bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation Requirements, Lytic Mechanism, and Development of a Novel Anti-CD8-Resistant CTL Population
Authors: McCarthy Susan A. , Mainwaring Michael S. , Dougall David S. , Lamouse-Smith Esi S.
Published: December 2022

Almost all conventional CD8+ CTL and their CD8+ precursors are inhibited by anti-CD8 mAb. This requirement for CD8 function reflects both an avidity-augmentation role and a signal-transduction role for CD8 on T cells. We have, however, previously identified and partially characterized a novel functional population of CD8+, but anti-CD8-resistant, MHC class I-allospecific CTL. These CTL have unusual activation requirements in that their efficient generation in vitro requires inhibition of the CD8 avidity contribution (but not the CD8 signaling contribution), by anti-CD8 mAb. In this study, we have investigated the relationship of anti-CD8-sensitive and anti-CD8-resistant CTL by several criteria. These CTL populations share the phenotypic markers we have tested to date, they have similar but not identical Ag-specific repertoires, and they both appear to be generated from naive unprimed T cells. However, anti-CD8-sensitive and anti-CD8-resistant CTL populations exhibit important functional differences. They differ in their kinetics of activation in vitro, their dependence on exogenous cytokines, their use of lytic effector mechanisms, and their tissue distribution during ontogeny. Based on these results, we favor the hypothesis that these CTL populations represent distinct T cell lineages or subsets, and not merely different TCR avidity ranges within a single T cell lineage or subset.
Direct Binding of the MHC Class I Molecule H-2Ld to CD8: Interaction with the Amino Terminus of a Mature Cell Surface Protein
Authors: Jelonek Marie T. , Classon Brendan J. , Hudson Peter J. , Margulies David H.
Published: December 2022

MHC class I molecules (MHC-I) display peptides from the intracellular pool at the cell surface for recognition by T lymphocytes bearing αβ TCR. Although the activation of T cells is controlled by the interaction of the TCR with MHC/peptide complexes, the degree and extent of the activation is influenced by the binding in parallel of the CD8 coreceptor with MHC-I. In the course of quantitative evaluation of the binding of purified MHC-I to engineered CD8, we observed that peptide-deficient H-2Ld (MHC-I) molecules bound with moderate affinity (Kd = 7.96 × 10−7 M), but in the presence of H-2Ld-binding peptides, no interaction was observed. Examination of the amino terminal sequences of CD8α and β chains suggested that H-2Ld might bind these protein termini via its peptide binding cleft. Using both competition and real-time direct assays based on surface plasmon resonance, we detected binding of empty H-2Ld to synthetic peptides representing these termini. These results suggest that some MHC molecules are capable of binding the amino termini of intact cell surface proteins through their binding groove and provide alternative explanations for the observed binding of MHC molecules to a variety of cell surface receptors and coreceptors.
Antiviral Activities of Individual Murine IFN-α Subtypes In Vivo: Intramuscular Injection of IFN Expression Constructs Reduces Cytomegalovirus Replication
Authors: Yeow Wen-Shuz , Lawson Cassandra M. , Beilharz Manfred W.
Published: December 2022

The IFN-α cytokines belong to a multigene family. However, the in vivo biological functions of each of the IFN-α subtypes is unknown. Recently, we developed an experimental model in which the tibialis anterior muscles of mice were transfected in situ with naked DNA plasmids encoding an IFN transgene. Here we use this model to investigate the in vivo effect of the expression of three murine IFN-α subtypes (A1, A4, and A9) on murine CMV replication in C57BL/6, BALB/c, and A/J mice. CMV was shown to replicate in the tibialis anterior muscles of mice for at least 6 days and induced an inflammatory infiltrate. However, mice expressing the IFN-α transgenes showed a marked reduction in the peak titers of virus replication, with less severe inflammation in the muscles compared with control mice that were inoculated with blank vectors. Moreover, mice expressing the IFN-α1 transgene had significantly lower CMV titers in the inoculated muscle than mice expressing either the IFN-α4 or the IFN-α9 transgenes. Furthermore, IFN-α/β receptor knockout mice had markedly higher levels of CMV replication in the tibialis anterior muscles than the wild-type parental strain (129/Sv/Ev) following IFN-α1 transgene inoculation, suggesting that the protection observed is due to host cell-mediated IFN signaling. These data provide the first evidence indicating that there are in vivo differences in the antiviral efficacy of the IFN-α subtypes.
Experimental Autoimmune Encephalomyelitis Is Exacerbated in Mice Lacking theNOS2Gene
Authors: Fenyk-Melody Judy E. , Garrison Augusta E. , Brunnert Steven R. , Weidner Jeffrey R. , Shen Frank , Shelton Beverly A. , Mudgett John S.
Published: December 2022

Nitric oxide is believed to be a prominent mediator of inflammation based in part on the correlative expression of the inducible nitric oxide synthase (iNOS) gene in various pathologies. The resulting high output of the highly reactive molecule nitric oxide is then believed to play an important role in the evolving inflammatory response. Studies have shown that iNOS and nitric oxide are present in the tissues of patients with multiple sclerosis (MS). In rodent models of MS, experimental autoimmune encephalomyelitis (EAE), it has been shown that nonspecific NOS inhibitors partially ameliorate the disease. To determine the importance of iNOS in this model of MS, we induced EAE in mice containing a disrupted iNOS (NOS2) gene. Surprisingly, by day 24, the NOS2 knockout mice had a greater incidence of EAE than wild-type control mice (75 vs 12%), and had a higher average severity score (2.42 vs 0.44). These differences appear to result largely from the failure of the disease to remit in NOS2 KO mice. Wild-type mice have a profound ability to reverse EAE (82%) compared with the knockout mice (19%). This result implies that iNOS may in some instances play a protective role in autoimmune-mediated tissue destruction.
Requirement for the Alternative Pathway as Well as C4 and C2 in Complement-Dependent Hemolysis Via the Lectin Pathway
Authors: Suankratay Chusana , Zhang Xiao-Hui , Zhang Yonghong , Lint Thomas F. , Gewurz Henry
Published: December 2022

