliferation. Phenotypic characterization of the EBV-induced, IL-2-responding cells shows them to express reduced levels of CD1 and a transitional CD4(high)8(low) phenotype. These data characterize the cellular response to EBV infection in thymocytes and may offer insight into EBV-associated T lineage malignancies and autoimmune disorders.
Isolation of low molecular mass polypeptide complementary DNA clones from primitive vertebrates. Implications for the origin of MHC class I-restricted antigen presentation.
Authors: Kandil E , Namikawa C , Nonaka M , Greenberg A S , Flajnik M F , Ishibashi T , Kasahara M
Published: December 2022

Proteasomes are the multisubunit proteases thought to be involved in the generation of peptides presented by MHC class I molecules. When cells are stimulated with IFN-gamma, two MHC encoded subunits, LMP2 and LMP7, are incorporated into the proteasomal complex, presumably by displacing the housekeeping subunits, designated Y and X, respectively. These changes in the subunit composition appear to facilitate class I-mediated Ag presentation, presumably bu altering the cleavage specificities of the proteasome. Here we show that the cartilaginous fish, the most primitive class of vertebrates in which the MHC has been identified, have both LMP7 and X genes. Interestingly, nurse sharks, a member of the cartilaginous fish, appear to have two LMP7 genes, one encoding a typical LMP7 subunit and the other encoding a less typical one. In contrast, only cDNA clones with residues characteristic of X were identified in hagfishes and lampreys, the two extant members of the jawless fish in which no MHC has been identified. Pairwise amino acid sequence comparison and phylogenetic tree analysis showed that the subunits encoded by these clones were nearly equidistant from LMP7 and X, suggesting that the LMP7 gene might have emerged after the appearance of the jawless fish. Sequence comparison of the LMP7 and X/X-like subunits isolated from various vertebrate species showed that, unlike the X/X-like subunit, the LMP7 subunit displays a striking interspecies sequence variability in the vicinity of its catalytic site.
Induction of CIITA and modification of in vivo HLA-DR promoter occupancy in normal thymic epithelial cells treated with IFN-gamma: similarities and distinctions with respect to HLA-DR-constitutive B cells.
Authors: Rigaud G , De Lerma Barbaro A , Nicolis M , Cestari T , Ramarli D , Riviera A P , Accolla R S
Published: December 2022

In this study, the IFN-gamma induction of MHC class II gene expression in primary cultures of thymic epithelial cells (TEC) was analyzed. This cellular system offers the advantage that MHC class II induction is studied in a "physiologic" cell lineage that, as a result of this expression within the thymus, is thought to participate to the selection and maturation of the T cells. It was found that the MHC class II gene expression was associated with the de novo transcription of the gene encoding the CIITA trans-activator, a crucial MHC class II gene regulatory factor. Furthermore, the anatomy of interaction between the MHC class II DRA promoter and corresponding binding factors was analyzed by in vivo DNAse I footprint. It was found that treatment with IFN-gamma induces changes in the occupancy of the DRA gene regulatory sequences by nuclear factors. The resulting occupancy displays strong similarities with the one observed in the MHC class II-constitutive B cells, represented by both the Burkitt lymphoma line Raji and normal tonsil- derived B cells. However, some peculiar differences were observed between the TEC, either IFN-gamma-induced or not, and the constitutive B cells. These results suggest that both common mechanisms, such as the one mediated by the CIITA trans-activator, and distinct tissue-specific constraints contribute to the transcriptional control of constitutive and IFN-gamma-induced MHC class II gene expression.
Cloning of the cDNA for human IFN-gamma-inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein.
Authors: Ushio S , Namba M , Okura T , Hattori K , Nukada Y , Akita K , Tanabe F , Konishi K , Micallef M , Fujii M , Torigoe K , Tanimoto T , Fukuda S , Ikeda M , Okamura H , Kurimoto M
Published: December 2022

We have recently reported that a novel molecule, murine IFN-gamma-inducing factor (IGIF) produced by mouse liver cells, possesses potent biologic activities, including the induction of IFN-gamma production by spleen cells and the enhancement of NK cell cytotoxicity. In this paper, we report on the isolation of human IGIF cDNA clones from normal human liver cDNA libraries using murine IGIF cDNA as a probe. The amino acid sequence deduced from the human cDNA clones indicated a 193-amino acid precursor peptide and revealed 65% homology with that of murine IGIF. The amino acid sequence of IGIF also included an IL-1 signature-like sequence. Subsequently, the cloned cDNA was expressed in Escherichia coli, and preliminary studies on the biologic activities of the recombinant protein were performed. The recombinant human IGIF induced IFN-gamma production by mitogen-stimulated PBMC and enhanced NK cell cytotoxicity, in a manner similar to murine IGIF. In addition, recombinant human IGIF also augmented granulocyte-macrophage-CSF production and decreased IL-10 production, but had no effect on IL-4 production by Con A-stimulated PBMC. Based on these pleiotropic effects of IGIF, we propose that this novel cytokine be designated as IL-18.
IL-6-deficient mice exhibit normal mucosal IgA responses to local immunizations and Helicobacter felis infection.
Authors: Bromander A K , Ekman L , Kopf M , Nedrud J G , Lycke N Y
Published: December 2022

