bluuebunny/crossref_metadata_2025 huggingface dataset:
Protection from Collagen-Induced Arthritis in Granulocyte-Macrophage Colony-Stimulating Factor-Deficient Mice
Authors: Campbell Ian K. , Rich Melissa J. , Bischof Robert J. , Dunn Ashley R. , Grail Dianne , Hamilton John A.
Published: December 2022

The involvement of granulocyte-macrophage CSF (GM-CSF) in collagen-induced arthritis (CIA) was examined using GM-CSF-deficient mice. Although CIA is generally considered to be restricted to mice of the H-2q or H-2r haplotypes, we examined the role of GM-CSF in the CIA model using GM-CSF-deficient (−/−) and wild-type (+/+) mice on a C57BL/6 (H-2b) background. Mice were immunized by intradermal injection at the base of the tail with chick type II collagen followed by a repeat injection 21 days later. We found, based on both clinical and histologic assessments, that wild-type mice on this background developed severe CIA, while the GM-CSF-deficient mice had virtually no disease. Mice that were heterozygous for the GM-CSF gene (+/−) collectively displayed an intermediate response between those of the GM-CSF+/+ and GM-CSF−/− groups, suggesting a gene dosage effect. GM-CSF+/+ and GM-CSF+/− mice exhibited CIA responses ranging from mild (single digits) to severe swelling of all four paws, while in the few GM-CSF−/− mice that developed CIA the disease was confined to single digits. Despite the putative role of GM-CSF in dendritic cell development, GM-CSF-deficient mice exhibited both humoral and cellular (delayed-type hypersensitivity) responses to type II collagen; however, the cellular response was significantly reduced in the GM-CSF-deficient mice compared with the wild-type controls. These findings suggest that GM-CSF is required for CIA development in mice and support the idea that GM-CSF is a key cytokine in inflammatory joint disease.
A Complex Element Regulates IFN-γ-Stimulated Monocyte Chemoattractant Protein-1 Gene Transcription
Authors: Valente Anthony J. , Xie Jing-feng , Abramova Margaret A. , Wenzel Ulrich O. , Abboud Hanna E. , Graves Dana T.
Published: December 2022

Monocyte chemoattractant protein-1 (MCP-1) is induced in chronic osseous inflammation, and is temporally and spatially correlated with monocyte recruitment. We investigated the mechanism of MCP-1 regulation in a human osteoblastic cell line in response to IFN-γ, a potent mediator of the immune inflammatory response. Nuclear run-on and stability studies demonstrated that IFN-γ stimulated MCP-1 transcription and did not enhance mRNA stabilization. Using MCP-1 promoter/reporter gene constructs, we determined that IFN-γ-enhanced MCP-1 transcription is regulated by a 29-bp element located at −227 relative to the ATG start codon. This element contains a 13-bp CT-rich sequence (GCTTCCCTTTCCT) adjacent to a IFN-γ activation site (GAS). Since deletion of the CT sequence enhanced both the magnitude and duration of IFN-γ-stimulated, GAS-mediated transcription, we have termed it the IFN response-inhibitory sequence (IRIS). The combined IRIS/GAS sequence is highly conserved in mouse, rat, and bovine MCP-1 genes. In gel-shift assays, nuclear extracts from IFN-γ-stimulated osteoblastic cells formed two specific inducible bands with labeled IRIS/GAS DNA. Both bands were supershifted by anti-STAT1 Abs, but not by Abs to STAT2, p48(ISGF-3γ), IFN-regulatory factor-1, or IFN-regulatory factor-2. Formation of one of the bands required the presence of the IRIS moiety. IRIS/GAS DNA also formed a number of specific complexes with constitutively expressed factors, none of which were affected by the above Abs. These studies establish a mechanism for IFN-γ-stimulated MCP-1 expression and identify a complex element that regulates MCP-1 gene transcription.
Evidence for IL-12-Activated Ca2+ and Tyrosine Signaling Pathways in Human Neutrophils
Authors: Collison Kate , Saleh Soad , Parhar Ranjit , Meyer Brian , Kwaasi Aaron , Al-Hussein Khalid , Al-Sedairy Sultan , Al-Mohanna Futwan
Published: December 2022

The cytokine IL-12 is proposed to play a bridging role between innate and adaptive immunity. Here we demonstrate that IL-12 binds specifically to human neutrophils. This binding leads to a transient increase in 1) intracellular free calcium due to its release from membrane-enclosed stores and its influx from extracellular medium, 2) actin polymerization, and 3) tyrosine phosphorylation. IL-12 treatment also leads to a concentration-dependent increase in reactive oxygen metabolite production. The effect of IL-12 is blocked by neutralizing Abs to IL-12. Inhibition of either calcium transient or tyrosine phosphorylation causes inhibition of reactive oxygen metabolite production. However, inhibition of actin polymerization enhances IL-12-induced oxidase activation. Our data suggest 1) a direct role for IL-12 in the activation of human neutrophils, and 2) a calcium-dependent signaling pathway for IL-12.
Immune Invasion of the Central Nervous System Parenchyma and Experimental Allergic Encephalomyelitis, But Not Leukocyte Extravasation from Blood, Are Prevented in Macrophage-Depleted Mice
Authors: Tran Elise H. , Hoekstra Karin , van Rooijen Nico , Dijkstra Christine D. , Owens Trevor
Published: December 2022