Mannan-binding lectin (MBL) is a C1q-like molecule opsonic for several micro-organisms. MBL can activate C4, C2, and later acting complement components in the presence of serine proteases similar to but distinct from C1r and C1s via the lectin pathway of complement activation. We report here that mannan-coated MBL-sensitized erythrocytes are lysed via the lectin pathway in human serum-Mg-EGTA. The surprising occurrence of MBL-initiated lysis in the absence of calcium contrasts with the calcium requirement for C1q-initiated activation of C4 and C2. C2 is required, and lysis is significantly enhanced when indicator cells presensitized with C4 and then coated with mannan (EAC4-M) are used. The alternative pathway also is required, since lysis is lost when either factor D or factor B is removed and is restored upon reconstitution with the purified protein. Even though MBL is a C-type lectin, it is retained on mannan-coated erythrocytes in the absence of calcium. This contrasts with the absence of calcium-independent retention on mannan immobilized on polystyrene plates or beads, and helps explain the MBL-initiated hemolysis in Mg-EGTA. These investigations show that the alternative pathway as well as C4 and C2 of the classical pathway are required for complement-dependent hemolysis via the lectin pathway and provide a method for assay of lectin pathway-mediated complement activity in human serum that should be useful in unraveling the molecular interactions of this pathway.
Inhibition of Inducible Nitric Oxide Synthase Prevents LPS-Induced Acute Lung Injury in Dogs
Authors: Numata Mari , Suzuki Shunsuke , Miyazawa Naoki , Miyashita Akira , Nagashima Yoji , Inoue Satoshi , Kaneko Takeshi , Okubo Takao
Published: December 2022

Nitric oxide (NO) is produced by inducible NO synthase (iNOS) after LPS stimulation, and reacts with superoxide to form peroxynitrite. We hypothesize that in LPS-induced lung injury, NO generated by iNOS plays a key role through the formation of peroxynitrite. We developed an acute lung injury dog model by injecting LPS, and examined the effects of selective iNOS inhibitors, aminoguanidine (AG) and S-methylisothiourea sulfate (SMT), on the LPS-induced lung injury. At 24 h after LPS injection, arterial oxygen tension and mean arterial pressure decreased, and shunt ratio and lung wet-to-dry weight ratio increased. On histology, the LPS group had marked neutrophil infiltration and widening of the alveolar septa. On immunohistochemistry, iNOS and nitrotyrosine, a major product of nitration of protein by peroxynitrite, were observed in the interstitium, capillary wall, and neutrophils in the airspaces of the LPS group. Treatments with AG and SMT prevented worsening of gas exchange, hemodynamics, and wet-to-dry weight ratio. On histology, AG and SMT treatments markedly suppressed lung injury, iNOS protein, and nitrotyrosine production. We conclude that NO released by iNOS may play a critical role in the pathogenesis of LPS-induced acute lung injury. This study suggests that iNOS inhibitors may have potential in the treatment of LPS-induced acute respiratory distress syndrome.
MHC Class I-Restricted Lysis of Human Oligodendrocytes by Myelin Basic Protein Peptide-Specific CD8 T Lymphocytes
Authors: Jurewicz Anna , Biddison William E. , Antel Jack P.
Published: December 2022

Multiple sclerosis (MS) is considered to be an autoimmune disease that is directed either at myelin or at its cell of origin, the oligodendrocytes (OL). The inflammatory lesions in the central nervous system contain multiple myelin Ag-restricted and nonrestricted cell populations with the potential to mediate tissue injury. Previous studies indicate that it is possible to generate MHC class I-restricted myelin peptide-specific cytotoxic CD8 T cells, and that human adult OLs express MHC class I molecules in vitro. The purpose of this study was to demonstrate that myelin basic protein peptide-specific CD8 T cells could induce OL injury. We generated CD8 T cell lines from six healthy donors and five MS patients, and all cell lines were HLA-A2 positive. The obtained CD8 cell lines induced lysis of HLA-A2- but not HLA-A3-transfected HMy2.C1R cells in the presence of myelin basic protein peptide 110–118. In the absence of exogenous peptide, the CD8 T cell lines were cytotoxic to HLA-A2 but not to non-HLA-A2 OLs. Cytotoxicity was blocked with anti-MHC class I-blocking Ab. These results support the postulate that autoreactive CD8 cytotoxic T cells can contribute to the tissue injury in MS.
Tissue-Specific Recognition of Mouse CD1 Molecules
Authors: Park Se-Ho , Roark Jessica H. , Bendelac Albert
Published: December 2022

Although there is evidence that some members of the CD1 gene family may present particular types of foreign Ags, such as mycobacterial lipid Ags or synthetic hydrophobic peptides, to αβ T cells, most CD1 isotypes share the unusual property of being recognized by a high frequency of naturally autoreactive αβ T cells. In the case of mouse CD1.1 and its human counterpart CD1d, a significant fraction of the autoreactive T cells express semi-invariant TCRs. CD1.1-specific T cells have a restricted tissue distribution and very promptly secrete a large panel of potent cytokines, including IL-4 and IFN-γ, upon primary activation through their TCR, suggesting that they might regulate some immune responses in these tissues. We show here that their autorecognition of mouse CD1.1 is highly dependent upon the cell type in which CD1.1 is expressed. For example, some of these T cells only respond to CD1.1 expressed by splenic dendritic cells, some respond preferentially to cortical thymocytes, and others respond to splenic B cells. Tissue specificity of CD1.1 recognition is also observed with various cell lines transfected with CD1.1 cDNA. These results show that different CD1.1 self Ags are expressed in different tissues and can be specifically recognized by autoreactive T cells. They suggest that CD1.1 may be naturally associated with a variety of self ligands that overlap only partially in different cell types.
Overexpression of the p80 TNF Receptor Leads to TNF-Dependent Apoptosis, Nuclear Factor-κB Activation, and c-Jun Kinase Activation
Authors: Haridas Valsala , Darnay Bryant G. , Natarajan K. , Heller Renu , Aggarwal Bharat B.
Published: December 2022