Using IL-6-deficient (IL-6 -/-) or wild-type mice, we investigated whether IL-6 is involved in the intestinal adjuvant activity of cholera toxin (CT) and to what extent IL-6 is required for mucosal IgA responses against soluble protein Ags or live Helicobacter felis infection. In naive IL-6 -/- mice we found normal total IgA levels in serum, bronchial and intestinal lavage and unaltered frequencies of IgA plasma cells in intestinal lamina propria. In Peyer's patches (PP) and mesenteric lymph nodes (MLN) IgA-producing cells were as frequent in IL-6 -/- as in wild-type mice. Immunohistochemical analysis of PP revealed germinal centers that co-localized IgA+ cells, indicating B cell activation and isotype switching in situ in the intestinal immune inductive site. Phenotypic analysis of the distribution of conventional B-2 cells (B220+CD5-/Mac-1-) and B-1 cells (B220+, CD5+/Mac-1+) in intestine-associated tissues gave comparable results in IL-6 -/- and wild-type mice. The ability to respond with mucosal IgA following oral and intranasal immunization with specific Ag, KLH or OVA, in the presence of CT adjuvant or to live H. felis infection was similar in IL-6 -/- and wild-type mice. CT exerted strong and comparable adjuvant functions in IL-6 -/- and wild-type mice. Repeated oral immunizations with CT alone stimulated immune protection against CT-induced diarrhea in ligated loops that was of similar magnitude in IL-6 -/- and wild-type mice. We conclude that, although IL-6 has been ascribed a crucial role in terminal differentiation of IgA B cells in vitro, we found no evidence to support the notion that IL-6 is critically required for IgA B cell development or specific mucosal IgA responses in vivo.
Antisense inhibition of vitamin D receptor expression induces apoptosis in monoblastoid U937 cells.
Authors: Hewison M , Dabrowski M , Vadher S , Faulkner L , Cockerill F J , Brickell P M , O'Riordan J L , Katz D R
Published: December 2022

The active vitamin D3 metabolite 1,25-dihydroxycholecalciferol (1,25(OH)2D3) acts as an antiproliferative and differentiating agent for the monoblastoid cell line U937 and as an important immunologic mediator implicated particularly in the function of cells belonging to the monocyte/macrophage lineage. These effects are controlled by the vitamin D receptor (VDR), a member of the steroid hormone receptor family. The objective of this study was to develop U937 transfectants expressing antisense VDR mRNA, and to use these to examine the role of 1,25(OH)2D3-VDR interaction in this lineage. A 2-kb VDR cDNA insert (including the complete VDR coding region) was cloned in an antisense orientation into the EBV episomal vector pMEP4 under the control of an inducible promoter and transfected into U937. The resultant cell line, DH42, was hygromycin resistant, contained VDR cDNA, expressed fewer VDRs than controls, and showed a substantial decrease in antiproliferative response to 1,25(OH)2D3. However, 1,25(OH)2D3 increased the number of cells expressing macrophage cell surface Ags, including CD14 and CD11b. A subpopulation of smaller cells did not express the differentiation markers after cadmium stimulation. Cell cycle analysis showed shifts in the distribution of cells from G1 to S phase, which were more pronounced after cadmium treatment. A considerable proportion of cells were outside the cycle and DNA fragmentation confirmed apoptosis. Thus, the functional outcome of the VDR antisense transfection suggests that in the myelomonocytic lineage, VDR expression may act as a protective mechanism against programmed cell death.
Release of leukemia inhibitory factor in primate sepsis. Analysis of the role of TNF-alpha.
Authors: Jansen P M , de Jong I W , Hart M , Kim K J , Aarden L A , Hinshaw L B , Taylor F B , Hack C E
Published: December 2022