Organ-specific autoimmune diseases are characterized by infiltrates, including T lymphocytes and activated macrophages. Macrophages and secondarily activated tissue resident counterparts can both present Ag to and contribute to cytokine secretion by T lymphocytes. We have previously shown a crucial role of peripheral macrophages in experimental allergic encephalomyelitis (EAE), a Th1-mediated demyelinating disease that serves as a an animal model for multiple sclerosis (MS), by their depletion using mannosylated liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP). Here we describe studies to investigate the mechanisms by which macrophages contribute to the lesion formation in EAE, by studying the effect of Cl2MDP-containing mannosylated liposomes (Cl2MDP-mnL) on adoptively transferred EAE in SJL/J mice. Adoptive transfer of EAE with myelin basic protein-reactive CD4+ T cells to SJL/J mice was abrogated by Cl2MDP-mnL treatment. CD4+ T cell and MHC II+ B220+ B cell extravasation from blood vessels and Th1 cytokine production were not inhibited. However, invasion of the central nervous system intraparenchymal tissues by lymphocytes, F4/80+, Mac-1+, and MOMA-1+ macrophages was almost completely blocked after treatment with Cl2MDP-mnL. Furthermore, in Cl2MDP-mnL-treated mice, the myelin sheaths appeared completely normal, whereas, in the control groups, marked demyelination occurred. Production of TNF-α and inducible nitric oxide synthase, both associated with macrophage/microglial activation, was inhibited. This intervention reveals a role for macrophages in regulating the invasion of autoreactive T cells and secondary glial recruitment that ordinarily lead to demyelinating pathology in EAE and multiple sclerosis.
Role of κII-A2 Light Chain CDR-3 Junctional Residues in Human Antibody Binding to theHaemophilus influenzaeType b Polysaccharide
Authors: Lucas Alexander H. , Moulton Karen D. , Reason Donald C.
Published: December 2022

Abs using the κII-A2 V gene segment predominate the human Ab repertoire to the Haemophilus influenzae b (Hib) polysaccharide (PS). All A2 anti-Hib PS Abs sequenced to date possess a 10-amino acid L chain complementarity-determining region-3 (CDR-3) having an insertional arginine (Arg) at position 95a, the V-J junction. These findings suggest an essential requirement for this conserved Arg residue in determining Hib PS-binding affinity. We examined this requirement by performing chain recombination experiments in which a series of A2 L chains, differing at position 95a, were combined individually with an Fd region known to generate a Hib PS-combining site when paired with an A2-Arg(95a)-Jκ1 V region. Hib PS binding of the recombinant Fabs was evaluated quantitatively using a radioantigen-binding assay. Fabs having A2 L chains with either Arg or lysine in position 95a in combination with Jκ1 gave equivalent and strongest binding to Hib PS. Fabs having A2-Jκ1 L chains with either tyrosine, glycine, alanine, leucine, serine, or threonine in position 95a, or having an A2-Arg(95a)-Jκ3 L chain, gave intermediate binding. Fabs having A2-Jκ1 L chains with glutamate or aspartate at 95a or with no junctional residue showed little or no Hib PS binding. These results demonstrate the importance of L chain junctional residue, as well as Jκ usage and CDR-3 length, in determining Hib PS-binding affinity. Contrary to expectation, an Arg junctional residue is not essential for generating either high or intermediate affinity-binding sites.
Cutting Edge: A Novel Viral TNF Receptor Superfamily Member in Virulent Strains of Human Cytomegalovirus
Authors: Benedict Chris A. , Butrovich Kris D. , Lurain Nell S. , Corbeil Jacques , Rooney Isabelle , Schneider Pascal , Tschopp Jurg , Ware Carl F.
Published: December 2022

The UL144 open reading frame found in clinical isolates of human CMV (HCMV) encodes a structural homologue of the herpesvirus entry mediator, a member of the TNFR superfamily. UL144 is a type I transmembrane glycoprotein that is expressed early after infection of fibroblasts; however, it is retained intracellularly. A YXXZ motif in the highly conserved cytoplasmic tail contributes to UL144 subcellular distribution. The finding that no known ligand of the TNF family binds UL144 suggests that its mechanism of action is distinct from other known viral immune evasion genes. Specific Abs to UL144 can be detected in the serum of a subset of HCMV seropositive individuals infected with HIV. This work establishes a novel molecular link between the TNF superfamily and herpesvirus that may contribute to the ability of HCMV to escape immune clearance.
Direct Effects on Antigen-Presenting Cells and T Lymphocytes Explain the Adjuvanticity of a Nontoxic Cholera Toxin Mutant
Authors: Yamamoto Masafumi , Kiyono Hiroshi , Yamamoto Shingo , Batanero Eva , Kweon Mi-Na , Otake Shigeo , Azuma Miyuki , Takeda Yoshifumi , McGhee Jerry R.
Published: December 2022

The present study has elucidated two distinct mechanisms that may explain how a mutant of cholera toxin (mCT), E112K, retains adjuvant effects though it lacks ADP-ribosyltransferase activity and associated toxicity. In the first mechanism, we show that mCT E112K, like native cholera toxin (nCT), enhances B7-2 expression, but, to some extent, also enhances B7-1 on Peyer’s patch B cells and macrophages. Cocultivation of CD4+ T cells with E112K- or nCT-treated B cells and macrophages in the presence of anti-CD3 stimulation resulted in the induction of T cell-proliferative responses. Further, the responses were blocked by mAbs to B7-1 and/or B7-2; however, the effect of anti-B7-1 was minimal. In the second mechanism, addition of mCT E112K or nCT to anti-CD3 mAb-stimulated Peyer’s patch CD4+ T cells inhibited proliferative responses, while recombinant CT-B subunit (rCT-B) did not. Analysis of cytokine responses showed that both mCT E112K and nCT preferentially inhibited IFN-γ production. Interestingly, however, nCT, but not mCT E112K, induced apoptosis in CD4+ T cells activated via the TCR-CD3 complex. These results indicate that CT uses at least two pathways for inhibition of Th1 responses and that, while nCT induces cAMP accumulation that in turn leads to apoptosis in Th1-type cells, mCT E112K, which lacks ADP-ribosyltransferase activity, inhibits IFN-γ synthesis by a separate mechanism. Thus, mCT E112K, like nCT, induces adjuvant responses via up-regulation of mainly B7-2 on APCs and through preferential inhibition of Th1-type CD4+ T cell responses in the absence of ADP-ribosyltransferase activity.
CD47 Signals T Cell Death
Authors: Pettersen Rolf D. , Hestdal Kjetil , Olafsen Mette Kløvstad , Lie Sverre O. , Lindberg Frederik P.
Published: December 2022