Because they have distinct intracellular domains, it has been proposed that the p60 and p80 forms of the TNF receptor mediate different signals. Several signaling proteins have been isolated that associate with either the p60 or the p80 receptor. By using TNF muteins specific to the p60 and p80 receptors, we have previously shown that cytotoxicity and nuclear factor-κB (NF-κB) activation are mediated through the p60 form of the endogenous receptor. What signals are mediated through the p80 receptor is less clear. This study was an effort to answer that question. HeLa cells, which express only p60 receptors, were transfected with p80 receptor cDNA and then examined for apoptosis, NF-κB activation, and c-Jun kinase activation induced by TNF and by p60 or p80 receptor-specific muteins. The p80 mutein, like TNF and the p60 mutein, induced apoptosis and activation of NF-κB and c-Jun kinase in cells overexpressing recombinant p80 receptor but had no effect on cells expressing a high level of endogenous p80 receptor. The apoptosis mediated through the p60 receptor was also potentiated after overexpression of the p80 receptor, suggesting a synergistic relationship between the two receptors. Interestingly, Abs to the p80 receptor blocked apoptosis induced by all ligands but by itself activated NF-κB in the p80-transfected cells. Overall, our results show that the p80 receptor, which lacks the death domain, mediated apoptosis, NF-κB activation, and c-Jun kinase activation, but only when it was overexpressed, whereas endogenous p60 receptor mediated similar signals without overexpression.
In Vivo β-Adrenergic Stimulation Suppresses Natural Killer Activity and Compromises Resistance to Tumor Metastasis in Rats
Authors: Shakhar Guy , Ben-Eliyahu Shamgar
Published: December 2022

The sympathetic nervous system has been implicated in mediating stress-induced alterations in NK cell activity, particularly through stimulation of β-adrenergic receptors. However, because catecholamines induce time-dependent alterations in the distribution of NK cells, the impact of β-adrenergic stimulation on individual NK cell cytotoxicity is not clear, nor are its implications regarding host resistance to metastatic spread. To address these issues, we used the β-adrenergic agonist, metaproterenol (MP), in F344 rats. The number of blood NK cells doubled within 10 min of MP administration and returned to baseline levels within 1 h. By this time, MP suppressed blood NK activity in a dose-dependent manner. Two β-adrenergic antagonists, propranolol, which crosses the blood-brain barrier, and nadolol, which does not, blocked this suppression. Corresponding findings were obtained using an NK-sensitive tumor model, the MADB106. MP caused an up to 10 times increase in the number of tumor cells retained in the lungs 1 day after inoculation and a similar rise in the number of consequent lung metastases detected 3 wk later. These effects were dose dependent and nadolol reversible. NK cells appear to play a central role in mediating the tumor-enhancing effects of MP because their selective depletion nearly abolished this effect. Overall, our findings suggest that independent of the transitory increase in numbers of blood NK cells, in vivo β-adrenergic stimulation suppresses NK activity in the rat. This suppression is induced peripherally and can compromise host resistance to NK-sensitive tumors. Homologies to studies in humans and clinical relevance are discussed.
Asymmetrical Phosphorylation and Function of Immunoreceptor Tyrosine-Based Activation Motif Tyrosines in B Cell Antigen Receptor Signal Transduction
Authors: Pao Lily I. , Famiglietti Sara J. , Cambier John C.
Published: December 2022

CD79a and CD79b function as transducers of B cell antigen receptor signals via a cytoplasmic sequence, termed the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs contain two conserved tyrosines that may become phosphorylated upon receptor aggregation and bind distinct effectors by virtue of the distinct preference of phosphotyrosyl-containing sequences for SH2 domains. To explore the function of CD79a and CD79b ITAM tyrosines, we created membrane molecules composed of MHC class II I-Ak extracellular and transmembrane domains, and CD79a or CD79b cytoplasmic domains in which one or both of the ITAM tyrosines were mutated to phenylalanine. Functional analysis revealed that both ITAM tyrosines are required for ligand-induced Syk phosphorylation. However CD79a-ITAM and CD79b-ITAM tyrosine phosphorylations were asymmetrical, with >80% of phosphorylation occurring on the N-terminal tyrosine (Y-E-G-L). Thus, these findings suggest that following receptor ligation, only a minor proportion of phosphorylated ITAMs are doubly phosphorylated and thus can engage Syk. Only the N-terminal ITAM tyrosine of CD79a was required for ligand-mediated phosphorylation of the receptor and a subset of downstream substrates, including p62, p110, and Shc, and for Ca2+ mobilization. However, responses mediated through CD79b exhibited a greater dependence on the presence of both tyrosines. Neither tyrosine in CD79a or CD79b appeared absolutely essential for Src family kinase phosphorylation. These results indicate that phosphorylations of the tyrosines in CD79a and CD79b occur with very different stoichiometry, and the respective tyrosyl residues have distinct functions.
Stimulation of Murine B Lymphocytes Induces a DNA Exonuclease Whose Activity on Switch-μ DNA Is Specifically Inhibited by Other Germ-Line Switch Region RNAs
Authors: Müller Jürgen R. , Marcu Kenneth B.
Published: December 2022