Leukemia inhibitory factor (LIF), a pleiotropic cytokine with many biologic effects overlapping with those of IL-6, has been implicated in the pathogenesis of sepsis. We here analyzed the kinetics of LIF in 13 baboons challenged with a lethal (n=6) or sublethal (n=7) dose of Escherichia coli. In addition, to assess the role of TNF-alpha in the induction of LIF in vivo, seven baboons were studied that had either received a bolus injection of recombinant human TNF-alpha (100 micrograms/kg, n=3), or to whom 15 mg/kg of an anti-TNF mAB before lethal E. coli challenge was administered (n=4). LIF levels increased 2 h after E coli challenge, and reached maximum values at 4 and 8 h after a sublethal (4.4 +/- 1.6 ng/ml) or lethal (40.9 +/- 3.8 ng/ml) dose, respectively. TNF-alpha injection induced a modest rise in LIF concentrations, peaking after 6 h (228 +/- 46 pg/ml). Circulating LIF correlated with plasma levels of IL-6, both after E. coli challenge (Spearman Rank coefficient of correlation (r) = 0.849, p<0.001), as well as upon TNF-alpha injection (r=0.863, p<0.001). Moreover, the E. coli-induced release of either cytokine was reduced 6- to 10-fold after pretreatment with anti-TNF mAb, except in one nonsurviving animal, which exhibited a progressive increase of LIF and IL-6 levels despite the absence of TNF immunoreactivity. These results show that TNF-alpha is an intermediate factor in concerted release of LIF and IL-6 in vivo, and indicate that the enhanced elaboration of these cytokines may predict disease outcome in severe sepsis.
Inhibition of the hemolytic activity of the first component of complement C1 by an Escherichia coli C1q binding protein.
Authors: van den Berg R H , Faber-Krol M C , van de Klundert J A , van Es L A , Daha M R
Published: December 2022

Molecular mimicry is a well established mechanism via which bacteria protect themselves from complement-mediated killing. We have previously demonstrated that a number of human cells express receptors for C1q (C1qR) and that the soluble form of this receptor inhibits activation of the classical pathway of complement. We now investigated whether Escherichia coli possesses a C1qR-like protein that protects these bacteria from complement-mediated injury. By FACS analysis it was shown that approximately 60% of the bacteria bound C1q directly in the absence of Abs. With ELISA we confirmed that the bacterial cell envelope was able to bind C1q in a dose-dependent fashion. We isolated a cell envelope associated C1q binding protein (C1qBP) by C1q affinity chromatography, then by anion exchange chromatography and gel filtration chromatography. On SDS-PAGE, the m.w. of C1qBP appeared to be 57 kDa and 51 kDa under reducing and nonreducing conditions, respectively. It was demonstrated that C1qBP specifically binds C1q and inhibits the hemolytic activity of C1q in both a dose- and time-dependent fashion. The binding of C1qBP to C1q is inhibited by C1q itself and also by the collagen-like stalks and the globular heads of C1q. In this respect, bacterial C1qBP is different from human C1qR because the binding of C1q to C1qR is only inhibited by the collagen-like stalks of C1q and not by the globular heads of C1q. C1qBP, when bound to C1q, prevents the assembly with C1r and C1s to form a functional C1 complex. The occurrence of C1qBP is not limited to certain E. coli strains, but is also found on Staphylococcus aureus, Citrobacter freundii, and Pseudomonas aeruginosa. Also, the binding of 125(I)-labeled C1q to these bacteria is specific because the binding of C1q to these bacteria is inhibitable with isolated soluble C1qBP. These findings provide evidence for the existence of a C1qR-like protein on bacteria that might protect them from complement-mediated damage.
Identification of multiple and distinct CD8+ T cell suppressor activities: dichotomy between infected and uninfected individuals, evolution with progression of disease, and sensitivity to gamma irradiation.
Authors: Barker T D , Weissman D , Daucher J A , Roche K M , Fauci A S
Published: December 2022

Using an in vitro model system that reflects the cellular interactions occurring in the microenvironment of lymphoid organs (i.e., the interaction between dendritic cells (DC) and CD4+ T lymphocytes), the ability of CD8+ T cells to inhibit HIV replication was investigated. DC, the most potent APC in the paracortical region of lymphoid organs, were cocultured with autologous, unstimulated CD4+ T cells resulting in viral replication in the absence of exogenous stimulation. Using two variations of DC cocultures, one an acute infection system and the other an endogenous infection system, two sets of activities were identified. One activity was expressed in both HIV-infected and -uninfected individuals, and a second was found only in HIV-infected individuals. These activities can be differentiated further by their evolution or lack thereof with disease progression in infected individuals and their sensitivity to gamma irradiation. Furthermore, the results indicate that CD8+ T cell modulation of HIV replication in CD4+ T cells is a multifactorial phenomenon involving both inhibitory and stimulatory effects on HIV replication.
Resting B cells from New Zealand Black mice demonstrate a defect in apoptosis induction following surface IgM ligation.
Authors: Kozono Y , Kotzin B L , Holers V M
Published: December 2022