Activation-induced death of T cells regulates immune responses and is considered to involve apoptosis induced by ligation of Fas and TNF receptors. The role of other receptors in signaling T cell death is less clear. In this study we demonstrate that activation of specific epitopes on the Ig variable domain of CD47 rapidly induces apoptosis of T cells. A new mAb, Ad22, to this site induces apoptosis of Jurkat cells and CD3ε-stimulated PBMC, as determined by morphological changes, phosphatidylserine exposure on the cell surface, uptake of propidium iodide, and true counts by flow cytometry. In contrast, apoptosis was not observed following culture with anti-CD47 mAbs 2D3 or B6H12 directed to a distant or closely adjacent region, respectively. CD47-mediated cell death was independent of CD3, CD4, CD45, or p56lck involvement as demonstrated by studies with variant Jurkat cell lines deficient in these signaling pathways. However, coligation of CD3ε and CD47 enhanced phosphatidylserine externalization on Jurkat cells with functional CD3. Furthermore, normal T cells required preactivation to respond with CD47-induced apoptosis. CD47-mediated cell death appeared to proceed independent of Fas or TNF receptor signaling and did not involve characteristic DNA fragmentation or requirement for IL-1β-converting enzyme-like proteases or CPP32. Taken together, our data demonstrate that under appropriate conditions, CD47 activation results in very rapid T cell death, apparently mediated by a novel apoptotic pathway. Thus, CD47 may be critically involved in controlling the fate of activated T cells.
TCR-Mediated Involvement of CD4+ Transgenic T Cells in Spontaneous Inflammatory Bowel Disease in Lymphopenic Mice
Authors: Koh Woon-Puay , Chan Elsie , Scott Kate , McCaughan Geoffrey , France Malcolm , Fazekas de St. Groth Barbara
Published: December 2022

Spontaneous colitis resembling ulcerative colitis developed in 3 of 10 independent TCR transgenic (Tg) mouse lines maintained under specific pathogen-free conditions. All three susceptible lines were CD4 lymphopenic, whereas resistant lines had normal numbers of CD4+ T cells. Thus, cytochrome c-specific 5C.C7 TCR Tg mice developed colitis only when crossed onto a SCID- or Rag-1-deficient background. A second line of lymphopenic cytochrome c-specific Tg mice bearing the AND TCR also developed colitis. In both cases, CD4+ T cells expressing the Tg-encoded TCR were preferentially activated in inflamed colons compared with lymph nodes or spleens. In contrast, Tg+CD4+ T cells remained quiescent in both inflamed and unaffected colons in another line of susceptible Tg mice carrying a TCR specific for myelin basic protein, suggesting a fortuitous cross-reactivity of the IEk-restricted cytochrome c-reactive AND and 5C.C7 TCRs with an Ag present in the gut. The percentage of CD4+ T cells expressing only endogenous TCR α-chains was increased consistently in inflamed colons in AND as well as 5C.C7 Rag-1−/− TCR Tg mice, suggesting that polyclonal CD4+ T cells were also involved in the pathogenesis of spontaneous colitis. Moreover, our data indicate that some α-chain rearrangement was still occurring in TCR Tg mice on a Rag-1−/− background, since activated CD4+ T cells expressing endogenously rearranged α-chains paired with the Tg-encoded β-chain were detected consistently in the colons of such mice.
Multiple NF-ATc Isoforms with Individual Transcriptional Properties Are Synthesized in T Lymphocytes
Authors: Chuvpilo Sergei , Avots Andris , Berberich-Siebelt Friederike , Glöckner Judith , Fischer Christian , Kerstan Andreas , Escher Cornelia , Inashkina Inna , Hlubek Falk , Jankevics Eriks , Brabletz Thomas , Serfling Edgar
Published: December 2022

The transcription factor NF-ATc that controls gene expression in T lymphocytes and embryonic cardiac cells is expressed in three prominent isoforms. This is due to alternative splice/polyadenylation events that lead to the predominant synthesis of two long isoforms in naive T cells and a shorter NF-ATc isoform in effector T cells. Whereas the previously described isoform NF-ATc/A contains a relatively short C terminus, the longer isoforms, B and C, span extra C-terminal peptides of 128 and 246 aa, respectively. We show here that in addition to the strong N-terminal trans-activation domain, TAD-A, which is common to all three NF-ATc isoforms, NF-ATc/C contains a second trans-activation domain, TAD-B, in its C-terminal peptide. Various stimuli of T cells that induce the activity of TAD-A also enhance the activity of TAD-B, but, unlike TAD-A, TAD-B remains unphosphorylated by protein from 12-O-tetradecanoyl 12-phorbol 13-acetate-stimulated T cells. The shorter C-terminal peptide of isoform NF-ATc/B exerts a suppressive transcriptional effect. These properties of NF-ATc/B and -C might be of importance for gene regulation in naive T lymphocytes in which NF-ATc/B and -C are predominantly synthesized.
Poxvirus-Encoded Serpins Do Not Prevent Cytolytic T Cell-Mediated Recovery from Primary Infections
Authors: Müllbacher Arno , Wallich Reinhard , Moyer Richard W. , Simon Markus M.
Published: December 2022