The Ig heavy chain class switch in B lymphocytes involves a unique genetic recombination that fuses specific regions within the Ig locus and deletes intervening sequences. Here we describe a novel exonuclease activity in nuclear lysates of B cells in an in vitro assay. This activity was induced in B lymphocytes after treatment with either LPSs or CD40 ligand/anti-δ-dextran, both of which induce switch recombination, and considerably less activity was detected in untreated or anti-δ-dextran-treated B cells, Con A-stimulated spleen cells, liver cells, or a number of cell lines. The exonuclease activity was dependent on divalent cations, and both 3′ and 5′ labels were efficiently removed from DNA substrates. The presence of RNase A, but not RNase H, inhibited exonucleolytic digestion, suggesting that a ribonucleoprotein is responsible for the exonucleolysis. The DNA digestion appears to be nonspecific, since DNA substrates with either switch-μ or unrelated sequence were hydrolyzed with comparable efficiency. Germ-line switch region transcripts (Igγ1, Igγ3, and Igα) strongly inhibited the exonucleolysis of switch-μ DNA but not that of unrelated control DNA, while switch antisense RNA or tRNA were much less effective inhibitors.
Identification of a Domain in Human Factor H and Factor H-Like Protein-1 Required for the Interaction with Streptococcal M Proteins
Authors: Kotarsky Heike , Hellwage Jens , Johnsson Eskil , Skerka Christine , Svensson Henrik G. , Lindahl Gunnar , Sjöbring Ulf , Zipfel Peter F.
Published: December 2022

The plasma protein factor H (FH) inhibits the alternative pathway of complement activation. Previous work has shown that FH binds to group A streptococci and that the interaction does not interfere with the complement-inhibitory capacity of FH. In this work, we report a molecular analysis of this interaction. In absorption experiments with human plasma, M protein-expressing group A streptococci bound both FH and FH-like protein-1 (FHL-1), an active 42-kDa splice product of the FH-gene transcript comprising the first 7 of its 20 short consensus repeat (SCR) domains. rFHL-1 also bound to M protein-expressing streptococci, but rFH fragments containing SCR 1–5 or SCR 1–6 did not. rFHL-1 bound to purified M5 protein with an affinity that was higher than the value calculated for the interaction between FH and M5 protein. The binding of radiolabeled rFHL-1 to immobilized M5 was blocked completely by unlabeled rFHL-1, but was inhibited only partially by SCR 1–6, emphasizing the importance of SCR 7 for the interaction. In experiments with the FH-related proteins FHR-3 and FHR-4, only the former bound to M protein-expressing streptococci, again pointing to an involvement of SCR 7, since FHR-3, but not FHR-4, contains a domain that is similar to SCR 7. Finally, the interaction between rFHL-1 and purified M5 protein was inhibited by heparin, which binds FH via SCR 7. Together, these data indicate that the interaction between streptococcal M proteins and FH or FHL-1 requires SCR 7.
Purification of L-Selectinlow Cells Promotes the Generation of Highly Potent CD4 Antitumor Effector T Lymphocytes
Authors: Kagamu Hiroshi , Shu Suyu
Published: December 2022

Successful adoptive immunotherapy of cancer requires the identification, isolation, and expansion of tumor-specific immune effector cells. A reliable source of tumor-immune lymphocytes is lymph nodes draining a growing tumor. After in vitro stimulation with anti-CD3 and expansion in IL-2, these cells are capable of mediating the regression of established tumors. In the absence of further Ag stimulation, we recently found that the down-regulation of the homing molecule L-selectin could serve as a surrogate marker for isolation of specific tumor-sensitized T cells. The L-selectinlow (L-selectin−) T cells proliferated more vigorously than unfractionated or L-selectinhigh cells. In adoptive immunotherapy of established intracranial MCA 205 tumors, L-selectin− cells displayed at least 30-fold greater therapeutic efficacy than unfractionated cells. L-selectinhigh cells did not demonstrate any antitumor effects. Activated L-selectin− cells secreted a number of cytokines, including IFN-γ, IL-2, IL-4, and IL-10, specifically when stimulated with cognate tumor cells. Further analysis revealed that CD4 T cells alone mediated tumor regression and secreted cytokines. Our results thus demonstrate that the purification of L-selectin− cells led to the generation of CD4 immune effector cells with unusually high therapeutic efficacy against chemically induced tumors. The lack of cytotoxicity and the ability to secrete cytokines suggest that these effector CD4 cells mediate antitumor effects through an indirect mechanism similar to the delayed hypersensitivity reaction.
Regulation of Cell Growth by IL-2: Role of STAT5 in Protection from Apoptosis But Not in Cell Cycle Progression
Authors: Zamorano José , Wang Helen Y. , Wang Rouxiang , Shi Yufang , Longmore Gregory D. , Keegan Achsah D.
Published: December 2022

Cytokines play an essential role in the regulation of lymphocyte survival and growth. We have analyzed the pathways activated by IL-2 that lead to protection from apoptosis and cell proliferation. IL-2 can act as a long-term growth factor in 32D cells expressing the wild-type human (hu)IL-2Rβ. By contrast, cells expressing a truncated form of the huIL-2Rβ, which is able to induce Bcl-2 and c-myc expression but not STAT5 activation, were not protected from apoptosis by IL-2; consequently, they could not be grown long term in the presence of IL-2. However, IL-2 promoted cell cycle progression in cells bearing the truncated huIL-2Rβ with percentages of viable cells in the G0/G1, S, and G2/M phases similar to cells expressing the wild-type huIL-2Rβ. Transplantation of a region from the erythropoietin receptor, which contains a docking site for STAT5 (Y343) to the truncated huIL-2Rβ, restored the ability of IL-2 to signal both activation of STAT5 and protection from apoptosis. By contrast, transplantation of a region from the huIL-4Rα containing STAT6 docking sites did not confer protection from apoptosis. These results indicate that the IL-2-induced cell cycle progression can be clearly distinguished from protection from apoptosis and that STAT5 participates in the regulation of apoptosis.
IL-17 Stimulates the Production and Expression of Proinflammatory Cytokines, IL-β and TNF-α, by Human Macrophages
Authors: Jovanovic Dragan V. , Di Battista John A. , Martel-Pelletier Johanne , Jolicoeur François C. , He Yulan , Zhang Mengkun , Mineau François , Pelletier Jean-Pierre
Published: December 2022