New Zealand Black (NZB) mice spontaneously develop autoimmune disease, usually characterized by an autoimmune hemolytic anemia, and NZB genes are essential for a severe systemic lupus-like disease in (NZB x NZW)F1 mice. We have found that resting B cells from NZB mice demonstrate a pronounced defect, compared with five normal strains, in apoptosis induction after cross-linking with anti-IgM Abs. In contrast, spontaneous apoptosis of NZB B cells in culture was similar to normal strains. B cells from young (NZB x SM/J)F1 and (NZB x NZW)F1 mice underwent apoptosis normally, indicating that the NZB defect in apoptosis is a recessive trait. However, older (8-32 wk) predisease (NZB x NZW)F1 mice manifested a similar defect in apoptosis induction. The analysis of NXSM recombinant inbred mice derived from NZB and SM/J, in addition to backcross mice, suggested that the NZB apoptosis defect is a multigenic trait. Interestingly, resting B cells form B6.lpr and B6gld mice underwent apoptosis following anti-IgM treatment at a level similar to that of the C57BL/6 parental strain. Thus, the induced apoptosis of resting B cells and the NZB defect are likely not related to either Fas or Fas ligand. We propose that this phenotypic defect in apoptosis induction, or the biochemical alteration that underlies the defect, may be casually related to autoimmune disease in NZB mice and its contribution to lupus-like disease in (NZB x NZW)F1 mice.
Human and mouse killer-cell inhibitory receptors recruit PTP1C and PTP1D protein tyrosine phosphatases.
Authors: Olcese L , Lang P , Vély F , Cambiaggi A , Marguet D , Bléry M , Hippen K L , Biassoni R , Moretta A , Moretta L , Cambier J C , Vivier E
Published: December 2022

NK cells express cell surface receptors for MHC class I proteins (KIR). Engagement of these receptors inhibits NK cell cytotoxic programs. KIR can be expressed on T cells, and their engagement also results in inhibition of effector functions initiated by the CD3/TCR complex. While human KIR genes belong to the Ig gene superfamily, mouse KIR belong to a family of dimeric lectins. Despite these distinct evolutionary origins, we show here that both HLA-Cw3-specific human p58.183 receptors and H-2D d/k-specific mouse Ly49A receptors recruit the same protein tyrosine phosphatases, PTP1C and PTP1D, upon phosphorylation of critical intracytoplasmic tyrosine residues. These results document a common pathway by which diverse KIR can down-regulate NK and T cell activation programs, and further define the sequence of the immunoreceptor tyrosine-based inhibitory motif (ITIM), initially described in FcgammaRIIB1, and expressed in both human and mouse KIR.
Intrathymic signals in thymocytes are mediated by p38 mitogen-activated protein kinase.
Authors: Sen J , Kapeller R , Fragoso R , Sen R , Zon L I , Burakoff S J
Published: December 2022

Thymocytes develop into mature functional T cells in the inductive environment of the thymus where thymocyte-stromal cell interactions and cytokines provide survival and differentiation signals as cues for thymocyte maturation. Disruption of the thymic microenvironment results in attenuation of T cell maturation, suggesting that intrathymic signals are essential for differentiation and repertoire selection. We have previously shown that several inducible nuclear factors such as AP-1, NF-AT, and NF-kappaB are activated in response to intrathymic signals. Here we demonstrate that in thymocytes p38 mitogen-activated protein (MAP) kinase, a member of the MAP kinase family of proteins that include the extracellular-signal regulated kinases and Jun aminoterminal kinases, is highly activated in response to intrathymic signals in vivo. These studies suggest a role for p38 MAP kinase in T cell survival and differentiation.
Dexamethasone inhibits the early steps of antigen receptor signaling in activated T lymphocytes.
Authors: Baus E , Andris F , Dubois P M , Urbain J , Leo O
Published: December 2022