Previous observations that the highly conserved poxvirus-encoded serpins inhibit cytotoxic activities of alloreactive CTL via granule and/or Fas-mediated pathways was taken to indicate their involvement in immune evasion by poxviruses. We now show that interference with 51Cr release from target cells by ectromelia and cowpoxvirus is limited to alloreactive but not MHC-restricted CTL. The data are in support of the paramount importance of CTL and its effector molecule perforin in the recovery from primary ectromelia virus infection and question the role of serpins in the evasion of poxviruses from killing by CTL. Further analysis of poxvirus interference with target cell lysis by alloreactive CTL revealed that suppression primarily affects the Fas-mediated, and to a lesser extent, the granule exocytosis pathway. Serpin-2 is the main contributor to suppression for both killing pathways. In addition, inhibition of lysis was shown to be both target cell type- and MHC allotype-dependent. We hypothesize that differences in TCR affinities and/or state of activation between alloreactive and MHC-restricted CTL as well as the quality (origin) of target cells are responsible for the observed phenomenon.
Human Polymorphonuclear Leukocytes Produce IL-12, TNF-α, and the Chemokines Macrophage-Inflammatory Protein-1α and -1β in Response toToxoplasma gondiiAntigens
Authors: Bliss Susan K. , Marshall Anthony J. , Zhang Yin , Denkers Eric Y.
Published: December 2022

The induction of a type 1 inflammatory cytokine response is a key event in the initiation of immunity to Toxoplasma gondii. Because polymorphonuclear leukocytes rapidly respond to infection by exiting the peripheral blood and accumulating at a site of infection, we sought to determine whether these cells produce cytokines in response to T. gondii. When human peripheral blood neutrophils were stimulated with parasite Ag, they produced both IL-12 (p70) and TNF-α. Similarly, up-regulated expression of macrophage-inflammatory protein-1α (MIP-1α) and MIP-1β gene transcripts was induced. Kinetic analysis of IL-12 and TNF-α production revealed distinct patterns following stimulation by T. gondii or LPS. Exogenous TNF-α alone also provided a potent stimulus of MIP-1α and MIP-1β expression, and when neutralizing anti-TNF-α antiserum was included in cultures of parasite-stimulated cells, expression of these CC-family chemokines was partially blocked. These results establish that T. gondii possesses the ability of driving neutrophil proinflammatory cytokine production, and they suggest that parasite-induced MIP-1α and MIP-1β partly results from autocrine stimulation through TNF-α.
Purine Metabolites Suppress Proliferation of Human NK Cells Through a Lineage-Specific Purine Receptor
Authors: Miller Jeffrey S. , Cervenka Tereza , Lund Jeanne , Okazaki Ian J. , Moss Joel
Published: December 2022

NK cell proliferation is suppressed in some patients with cancer by unknown mechanisms. Because purine metabolites released into the extracellular space during cell lysis may affect cell function, we hypothesized that these metabolites could serve as feedback regulators of NK cell proliferation. Sorted NK (CD56+/CD3−) cells were incubated with IL-2 (1000 U/ml) in a 4-day thymidine uptake assay with or without 10–10,000 μM of nucleotides. Adenine nucleotides inhibited NK cell proliferation, with ATP = ADP > 5′-adenylylimidodiphosphate > AMP = adenosine; ADP-ribose and nicotinamide adenine dinucleotide, but not nicotinamide or UTP, caused a dose-dependent suppression of thymidine uptake. A total of 100 μM ATP, a concentration that induced a maximal (80%) inhibition of thymidine uptake, did not inhibit cytotoxic activity against K562 targets. Because NK cells retained the ability to lyse K562 targets 4 days after exposure to 500 μM ATP or 1000 μM adenosine, inhibition of thymidine uptake was not due to cell death. Incubation of NK cells with dibutyryl cAMP and forskolin also suppressed thymidine uptake. Cholera toxin and pertussis toxin suppressed NK cell proliferation. Pertussis toxin did not block the adenine nucleotide effects. Further, ATP, but not adenosine or other nucleotides, markedly increased intracellular cAMP in a dose-dependent manner. The ATP-induced increase in cAMP was specific to cytolytic cells, because CD19+ B cells and CD4+ T cells did not increase their intracellular cAMP. These studies demonstrate that NK proliferation is regulated through purine receptors by adenine nucleotides, which may play a role in decreased NK cell activity. The response to adenine nucleotides is lineage-specific.
POMC Gene-Derived Peptides Activate Melanocortin Type 3 Receptor on Murine Macrophages, Suppress Cytokine Release, and Inhibit Neutrophil Migration in Acute Experimental Inflammation
Authors: Getting Stephen J. , Gibbs Linda , Clark Adrian J. L. , Flower Roderick J , Perretti Mauro
Published: December 2022