IL-17 is a newly described, T cell-derived cytokine with ill-defined physiologic properties. As such, we examined the release of proinflammatory mediators by human macrophages in response to recombinant human (rh) IL-17. IL-1β and TNF-α expression and synthesis were up-regulated by rhIL-17 in a dose (ED50 was 50 ± 9 ng/ml)- and time-dependent fashion, with cytokine accumulation reaching a zenith after 9 h. Release of IL-6, PGE2, IL-10, IL-12, IL-1R antagonist, and stromelysin was also stimulated by rhIL-17. IL-1β and TNF-α mRNA expression levels were controlled by rhIL-17 in a complex manner with an initial 30-min inhibitory phase, and then up-regulation beginning at 1 h and reaching a plateau at about 3 h. The latter expression pattern closely mirrored the nuclear accumulation of the transcription factor nuclear factor-κB. cAMP mimetics isobutyl-1-methylxanthine (IBMX), forskolin, PGE2, and cholera toxin reversed rhIL-17-induced release of TNF-α, but had no consistent effect on induced IL-1β synthesis. Induced release of TNF-α was also inhibited by serine/threonine protein kinase inhibitors KT-5720 (protein kinase A) and Calphostin C (protein kinase C), mitogen-activated protein kinase kinase inhibitor PD098059, and a nonspecific tyrosine kinase inhibitor, genistein. Calphostin C alone abrogated the rhIL-17-induced release of IL-1β. The antiinflammatory cytokines IL-4 (p < 0.01) and IL-10 (p < 0.02) completely reversed rhIL-17-stimulated IL-1β release, while IL-13 and TGF-β2 were partially effective (59 and 43% diminution, respectively). IL-10 exerted a significant suppressive effect on IL-17-induced TNF-α release (99%, p < 0.02), while the inhibitory effects of IL-4, IL-13, and TGF-β2 on TNF-α secretion were partial (48, 10, and 23%, respectively). The data suggest a pivotal role for IL-17 in initiating and/or sustaining an inflammatory response.
Involvement of NK1+ T Cells and Their IFN-γ Production in the Generalized Shwartzman Reaction
Authors: Ogasawara Kouetsu , Takeda Kazuyoshi , Hashimoto Wataru , Satoh Masayuki , Okuyama Ryuhei , Yanai Nobuaki , Obinata Masuo , Kumagai Katsuo , Takada Haruhiko , Hiraide Hoshio , Seki Shuhji
Published: December 2022

IL-12 (or LPS) priming and subsequent challenge by LPS produces the generalized Shwartzman reaction. IFN-γ induced by IL-12 is a crucial cytokine in the priming phase. In vivo depletion of both NK cells and NK1+ αβ T cells of mice by anti-NK1.1 Ab greatly reduced the elevation of serum IFN-γ induced by IL-12 and significantly reduced mortality after subsequent injection of LPS, whereas depletion of NK cells alone by anti-asialo GM1 Ab only partially decreased serum IFN-γ, and lethality was not changed. Cell sorting and culture experiments confirmed that liver NK1+ αβ T cells of IL-12-injected mice produced greater amounts of IFN-γ than did liver NK cells. MHC class I-deficient mice of C57BL/6 background, which lack a majority of NK1+ αβ T cells, produced low amounts of IFN-γ by IL-12; no mortality was observed after the LPS challenge. However, production of TNF-α in the second phase (after LPS challenge) was not inhibited by depletion of NK cells alone or both subsets. IL-12 and subsequent LPS challenge activated NK1+ αβ T cells in the liver and induced strong cytotoxicity of these cells not only against tumor cells (including Fas-negative tumors) but also against a syngeneic hepatocyte cell line. Our findings show that IFN-γ produced by NK1+ αβ T cells is essential for the IL-12 priming of the Shwartzman reaction, and the autoreactivity of NK1+ αβ T cells in the liver is involved in the hepatic disorders that are sometimes caused by IL-12, LPS, or the generalized Shwartzman reaction.
IgE Versus IgG4 Production Can Be Differentially Regulated by IL-10
Authors: Jeannin Pascale , Lecoanet Sybille , Delneste Yves , Gauchat Jean-François , Bonnefoy Jean-Yves
Published: December 2022

Allergen-specific IgE plays a key role in the physiopathology of allergic disorders. This IgE response is usually accompanied by a production of IgG4. Indirect evidence suggests that IgG4 may not be a sensitizing Ab but, in contrast, could be protective. As such, it may be of potential therapeutic interest to selectively modulate IgE vs IgG4 production. To date, IgE and IgG4 switching seems to be controlled by common mechanisms. We report here that IL-10 has a differential effect on IgE vs IgG4 production by PBMC. IL-10 decreases ε transcript expression and IgE production induced by IL-4 when added during the first 3 days of in vitro culture, suggesting that IL-10 decreases IL-4-induced IgE switching. In contrast, if added later on B cells that are already IgE switched, IL-10 potentiates IgE production. Interestingly, whatever the time of addition, IL-10 augments IL-4-induced γ4 transcript expression and IgG4 production, with a maximal effect when added during the first 3 days. As IL-10 is not a switch factor for IgG4, it is likely that IL-10 enhances IgG4 production by potentiating IL-4-induced IgG4 switching. However, IL-10 may also act by enhancing the growth and/or differentiation of cells that are already IgG4 committed. Finally, CD40 ligation reverses the early down-regulating effect of IL-10 on IgE production. These results are the first evidence of a molecule that differentially regulates IgE vs IgG4 production, thereby suggesting the existence of a pathway(s) selectively controlling their production.
Dendritic Cells and Macrophages Are the First and Major Producers of TNF-α in Pancreatic Islets in the Nonobese Diabetic Mouse
Authors: Dahlén Eva , Dawe Kim , Ohlsson Lennart , Hedlund Gunnar
Published: December 2022