Glucocorticoids are very effective in inhibiting inflammatory and immune responses. In particular, the synthetic analogue dexamethasone has been shown to inhibit T cell proliferation and IL-2 production by interfering with the transcriptional activation of the IL-2 gene. The experiments described in this report were performed to determine whether dexamethasone treatment affects the early steps of TCR signal transduction in T cell hybrids. Incubation of murine T cell hybrids in the presence of dexamethasone prevents the intracellular calcium increase that normally follows TCR/CD3 aggregation. Accordingly, dexamethasone treatment decreases inositol phosphates production and phospholipase-Cgamma1 tyrosine phosphorylation induced after TCR/CD3 stimulation. Dexamethasone has no effect on cell surface expression of TCR-associated structures nor does it inhibit calcium responses induced by a heterologous G protein-coupled muscarinic receptor, suggesting that this hormone analogue specifically inhibits the TCR signaling pathway at a postreceptor stage. We also show that inhibition of membrane proximal events by dexamethasone requires binding to the intracellular glucocorticoid receptor and de novo protein synthesis. When splenic T cells were assayed, only activated but not resting T cells were found to be sensitive to this new immunomodulatory effect of dexamethasone. These findings indicate that in addition to their previously described inhibitory effects on cytokine gene transcription, glucocorticoids block IL-2 production in activated T cells by interfering with an early step of the signal transduction cascade initiated by TCR/CD3 cross-linking.
Purification of a novel MHC class I element binding activity from thymus nuclear extracts reveals that thymic RBP-Jkappa/CBF1 binds to NF-kappaB-like elements.
Authors: Shirakata Y , Shuman J D , Coligan J E
Published: December 2022

We purified a DNA binding protein that recognizes a portion of the MHC class I regulatory element region 1/NF-kappaB binding site whose expression correlates with the expression of a MHC class I transgene in the thymus. The N-terminal amino acid sequence and the molecular size matched the RBP-Jkappa protein, also known as the EBV C-promoter binding factor, CBF1. Anti-peptide sera reactive with RBP-Jkappa/CBF1 also reacted with this protein in gel mobility shift assays. Although RBP-Jkappa/CBF1 is ubiquitously expressed, binding to the MHC class Ia NF-kappaB site was limited to the thymus. Comparison of the DNA binding specificities of RBP-Jkappa/CBF1 in thymic and splenic nuclear extracts revealed strong binding from both extracts to an IFN-beta kappaB site containing the RBP-Jkappa/CBF1 consensus sequence (CGTGGGAA). In contrast, only the thymic nuclear extract showed strong DNA binding activity with probes containing the NF-kappaB recognition sequences present in the MHC class Ia, IL-2Ralpha, and granulocyte-macrophage CSF promoters. Thus, RBP-Jkappa/CBF1 in thymic extracts demonstrates a clearly distinguishable DNA binding specificity that correlates with tissue-specific expression of a class I transgene. This, coupled with the fact that our previous study showed enhanced expression of the transgene in CD4+CD8+ thymocytes, suggests that RBP-Jkappa/CBF1 may play a role in the development of the immune system.
Induction of Th1 cell-mediated protective immunity to Schistosoma mansoni by co-administration of larval antigens and IL-12 as an adjuvant.
Authors: Mountford A P , Anderson S , Wilson R A
Published: December 2022

In this study, rIL-12, which is a powerful inducer of Th1 lymphocyte development, was administered to mice as an adjuvant in conjunction with a soluble lung-stage Ag preparation (SLAP) derived from lung-stage larvae of Schistosoma mansoni to potentiate Th1-mediated immune responses and induce resistance to reinfection. Immunization of mice with one or two doses of SLAP + IL-12 elicited a dominant population of Ag-specific Th1 lymphocytes in the draining lymph nodes, as judged by the secretion of abundant IFN-gamma but undetectable levels of IL-4, upon antigenic restimulation in vitro. In contrast, SLAP alone induced a mixed population of Th1 and Th2 cells with secretion of IFN-gamma, IL-4, and IL-10. The development of a biased Th1 cell population in mice immunized with SLAP + IL-12 was reflected in enhanced levels of Ag-specific IgG2a but decreased levels of IgG1 and total IgE serum Abs. Ablation of NK1.1+ cells before the administration of a single dose of SLAP + IL-12 reduced Th cell proliferation and almost completely inhibited secretion of IFN-gamma by in vitro-cultured lymph node cells. This indicates that NK cells stimulated by IL-12 shortly after vaccination are critical to the subsequent development of Ag-specific Th1 cells. Finally, it is demonstrated that the delivery of two doses of SLAP + IL-12 to mice is sufficient to elicit moderate but highly significant levels of protective immunity against challenge infection.
Resistance to murine AIDS in offspring of mice infected with LP-BM5. Role of CD8 T cells.
Authors: Pavlovitch J H , Hulier E , Rizk-Rabin M , Marussig M , Mazier D , Joffret M L , Hoos S , Papiernik M
Published: December 2022