To investigate the relevance of adrenocorticotrophic hormone (ACTH) therapy in human gouty arthritis, we have tested the effect of several ACTH-related peptides in a murine model of experimental gout. Systemic treatment of mice with ACTH4–10 (MEHFRWG) (10–200 μg s.c.) inhibited neutrophil accumulation without altering peripheral blood cell counts or circulating corticosterone levels. A similar effect was seen with α- and β-melanocyte stimulating hormones (1–30 μg s.c.). In vivo release of the chemokine KC-(detected in the lavage fluids before maximal influx of neutrophils) was significantly reduced (−50 to −60%) by ACTH4–10. Macrophage activation in vitro, determined as phagocytosis and KC release, was inhibited by ACTH and ACTH4–10 with approximate IC50 values of 30 nM and 100 μM, respectively. The melanocortin receptor type 3/4 antagonist SHU9119 prevented the inhibitory actions of ACTH4–10 both in vitro and in vivo. However, melanocortin type 3, but not type 4, receptor mRNA was detected in mouse peritoneal macrophages by RT-PCR. Therefore, we propose that activation of this receptor type by ACTH4–10 and related amino acid sequences attenuates KC release (and possibly production of other cytokines) from macrophages with consequent inhibition of the host inflammatory response, thus providing a notional anti-inflammatory mechanism for ACTH that is unrelated to stimulation of glucocorticoid release.
Rhinovirus Regulation of IL-1 Receptor Antagonist In Vivo and In Vitro: A Potential Mechanism of Symptom Resolution
Authors: Yoon Ho Joo , Zhu Zhou , Gwaltney Jack M. , Elias Jack A.
Published: December 2022

Rhinovirus (RV) upper respiratory tract infections are prototypic transient inflammatory responses. To address the mechanism of disease resolution in these infections, we determined if RV stimulated the production of the IL-1 receptor antagonist (IL-1ra) in vivo and in vitro. In contrast to IL-1α and IL-1β, immunoreactive IL-1ra was readily detected in the nasal washings of normal human volunteers. Symptomatic RV infection caused a small increase in IL-1α, a modest increase in IL-1β, and an impressive increase in IL-1ra. Maximal induction of IL-1α and IL-1β was transiently noted 48 h after RV infection. In contrast, maximal induction of IL-1ra was prolonged appearing 48–72 h after RV infection. These time points corresponded to the periods of peak symptomatology and the onset of symptom resolution, respectively. Western analysis of nasal washings demonstrated that RV stimulated the accumulation of intracellular IL-1ra type I in all and secreted IL-1ra in a subset of volunteers. Unstimulated normal respiratory epithelial cells contained intracellular IL-1ra type I mRNA and protein. RV infection increased the intracellular levels and extracellular transport of this IL-1ra moiety without causing significant changes in the levels of IL-1ra mRNA. IL-1ra may play an important role in the resolution of RV respiratory infections. RV stimulates epithelial cell IL-1ra elaboration, at least in part, via a novel translational and/or posttranslational mechanism.
Effect of an Orally Active Th1/Th2 Balance Modulator, M50367, on IgE Production, Eosinophilia, and Airway Hyperresponsiveness in Mice
Authors: Kato Yutaka , Manabe Tadashi , Tanaka Yukiko , Mochizuki Hidenori
Published: December 2022

We have found a novel anti-allergic agent, M50367, which suppresses IgE biosynthesis and eosinophil accumulation in vivo. In this study, we evaluated the ability of M50367 to modulate Th1/Th2 balance in Th2-background BALB/c mice and to inhibit airway hyperresponsiveness in a murine model of atopic asthma. Oral M50367 at 3–30 mg/kg/day exhibited 51 to 73% reduction of IL-4/IL-5 production and 2- to 5-fold augmentation of IFN-γ production by Ag-stimulated cultured splenocytes of the mice sensitized with DNP-Ascaris. These alterations in Th1/Th2 cytokine production were accompanied by 55–85% suppression of plasma IgE level. Oral M50367 at a dose of 10 mg/kg/day significantly inhibited Ig-independent peritoneal eosinophilia by 54%, which was induced by repeated i.p. injections of Ascaris suum extract. To develop airway hyperresponsiveness caused by allergic airway inflammation, BALB/c mice were sensitized with i.p. OVA injections, followed three times by OVA inhalation. Oral M50367 significantly inhibited the increase in airway reactivity to acetylcholine, together with the elevation of plasma IgE level and pulmonary eosinophilia, which were observed in vehicle-treated mice 1 day after the last inhalation. Moreover, M50367 treatment reduced IL-4 and IL-5 production and tended to enhance IFN-γ production, not only by cultured splenocytes, but also in bronchoalveolar lavage fluid. These results suggest that M50367 has a modulating ability of Th1/Th2 balance to down-regulate Th2 response in the circulating system as well as at the sites of inflammation, and may be beneficial for the treatment of allergic disorders such as atopic asthma.
IL-13 and IFN-γ Secretion by Activated T Cells in HIV-1 Infection Associated with Viral Suppression and a Lack of Disease Progression
Authors: Bailer Robert T. , Holloway Alvy , Sun Junwei , Margolick Joseph B. , Martin Melissa , Kostman Jay , Montaner Luis J.
Published: December 2022

The immunopathology of HIV-1 infection includes immune defects in T cell cytokine secretion, resulting in decreased Ag-specific responses. In this report, IL-13 and IFN-γ were analyzed in progressive HIV-1 disease. Both cytokines exert positive effects on Ag presentation and inhibit HIV-1 infection of macrophages. Anti-CD3/anti-CD28-activated PBMC from HIV-1-infected individuals (n = 74) compared with uninfected subjects (n = 30) secreted significantly less IL-13 (median, 0.64 ng/ml vs 2.07 ng/ml; p < 0.001) and IFN-γ (median, 40.96 ng/ml vs 129.5 ng/ml; p < 0.005). Decreased IL-13 and IFN-γ secretion in HIV infection was present in sorted CD4+ and CD8+ T cell subsets, and additional analysis determined concurrent deficiency at the protein and transcriptional level. Longitudinal analysis showed that cytokine secretion levels correlated positively with CD4 count and negatively with plasma HIV-1 viral load. Patients changing to suppressive antiretroviral therapy during the study showed increases in IL-13 and IFN-γ secretion. Overall, results show a decline in IL-13 and IFN-γ secretion in progressive HIV-1 infection and suggest a role for both cytokines as part of T cell adaptive responses associated with a lack of disease progression.
Complement Receptor 1 (CD35) on Human Reticulocytes: Normal Expression in Systemic Lupus Erythematosus and HIV-Infected Patients
Authors: Lach-Trifilieff Estelle , Marfurt Jutta , Schwarz Sybille , Sadallah Salima , Schifferli Jürg A.
Published: December 2022