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune insulin-dependent diabetes mellitus (IDDM) and serves as an animal model for human type I diabetes. TNF-α is known to be produced by islet-infiltrating mononuclear cells during insulitis and subsequent β cell destruction and has been implicated in the pathogenesis of IDDM. Previously, T cells have been suggested as the main source of TNF-α in the islet infiltrate. However, on immunohistochemical analysis of TNF-α expression in islets, we are able to show that the staining pattern of TNF-α resembles that of dendritic cells (DC) and macrophages (Mφ) rather than T cells and that TNF-α is expressed in islets at the very early stages of insulitis when no T cells are detected. On double staining for TNF-α and cell surface markers, we can demonstrate that TNF-α staining clearly correlates with DC and Mφ, whereas there is a poor correlation with T cells. This feature was observed at both early and late stages of insulitis. TNF-α expression was also seen in NOD-SCID islets, in addition to a peri-islet infiltration consisting of DC and Mφ, indicating that T cells are not required for the early DC and Mφ infiltration and TNF-α expression in islets. In conclusion, our results show that DC and Mφ are the major, early source of TNF-α in the NOD islet infiltrate and that TNF-α can be expressed independently of T cells, indicating that the early DC and Mφ infiltration and expression of TNF-α are crucial in initiation of diabetes.
Specific Suppression of Human CD4+ Th Cell Responses to Pig MHC Antigens by CD8+CD28− Regulatory T Cells
Authors: Ciubotariu Rodica , Colovai Adriana I. , Pennesi Giuseppina , Liu Zhouru , Smith Douglas , Berlocco Pasquale , Cortesini Raffaello , Suciu-Foca Nicole
Published: December 2022

Evidence that T cells can down-regulate the immune response by producing or consuming certain cytokines or by lysing APCs or Th cells has been provided in various systems. However, the generation and characterization of suppressor T cell lines have met with limited success. Here we show that xenospecific suppressor T cells can be generated by in vitro stimulation of human T cells with pig APCs. Similar to allospecific suppressors, these xenospecific suppressor T cells carry the CD8+CD28− phenotype and react to MHC class I Ags expressed by the APCs used for priming. TCR spectratyping of T suppressor cells showed oligoclonal usage of TCR-Vβ families, indicating that xenostimulation of CD8+CD28− T cells results in Ag-driven selection of a limited Vβ repertoire. Xenospecific T suppressor cells prevent the up-regulation of CD154 molecules on the membrane of Th cells, inhibiting their ability to react against the immunizing MHC class II xenoantigens. The mechanism of this suppression, therefore, appears to be blockade of CD154/CD40 interaction required for efficient costimulation of activated T cells.
Experimental MurineTrypanosoma congolenseInfections. I. Administration of Anti-IFN-γ Antibodies Alters Trypanosome-Susceptible Mice to a Resistant-Like Phenotype
Authors: Uzonna Jude E. , Kaushik Radhey S. , Gordon John R. , Tabel Henry
Published: December 2022

The mechanisms regulating resistance or susceptibility to African trypanosomes have been enigmatic. In this study, we assessed the production of several cytokines (IL-4, IFN-γ, and TNF-α) in vivo and in vitro using genetically susceptible (BALB/c) or resistant (C57BL/6) mice infected with cloned Trypanosoma congolense and the role of these cytokines in pathogenesis of this infection. Plasma of infected BALB/c mice contained higher levels of IL-4 and IFN-γ than the plasma of infected C57BL/6 mice. Conversely, plasma TNF-α levels were elevated significantly in the resistant mice relative to the susceptible ones. Splenic IFN-γ mRNA appeared earlier and were maintained at higher levels in infected BALB/c than in C57BL/6 mice. Both spontaneous and Con A-induced secretions of IL-4 and IFN-γ by splenocytes from infected BALB/c mice were significantly higher than those from their C57BL/6 counterparts. Con A-induced proliferation of splenocytes from infected BALB/c mice was progressively suppressed. Nitric oxide was not involved in this suppression, but the suppression was positively correlated with IFN-γ secretion. Addition of neutralizing Abs to IFN-γ to cultures of Con A-stimulated spleen cells from infected BALB/c mice effectively reversed this suppression. Furthermore, administration of anti-IFN-γ Abs to BALB/c mice early during infection dramatically shifted the phenotype of these susceptible mice to a more resistant-like phenotype, as expressed by a low and undulating parasitemia and a >300% increase in survival period. These results strongly suggest that the enhanced induction and secretion of IFN-γ during T. congolense infections contribute to the relative susceptibility of BALB/c mice to the disease.
Suppressive Effect on Theiler’s Murine Encephalomyelitis Virus-Induced Demyelinating Disease by the Administration of Anti-IL-12 Antibody
Authors: Inoue Atsushi , Koh Chang-Sung , Yamazaki Masashi , Yahikozawa Hiroyuki , Ichikawa Motoki , Yagita Hideo , Kim Byung S.
Published: December 2022