The murine-acquired immunodeficiency syndrome (MAIDS) is caused by a mixture of murine leukemia viruses (LP-BM5 MuLV). The influence of perinatal contact with retroviruses or their Ags on the response to infection was tested by infecting with LP-BM5 (MuLV) the adult offspring of mice with MAIDS. These offspring were resistant to disease after virus challenge. Most of them were free of defective viral DNA, and even those with molecular evidence of infection had lymphoid cells with a lower infectious capacity to cause MAIDS in naive recipients. No ecotropic, xenotropic, or mink cell focus-forming (MCF) virus expression was found at the age of 5 wk, which is the time of LP-BM5 (MuLV) challenge. However, at 22 wk of age, one-half of the offspring from MAIDS mothers had ecotropic virus-expressing cells in their spleens. At the time of suckling, offspring from infected mothers had enhanced percentages of B cells and CD4 and CD8 T cells in the spleen, possibly followed by a slight persistent splenomegaly. These results suggest that immune reactivity, rather than tolerance to the virus, is responsible for resistance to disease after challenge. The offspring of MAIDS mice could clear the virus after challenge. This clearance was mediated by CD8 T cells, as continuous CD8 T cell depletion initiated at the time of viral challenge abrogated the resistance of these mice to MAIDS.
Mycobacterium avium- and Mycobacterium tuberculosis-containing vacuoles are dynamic, fusion-competent vesicles that are accessible to glycosphingolipids from the host cell plasmalemma.
Authors: Russell D G , Dant J , Sturgill-Koszycki S
Published: December 2022

The vacuoles inhabited by viable Mycobacterium avium and Mycobacterium tuberculosis show limited fusion with endosomal and lysosomal compartments. This ability to regulate the maturation of their phagosomal compartments and restrict their differentiation into hydrolytically active vacuoles appears to correlate with the survival of the bacilli. Data presented in this current study demonstrate that despite the apparent isolation of mycobacterial vacuoles from the lysosomal network, they are dynamic, fusion-competent vesicles. Exploiting the ability of cholera toxin B subunit to bind to GM1 ganglioside on the macrophage plasmalemma, we demonstrate that these glycosphingolipids have ready access to the mycobacterial vacuoles. Entry into mycobacterial vacuoles is rapid, within 5 min of addition to the cells, and does not proceed through a brefeldin A-sensitive pathway. Furthermore, the gangliosides follow a route that differs from that taken by fluid-phase markers. TLC analysis gangliosides isolated from Mycobacterium-containing vacuoles, and IgG-bead phagosomes reveal similar profiles. These data indicate that rather than being fusion incompetent, mycobacterial vacuoles are actually highly dynamic, fusion-competent vesicles that behave like an extension of the recycling endosomal apparatus.
The effect of acute phase proteins on clearance of chromatin from the circulation of normal mice.
Authors: Burlingame R W , Volzer M A , Harris J , Du Clos T W
Published: December 2022

The clearance of nucleosome core particles and H1-stripped chromatin from the circulation of mice was examined. Radiolabeled chromatin preparations were injected into mice, and blood samples were obtained over 60 min. The animals were then killed, and the selected organs were collected and radioactivity was measured. The acute phase response (APR) was induced by i.p. injections of casein before some clearance studies. Serum amyloid P component, the major acute phase protein in mice, increased from 27 microg/ml to 339 microg/ml during the acute phase. The rate of chromatin clearance decreased during the acute phase in C57BL/10J mice. At 5 min, 18% +/- 3% of the originally measured radioactivity remained in control animals compared with 49% +/- 2% in acute phase animals (p < 0.001). Co-injection of either serum amyloid P component or C-reactive protein, the major acute phase protein in humans, caused a decrease in the rate of chromatin clearance similar to that observed following the induction of the APR. APR induction also caused a higher percentage of the chromatin to localize in the liver compared with the spleen, with the ratio changing from 10.2 +/- 0.7 to 16.1 +/- 1.9 (p < 0.004). In addition, the APR caused a decrease in the percentage of chromatin localized in the kidney. The lack of radioactivity associated with cells in the circulation indicates that complement is not a major factor in the clearance mechanism of chromatin. These findings suggest that the APR produces major changes in the rate and path of chromatin clearance. These changes may protect against deposition of chromatin in target organs of systemic lupus erythematosus.
Expression of C-reactive protein by alveolar macrophages.
Authors: Dong Q , Wright J R
Published: December 2022