The low levels of complement receptor 1 (CR1) on erythrocytes in autoimmune diseases and AIDS may be due to accelerated loss in the circulation, or to a diminished expression of CR1 on the red cell lineage. Therefore, we analyzed the expression of CR1 on reticulocytes (R) vs erythrocytes (E). Healthy subjects had a significant higher CR1 number per cell on R (919 ± 99 CR1/cell) than on E (279 ± 30 CR1/cell, n = 23), which corresponded to a 3.5- ± 1.3-fold loss of CR1. This intravascular loss was confirmed by FACS analysis, which showed that all R expressed CR1, whereas a large fraction of E was negative. The systemic lupus erythematosus (SLE), HIV-infected, and cold hemolytic Ab disease (CHAD) patients had a CR1 number on R identical to the healthy subjects, contrasting with a lower CR1 on their E. The data indicated a significantly higher loss of CR1 in the three diseases, i.e., 7.0- ± 3.8-, 6.1- ± 2.9-, and 9.6- ± 5.6-fold, respectively. The intravascular loss was best exemplified in a patient with factor I deficiency whose CR1 dropped from 520 CR1/R to 28 CR1/E, i.e., 18.6-fold loss. In one SLE patient and in the factor I-deficient patient, the FACS data were consistent with a loss of CR1 already on some R. In conclusion, CR1 is lost progressively from normal E during in vivo aging so that old E are almost devoid of CR1. The low CR1 of RBC in autoimmune diseases and HIV-infection is due to a loss occurring in the circulation by an active process that remains to be defined.
C1.7 Antigen Expression on CD8+ T Cells Is Activation Dependent: Increased Proportion of C1.7+CD8+ T Cells in HIV-1-Infected Patients with Progressing Disease
Authors: Peritt David , Sesok-Pizzini Deborah A. , Schretzenmair Richard , Macgregor Rob R. , Valiante Nicholas M. , Tu Xin , Trinchieri Giorgio , Kamoun Malek
Published: December 2022

The C1.7 Ag is a surface marker previously shown to be expressed on all NK cells and on a subset of CD8+ T cells. We report in this study that C1.7 Ag expression on peripheral blood-derived CD8+ T cells overlaps with activation markers S6F1high and CD29high and is reciprocally expressed with CD62L. C1.7 Ag expression can be induced in vitro on CD8+ T cells by anti-CD3 cross-linking, suggesting that C1.7 Ag is activation dependent. In contrast to NK cells, C1.7 Ag does not signal on CD8+ T cells, nor does it induce redirected lysis upon ligation. The proportion of C1.7 Ag+CD8+ T cells is increased in HIV-infected patients compared with healthy donors. In 69 HIV-infected patients, we observed a significant inverse correlation between the percentage of C1.7 Ag-expressing CD8+ T cells and the absolute CD4+ T cell count. Two-year clinical follow-up of patients with initial CD4+ T cell count of >400 cells/mm3 and a normal proportion of C1.7 Ag+CD8+ T cells revealed that these patients were clinically stable with minimal HIV-associated symptoms. In contrast, 10 of 12 patients with CD4+ T cell counts of >400 cells/mm3 and an elevated proportion of C1.7 Ag+CD8+ T cells were symptomatic. ANOVA analysis of patients indicates that C1.7 Ag is a better predictor of disease progression than CD4 count. Overall, our findings indicate that C1.7 Ag is the first described marker for activated/memory CD8+ T cells and a useful parameter for evaluating the level of CD8+ T cell activation in vivo.
Cutting Edge: Absence of Expression of RAG1 in Peritoneal B-1 Cells Detected by Knocking into RAG1 Locus with Green Fluorescent Protein Gene
Authors: Kuwata Naomi , Igarashi Hideya , Ohmura Takafumi , Aizawa Shinichi , Sakaguchi Nobuo
Published: December 2022

It has been proposed that Ig gene rearrangement in the peritoneal cavity (Pc) B-1 cells might be involved in autoantibody generation. To study possible secondary B cell maturation, we prepared mice carrying a target integration of gfp gene into a rag1 locus (rag1/gfp mice). The GFP+ cells express rag1 mRNA and are undergoing Ig gene rearrangement. RAG1 expression was studied in Pc B-1 cells to detect cells during the stage of Ig gene rearrangement. In contrast to previous reports, Pc B-1 cells did not show RAG1 expression in adolescent or elderly mice. RAG1 expression was not induced in Pc B-1 cells in vivo after stimulation by oral or i.p. administration of LPS. Our results suggest that RAG1 expression in Pc B-1 cells is inhibited for a long period under normal condition and that this suppression is an essential state which maintains allelic exclusion of Ig genes.
Immunogenicity. I. Use of Peptide Libraries to Identify Epitopes That Activate Clonotypic CD4+ T Cells and Induce T Cell Responses to Native Peptide Ligands
Authors: Wilson Darcy B. , Pinilla Clemencia , Wilson Dianne H. , Schroder Kim , Boggiano César , Judkowski Valeria , Kaye Jonathan , Hemmer Bernhard , Martin Roland , Houghten Richard A.
Published: December 2022