We examined the role of IL-12, a cytokine critical to the evolution of cellular responses, in the development of Theiler’s murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to IL-12, especially during the effector phase, resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of inflammatory and Th1-derived cytokines such as TNF-α and IFN-γ in the spleen cells was decreased, and that of Th2-derived cytokines such as IL-4 and IL-10 was increased. The delayed type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. These data suggest that IL-12 is critically involved in the pathogenesis of TMEV-IDD and that Abs to IL-12 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.
Induction of Keratinocyte Proliferation and Lymphocytic Infiltration by In Vivo Introduction of the IL-6 Gene into Keratinocytes and Possibility of Keratinocyte Gene Therapy for Inflammatory Skin Diseases Using IL-6 Mutant Genes
Authors: Sawamura Daisuke , Meng Xianmin , Ina Shinsuke , Sato Masanori , Tamai Katsuto , Hanada Katsumi , Hashimoto Isao
Published: December 2022

To understand biological function of IL-6 in the skin in vivo, we constructed a vector that strongly expressed human IL-6 in keratinocytes and introduced it into rat keratinocytes in vivo by the naked DNA method. The overexpression of IL-6 induced macroscopic erythema and histologically evident keratinocyte proliferation and lymphocytic infiltration in the treated area of rat skin. Since previous studies using IL-6 transgenic mice have not shown skin inflammation of these mice, our result provides the first evidence that IL-6 is related to the pathogenesis of inflammatory skin diseases. ELISA suggested that a certain degree of transgenic IL-6 expression in keratinocytes was required for inducing skin inflammation. Cytokine profile in rat keratinocytes after the gene introduction was examined by reverse transcriptase-PCR assay and revealed that gene expression of rat IL-1α and TNF-α showed no marked change until 24 h, whereas that of rat IL-6 and TGF-α increased with time. We then introduced and expressed the IL-6 mutant genes, which were designed to behave as IL-6Rα antagonists, and found that their ability to induce erythema was lower than that of the wild-type gene. Furthermore, preintroduction of some mutant genes delayed the erythema induced by postintroduction of the wild-type IL-6 gene, suggesting that the mutant forms of IL-6 prevent wild-type IL-6 from binding to IL-6Rα. This result indicates that keratinocyte gene therapy may be possible for inflammatory skin diseases using IL-6 mutant genes.
Endogenous Superallergen Protein Fv Induces IL-4 Secretion from Human FcεRI+ Cells Through Interaction with the VH3 Region of IgE
Authors: Patella Vincenzo , Giuliano Ada , Bouvet Jean-Pierre , Marone Gianni
Published: December 2022

We investigated the mechanism whereby protein Fv (pFv), a human sialoprotein found in normal liver and largely released in the intestinal tract in patients with viral hepatitis, induces mediator release from basophils and mast cells and evaluated whether it also induces IL-4 synthesis and secretion in basophils. pFv is a potent stimulus for histamine and IL-4 release from purified basophils. Histamine and IL-4 secretion from basophils activated by pFv was significantly correlated (rs = 0.70; p < 0.001). There was also a correlation (rs = 0.58; p < 0.01) between the maximum pFv- and anti-IgE-induced IL-4 release from basophils. The average t1/2 for pFv-induced histamine release was lower (3.5 ± 1.5 min) than for IL-4 release (79.5 ± 8.5 min; p < 0.01). IL-4 mRNA, constitutively present in basophils, was increased after stimulation by pFv and was inhibited by cyclosporin A and tacrolimus. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released IL-4 in response to pFv and anti-IgE. The response to an mAb cross-linking the α-chain of FcεRI was unaffected by this treatment. Three human VH3+ monoclonal IgM concentration-dependently inhibited pFv-induced secretion of IL-4 and histamine from basophils and of histamine from human lung mast cells. In contrast, VH6+ monoclonal IgM did not inhibit the release of IL-4 and histamine induced by pFv. These results indicate that pFv, which acts as an endogenous superallergen, interacts with the VH3 domain of IgE to induce the synthesis and release of IL-4 from human FcεRI+ cells.
IL-7 Deficiency Prevents Development of a Non-T Cell Non-B Cell-Mediated Colitis
Authors: von Freeden-Jeffry Ursula , Davidson Natalie , Wiler Rhonda , Fort Madeline , Burdach Stefan , Murray Richard
Published: December 2022

IL-7 is a stromal cell-derived cytokine with a well-established physiologic role in lymphocyte biology. This report describes an unexpected role for IL-7 in the development of colitis in a T and B cell-deficient environment. Recombination-activating gene-2 (RAG-2)-deficient mice (RAG-2−/−) were exposed to and subsequently maintained a horizontally transmitted microbial flora that included Helicobacter hepaticus. These animals mounted a strong myeloid cell response and developed both systemic and local signs of a severe colitis. A striking infiltration of F4/80 and MHC class II-positive cells was seen in the colon and cecum of animals undergoing the disease. Mice mutant for both IL-7 and RAG-2 (IL-7/RAG-2−/−) that were colonized by the same flora showed no signs of myeloid responses or colitis, indicating that IL-7 plays a critical role in exacerbating a non-T cell/non-B cell-mediated chronic inflammatory response. Recombinant IL-10 protein therapy was able to prevent the occurrence of colitis in susceptible mice, suggesting a pivotal role for macrophages. The implications of a role for IL-7 in this disease model with respect to human inflammatory bowel disease are discussed.
Caspase-Mediated Inhibition of Human Cytosolic Phospholipase A2 During Apoptosis
Authors: Adam-Klages Sabine , Schwandner Ralf , Lüschen Silke , Ussat Sandra , Kreder Dirk , Krönke Martin
Published: December 2022