C-reactive protein (CRP) is well characterized as one of the serum acute phase proteins, the levels of which increase dramatically after infection. CRP has been shown to be involved in multiple immunoregulatory functions. For example, it activates the classical complement cascade, opsonizes bacteria for phagocytosis, and stimulates phagocytic cells. Although CRP is predominantly produced and secreted by hepatocytes, other cells including subsets of lymphocytes, Kupffer cells, and blood monocytes have been shown to synthesize this protein as well. We hypothesized that CRP may be produced in the lung, and therefore it could function directly in pulmonary host defense. Western blot analysis showed that CRP was present in the lung tissue, lung lavage, and alveolar macrophages. This result was further confirmed by immunohistochemical staining of lung sections that showed the localization of CRP in alveolar macrophages. The CRP mRNA was detected subsequently by reverse-transcriptase PCR (RT-PCR), and a single amplified product was obtained from alveolar macrophages as well as from whole lung tissue. Both were the same size as the amplified product obtained from liver mRNA. Furthermore, in situ hybridization with CRP riboprobe demonstrated specific staining of alveolar macrophages both in lung sections and isolated cells. In addition, in situ hybridization showed that CRP mRNA levels in isolated alveolar macrophages were up-regulated by in vitro LPS stimulation. In summary, these results indicate that CRP is produced by alveolar macrophages, and suggest that CRP may be involved in the pulmonary immune response.
IL-13 released by and localized in human basophils.
Authors: Li H , Sim T C , Alam R
Published: December 2022

We and others have shown that human basophils can synthesize and release IL-4. However, IL-13, a cytokine that closely resembles IL-4, has not hitherto been described as a basophil product. The production of IL-13 by basophils was demonstrated by immunocytochemistry. Approximately 70% of basophils stimulated with anti-FcepsilonRIalpha (antibody to the alpha subunit of IgE receptor type I) stained for IL-13. Under similar experimental conditions, mononuclear cells failed to stain for IL-13. The cytokine was localized to basophilic granules by electron microscopic examination of immunogold staining. The secretion of IL-13 into the culture supernatant was assayed by ELISA. Kinetic studies showed detectable IL-13 release at 3 h, which steadily increased up to 24 h. This is significantly different from the kinetics of basophil histamine and IL-4 release. IL-13 production was also observed upon stimulation with anti-IgE, anti-FcepsilonRIalpha, IL-3, and A23187 in a dose-dependent manner. PBMC, neutrophils, and eosinophils isolated from the same donors did not release IL-13 after anti-IgE stimulation. The anti-IgE-induced basophil IL-13 synthesis could be enhanced by IL-3 preincubation (with and without IL-3 preincubation, anti-IgE-induced IL-13 production was 227 +/- 99 and 42 +/- 13 pg/10(6) basophils, respectively). PBMC produced a significant amount of IL-1 3 upon stimulation with PHA, but a low level of IL-13 in response to A23187 and/or PMA. Eosinophils and neutrophils did not produce IL-13 when cultured with A23187, IL-5, and anti-FcepsilonRIalpha. This is the first demonstration of IL-1 3 production by basophils. Our data suggest that basophils, in addition to secreting mediators, can represent an important source of proallergic cytokines.
Functional contributions of the FcepsilonRIalpha and FepsilonRIgamma subunit domains in FcepsilonRI-mediated signaling in mast cells.
Authors: Repetto B , Bandara G , Kado-Fong H , Larigan J D , Wiggan G A , Pocius D , Basu M , Gilfillan A M , Kochan J P
Published: December 2022

The functional contributions of the alpha and gamma subunit domains of the high affinity receptor for IgE (Fcepsilon-RI) were determined following chimeric receptor aggregation. Chimeric receptors of the extracellular (EC) and cytoplasmic tail (CT) domains of FcepsilonRI and the IL-2R p55 subunit (I) were constructed and stably expressed in RBL-2H3 cells. Signaling (inositol phosphate production, tyrosine phosphorylation, Ca2+ mobilization, and secretion of histamine and arachidonic acid metabolites) via alpha/gamma/gamma or I/gamma/gamma was similar to the native rat receptor, and both were shown to associate with endogenous FcepsilonRIbeta and FcepsilonRIgamma subunits. Therefore, the contributions of the EC domains could not be evaluated. The chimeras alpha/I/gamma and I/I/gamma were found to be single polypeptide chains, as they did not associate with beta and gamma. Signaling via alpha/I/gamma resulted in the appearance of biochemical events common to the native receptor. Cross-linking I/I/gamma elicited histamine release, [14C]arachidonic acid metabolites, tyrosine phosphorylation, Ca2+ mobilization, and only inositol trisphosphate production, which were not of a similar magnitude to the native FcepsilonRI. No biochemical events were elicited by cross-linking alpha/I/I or I/I/I. These results demonstrate that both the FcepsilonRIalpha EC domain and the FcepsilonRIgamma CT domain are essential for the FcepsilonRI signaling process, and that while FcepsilonRIIgamma CT plays a critical role in FepsilonRI signaling, the EC domain of FcepsilonRIalpha has a major contribution in signaling, as well as a role in modulating the magnitude of the biochemical events.
Pathogenic role of anti-endothelial cell antibodies in vasculitis. An idiotypic experimental model.
Authors: Damianovich M , Gilburd B , George J , Del Papa N , Afek A , Goldberg I , Kopolovic Y , Roth D , Barkai G , Meroni P L , Shoenfeld Y
Published: December 2022