Recent studies have demonstrated the utility of synthetic combinatorial libraries for the rapid identification of peptide ligands that stimulate clonotypic populations of T cells. Here we screen a decapeptide combinatorial library arranged in a positional scanning format with two different clonotypic populations of CD4+ T cells to identify peptide epitopes that stimulate proliferative responses by these T cells in vitro. An extensive collection of mimic peptide sequences was synthesized and used to explore the fine specificity of TCR/peptide/MHC interactions. We also demonstrate that many of these deduced ligands are not only effective immunogens in vivo, but are capable of inducing T cell responses to the original native ligands used to generate the clones. These results have significant implications for considerations of T cell specificity and the design of peptide vaccines for infectious disease and cancer using clinically relevant T cell clones of unknown specificity.
Negative Regulation of CD4 Lineage Development and Responses by CD5
Authors: Peña-Rossi Claudia , Zuckerman Linda A. , Strong Julie , Kwan Joanne , Ferris William , Chan Susan , Tarakhovsky Alexander , Beyers Albert D. , Killeen Nigel
Published: December 2022

CD5 deficiency results in a hyper-responsive phenotype to Ag receptor stimulation. Here we show that the development and responses of CD4 lineage T cells are regulated by the function of CD5. Thymocytes expressing the I-Ad-restricted DO11.10 TCR undergo abnormal selection without CD5. In H-2d mice, the absence of CD5 causes deletion of double-positive thymocytes, but allows for efficient selection of cells expressing high levels of the DO11.10 clonotype. By contrast, there is enhanced negative selection against the DO11.10 clonotype in the presence of I-Ab. T cell hybridomas and DO11.10 T cells are more responsive to TCR stimulation in the absence of CD5. Such hypersensitivity can be eliminated by expression of wild-type CD5, but not by a form of CD5 that lacks the cytoplasmic tail. Finally, CD5 deficiency partially suppresses the block of CD4 lineage development in CD4-deficient mice. Taken together, the data support a general role for CD5 as a negative regulator of Ag receptor signaling in the development and immune responses of CD4 lineage T cells.
Stat6-Dependent and -Independent Pathways for IL-4 Production
Authors: Kaplan Mark H. , Wurster Andrea L. , Smiley Stephen T. , Grusby3 Michael J.
Published: December 2022

Stat6 has been shown to have a crucial role in the IL-4-dependent differentiation of Th2 cells. In this report, we explore whether in vitro Th2 differentiation driven by altered costimulatory signals or Ag dose is Stat6 dependent. We find that blocking B7-1 signaling in vitro promotes the differentiation of IL-4-secreting Th2 cells in wild-type but not Stat6-deficient T cell cultures. Additionally, stimulation with peptide Ag doses that normally result in the production of Th2 cells in vitro fails to do so in cultures of Stat6-deficient cells. We also demonstrate that Stat6 is required for the in vitro differentiation of CD8+ T cells into IL-4-secreting cytotoxic T cell type 2 cells. However, IL-4 expression is not absolutely dependent on Stat6. We demonstrate that populations of T cells that do not require IL-4 for their development, such as NK T cells, are still competent to secrete IL-4 in the absence of Stat6. These results demonstrate that Stat6 is required for the differentiation program leading to the generation of Th2 and cytotoxic T cell type 2 cells but not for IL-4 expression in cells that do not undergo differentiation in response to IL-4.
Distinct Subcellular Localization and Substrate Specificity of Extracellular Signal-Regulated Kinase in B Cells upon Stimulation with IgM and CD40
Authors: Shirakata Yumiko , Ishii Kumiko , Yagita Hideo , Okumura Ko , Taniguchi Masaru , Takemori Toshitada
Published: December 2022

We and others previously observed that IgM and CD40 stimulation in murine B cells resulted in activation of extracellular signal-regulated kinase (ERK), a subfamily of mitogen-activated protein kinase. The present study demonstrated that ERK was rapidly phosphorylated and translocated to the nucleus in murine B cells upon stimulation with CD40, whereas it was preferentially localized within the cytosol after stimulation with IgM, suggesting that signaling through CD40 and IgM differentially regulates ERK subcellular localization. Costimulation with CD40 and IgM (CD40/IgM) resulted in subcellular localization of ERK within the cytosol, supporting the notion that stimulation with IgM delivers the signal responsible for inhibition of ERK nuclear transport. Consistent with these observations, IgM and CD40/IgM stimulation resulted in activation of ribosomal S6 kinase, which is a cytoplasmic substrate for ERK, whereas CD40 stimulation had little effect on its activity. Disruption of the microtubule by colchicine in WEHI231 cells resulted in reduction of ERK activity in IgM signaling, but not in CD40 signaling, compatible with the notion that the microtubule network may hold cytoplasmic ERK activity mediated by IgM stimulation. These results support the notion that ERK could mediate different effector functions in B cells upon stimulation with IgM and CD40.
Ligation of Microglial CD40 Results in p44/42 Mitogen-Activated Protein Kinase-Dependent TNF-α Production That Is Opposed by TGF-β1 and IL-10
Authors: Tan Jun , Town Terrence , Saxe Michael , Paris Daniel , Wu Yajuan , Mullan Michael
Published: December 2022