Activation of cytosolic phospholipase A2 (cPLA2) is an essential step in the initiation of the cascade of enzymatic reactions leading to the generation of proinflammatory lipid mediators. Hence, the regulation of cPLA2 is a key event in the induction of inflammatory responses. cPLA2 is activated, in part, by apoptotic stimuli such as TNF or Fas ligand. Apoptosis, however, does not provoke an inflammatory response. Here, we demonstrate that cPLA2 is cleaved by caspase-3 and/or a related caspase in HeLa cells undergoing apoptosis. Mutation of a predicted caspase-3 cleavage site abolishes cPLA2 processing both in vitro and in intact cells. The 70-kDa cleavage product of cPLA2 itself has no catalytic function, while inhibition of cleavage results in an increased enzymatic activity. Additionally, overexpression of the 70-kDa fragment appears to produce a dominant negative effect on endogenous cPLA2 activity. In HeLa cells, cPLA2 activity was dispensable for the course of apoptosis. We cannot rule out, however, that cPLA2 activity is involved in the induction of apoptosis in other cell types. Taken together, our results suggest that the enzymatic activity of cPLA2 is specifically inhibited by caspase-mediated cleavage during apoptosis. The inactivation of cPLA2 represents a previously unrecognized mechanism for avoiding an inflammatory reaction against apoptotic cells.
A Novel Model of Inflammatory Bowel Disease: Mice Deficient for the Multiple Drug Resistance Gene,mdr1a, Spontaneously Develop Colitis
Authors: Panwala Chetan M. , Jones Jon C. , Viney Joanne L.
Published: December 2022

The murine multiple drug resistance (mdr) gene, mdr1a, encodes a 170-kDa transmembrane protein that is expressed in many tissues including intestinal epithelial cells, a subset of lymphoid cells and hematopoietic cells. We report that mdr1a knockout (mdr1a−/−) mice are susceptible to developing a severe, spontaneous intestinal inflammation when maintained under specific pathogen-free animal facility conditions. The intestinal inflammation seen in mdr1a−/− mice has a pathology similar to that of human inflammatory bowel disease (IBD) and is defined by dysregulated epithelial cell growth and leukocytic infiltration into the lamina propria of the large intestine. Treating mdr1a−/− mice with oral antibiotics can both prevent the development of disease and resolve active inflammation. Lymphoid cells isolated from mice with active colitis are functionally reactive to intestinal bacterial Ags, providing evidence that there is enhanced immunologic responsiveness to the normal bacterial flora during IBD. This study is the first description of spontaneous colitis in a gene knockout mouse with an apparently intact immune system. This novel model of spontaneous colitis may provide new insight into the pathogenesis of IBD, the nature of dysregulated immune reactivity to intestinal bacterial Ags, and the potential functional role of mdr genes expressed in the cells and tissues of the colonic microenvironment.
Acute Hepatotoxicity ofPseudomonas aeruginosaExotoxin A in Mice Depends on T Cells and TNF
Authors: Schümann Jens , Angermüller Sabine , Bang Renate , Lohoff Michael , Tiegs Gisa
Published: December 2022

The most potent virulence factor of Pseudomonas aeruginosa, its exotoxin A (PEA), inhibits protein synthesis, especially in the liver, and is a weak T cell mitogen. This study was performed to correlate hepatotoxic and possible immunostimulatory features of PEA in vivo. Injection of PEA to mice caused hepatocyte apoptosis, an increase in plasma transaminase activities, and the release of TNF, IFN-γ, IL-2, and IL-6 into the circulation. Most strikingly, liver damage depended on T cells. Athymic nude mice or mice depleted of T cells by anti-Thy1.2 mAb pretreatment failed to develop acute hepatic failure, and survival was significantly prolonged following T cell depletion. Neutralization of TNF or lack of TNF receptors prevented liver injury. In the liver, TNF was produced by Kupffer cells before hepatocellular death occurred. After T cell depletion, Kupffer cells failed to produce TNF. Transaminase release was significantly reduced in perforin knockout mice, and it was even elevated in lpr/lpr mice. These results demonstrate that PEA induces liver damage not only by protein synthesis inhibition but also by TNF- and perforin-dependent, Fas-independent, apoptotic signals.
Direct Binding and Functional Transfer of NK Cell Inhibitory Receptors Reveal Novel Patterns of HLA-C Allotype Recognition
Authors: Winter Christine C. , Gumperz Jenny E. , Parham Peter , Long Eric O. , Wagtmann Nicolai
Published: December 2022

Cytotoxicity of human NK cells is under negative control of killer cell Ig-like receptors (KIR) specific for HLA class I. To determine the specificity of five KIR containing two Ig domains (KIR2D), direct binding of soluble recombinant KIR2D to a panel of HLA class I transfectants was assayed. One soluble KIR2D, derived from an inhibitory receptor with a long cytoplasmic tail (KIR2DL1), bound to HLA-C allotypes containing asparagine 77 and lysine 80 in the heavy chain, as expected, since these allotypes inhibit lysis by NK cells expressing KIR2DL1. Surprisingly, another KIR2D (KIR2DL2), which inhibits NK lysis of cells expressing HLA-C molecules with serine 77 and asparagine 80, bound to HLA-C allotypes carrying either amino acid motif. Expression of the KIR2DL receptors in NK cells using recombinant vaccinia viruses confirmed these patterns of recognition, and identified KIR2DL3 as another KIR reacting with both groups of HLA-C allotypes. Mutagenesis of amino acid 44 in KIR2DL1 and KIR2DL2 suggested this residue controls the affinity of KIR for the 77/80 motif of HLA-C molecules. Two other soluble KIR2D, derived from noninhibitory receptors with short cytoplasmic tails (KIR2DS), did not bind to any of the HLA class I allotypes tested. One of these receptors (KIR2DS2) is closely related in sequence to KIR2DL2. Substitution of tyrosine 45 with the phenylalanine conserved in other KIR was sufficient to permit specific binding of KIR2DS2 to HLA-C. These results show that KIR2DL receptors are specific for HLA-C, but that recognition of HLA-C allotypes appears more permissive than indicated by previous functional experiments.