Idiotypic manipulation of naive mice has previously been used for induction of systemic autoimmune diseases (e.g., antiphospholipid syndrome, systemic lupus erythematosus, and Wegener's granulomatosis). The aim of this study focused on the utilization of this technique to induce the production of anti-endothelial cells Abs (AECA) and autoimmune vasculitis in a murine model. AECA were derived from a Wegener's granulomatosis patient's plasma. IgG was purified by absorption on a proteinase-3 affinity column resulting in the depletion of anti-neutrophil cytoplasmic Ab activity. The absorbed IgG fraction displayed a high titer of AECA as evidenced by a cyto-ELISA against unfixed human umbilical vein endothelial cells. BALB/c mice were actively immunized with the purified AECA. Three months after a boost injection with the human AECA, mice developed endogenous AECA (AB), but not Abs to proteinase-3, cardiolipin, or DNA. Histologic examination of lungs and kidneys revealed both lymphoid cell infiltration surrounding arterioles and venules; as well as deposition of Igs at the outer part of blood vessel walls. This experimental animal model of vasculitis, a product of our method of idiotypic manipulation, provides the first direct proof for the pathogenicity of AECA.
Delineation of IL-5 domains predicted to engage the IL-5 receptor complex.
Authors: Dickason R R , Huston M M , Huston D P
Published: December 2022

IL-5 is an interdigitating homodimeric glycoprotein and a member of the helical bundle family of cytokines. IL-5 is a potent activator of eosinophils and a specific promoter of their differentiation. This activity has implicated IL-5 in the pathogenesis of asthma and allergic disease. A detailed understanding of IL-5 structure and function is required to develop immunomodulators of IL-5-mediated inflammatory responses. We generated a panel of neutralizing anti-IL-5 mAbs which were used to map functional domains on IL-5. In addition, the nucleotide sequences for human IL-5, murine IL-5, rat IL-5, and eight human/murine IL-5 chimeras were engineered and expressed in COS-7 cells. These recombinant cytokines and mAbs were used in TF-1 bioassays to identify five functional epitopes on the tertiary structure of IL-5. Residues responsible for the species-specific activity of human IL-5 were identified with the murine BCL1 bioassay. One set of epitopes cluster around the helix A-loop 2 region, which is predicted to engage the IL-5 receptor beta-chain. The second set of epitopes as well as the species specificity domain cluster around the loop 3-helix D region, which is predicted to engage the IL-5 receptor alpha-chain. Together, these analyses target the A/D helical face of IL-5 as the region involved in receptor engagement.
Inhibition of IFN-stimulated gene expression and IFN induction of cytolytic resistance to natural killer cell lysis correlate with E1A-p300 binding.
Authors: Routes J M , Li H , Bayley S T , Ryan S , Klemm D J
Published: December 2022

Treatment of target cells with IFN induces resistance to NK cell lysis. This process is blocked by expression of E1A gene products in adenovirus (Ad)-infected and Ad-transformed cells. We compared the ability of adenovirus serotype 5 (Ad5) E1A exon 1 mutants to inhibit the induction of cytolytic resistance by IFN and block IFN-stimulated gene expression with their capacity to bind the cellular proteins p105 (retinoblastoma gene product), p107, and p300. E1A mutants that did not express conserved region 3 (CR3; residues 138-184) or contained deletions in the nonconserved regions between residues 26-35 or 86-120, bound p105, p107, and p300 and were not impaired in their capacity to block IFN-stimulated gene expression or IFN's induction of cytolytic resistance. E1A mutants with deletions in CR2 (residues 121-138) could not bind p105 or p107, but blocked IFN-stimulated gene expression and IFN's induction of cytolytic resistance. In contrast, mutants in CR1 or the N-terminal nonconserved region (residues 2, 4-25, and 48-60), which define E1A's binding site for p300, were unable to block either IFN-stimulated gene expression or IFN's induction of cytolytic resistance. We conclude that E1A's capacity to block both IFN-stimulated gene expression and IFN's induction of cytolytic resistance appears to be transduced through a pathway that involves E1A-p300 binding. The capacity of E1A to block IFN's induction of cytolytic resistance is probably secondary to E1A's more general ability to inhibit IFN-stimulated gene expression.