Recently, it has been demonstrated that the CD40 receptor is constitutively expressed on cultured microglia at low levels. Ligation of CD40 by CD40 ligand on these cells results in microglial activation, as measured by TNF-α production and neuronal injury. However, the intracellular events mediating this effect have yet to be investigated. We report that ligation of microglial CD40 triggers activation of p44/42 mitogen-activated protein kinase (MAPK). This effect is evident 30 min posttreatment, and progressively declines thereafter (from 30 to 240 min). Phosphorylated p38 MAPK is not observed in response to ligation of microglial CD40 across the time course examined. Inhibition of the upstream activator of p44/42 MAPK, mitogen-activated protein/extracellular signal-related kinase kinase 1/2, with PD98059, decreases phosphorylation of p44/42 MAPK and significantly reduces TNF-α release following ligation of microglial CD40. Furthermore, cotreatment of microglial cells with CD40 ligand and TGF-β1 or IL-10, or both, inhibits CD40-mediated activation of p44/42 MAPK and production of TNF-α in a statistically interactive manner. Taken together, these data show that ligation of microglial CD40 triggers TNF-α release through the p44/42 MAPK pathway, an effect that can be opposed by TGF-β1 and IL-10.
T Cell Activation Up-Regulates the Expression of the Focal Adhesion Kinase Pyk2: Opposing Roles for the Activation of Protein Kinase C and the Increase in Intracellular Ca2+
Authors: Tsuchida Masahiro , Manthei Eric R. , Alam Tausif , Knechtle Stuart J. , Hamawy Majed M.
Published: December 2022

T cell activation initiates signals that control gene expression of molecules important for T cell function. The focal adhesion kinase Pyk2 has been implicated in T cell signaling. To further analyze the involvement of Pyk2 in T cell processes, we examined the effect of T cell stimulation on the expression of Pyk2. We found that TCR ligation or PMA increased Pyk2 expression in Jurkat T cells and in normal T cells. In contrast, TCR ligation and PMA failed to induce any detectable increase in the expression of the other member of the focal adhesion kinase family, Fak, in Jurkat T cells and induced only a weak increase in Fak expression in normal T cells. The serine/threonine kinases, protein kinase C and mitogen-activated protein/extracellular signal-related kinase kinase (MEK), regulated Pyk2 expression, as inhibitors of these kinases blocked stimulus-induced Pyk2 expression. Cyclosporin A, FK506, and KN-62 did not block Pyk2 expression; thus, calcineurin and Ca2+/calmodulin-activated kinases are not critical for augmenting Pyk2 expression. TCR ligation increased Pyk2 mRNA, and the transcriptional inhibitor actinomycin D blocked Pyk2 expression. Strikingly, Ca2+ ionophores, at concentrations that in combination with other stimuli induced IL-2 expression, blocked TCR- and PMA-induced up-regulation of Pyk2 expression. Thus, the increase in Ca2+ has opposing effects on IL-2 and Pyk2 expression. Cyclosporin A and FK506, but not KN-62, blocked Ca2+ ionophore-mediated inhibition of Pyk2 expression, implicating calcineurin in down-regulating Pyk2 expression. These results show that TCR-triggered intracellular signals increase Pyk2 expression and shed light on the molecular mechanisms that regulate Pyk2 expression in T cells.
Microphthalmic Mice Display a B Cell Deficiency Similar to that Seen for Mast and NK Cells
Authors: Roundy Kirstin , Kollhoff Angela , Eichwald E. J. , Weis Janis J. , Weis John H.
Published: December 2022

The microphthalmic mouse (mi) possesses a 3-bp deletion of the Mi gene that alters the DNA binding site of the transcription factor gene product. This animal has diminished numbers of NK and mast cells (MC) and is osteopetrotic due to a lack of the normal complement of functional osteoclasts. The reduction of MC has been proposed to be due to the lack of adequate c-Kit expression that is required for MC differentiation. However, data from other labs has questioned this interpretation. In this report, we present data suggesting bone marrow-derived deficiencies of the mi mouse are not due to a lack of c-Kit expression and function, but instead due to an inhospitable environment within the bone marrow itself. Specifically, we have found that such animals also lack virtually all B cell precursors within the marrow and rely upon other lymphatic sites, such as the spleen, for B cell development and maturation. Although the animal has depressed numbers of NK cells, B cells, and MC, it still possesses a normal thymus and peripheral T cells. Therefore, the block in cellular differentiation must be within the marrow environment, which is essential for maturing B cells, NK cells, and MC but not T cells.
Expression and Regulation of the Human β-Defensins hBD-1 and hBD-2 in Intestinal Epithelium
Authors: O’Neil Deborah A. , Porter Edith Martin , Elewaut Dirk , Anderson G. Mark , Eckmann Lars , Ganz Tomas , Kagnoff Martin F.
Published: December 2022

The intestinal epithelium forms a physical barrier to limit access of enteric microbes to the host and contributes to innate host defense by producing effector molecules against luminal microbes. To further define the role of the intestinal epithelium in antimicrobial host defense, we analyzed the expression, regulation, and production of two antimicrobial peptides, human defensins hBD-1 and hBD-2, by human intestinal epithelial cells in vitro and in vivo. The human colon epithelial cell lines HT-29 and Caco-2 constitutively express hBD-1 mRNA and protein but not hBD-2. However, hBD-2 expression is rapidly induced by IL-1α stimulation or infection of those cells with enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-κB target gene in the intestinal epithelium as blocking NF-κB activation inhibits the up-regulated expression of hBD-2 in response to IL-1α stimulation or bacterial infection. Caco-2 cells produce two hBD-1 isoforms and a hBD-2 peptide larger in size than previously described hBD-2 isoforms. Paralleling the in vitro findings, human fetal intestinal xenografts constitutively express hBD-1, but not hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in xenografts infected intraluminally with Salmonella. hBD-1 is expressed by the epithelium of normal human colon and small intestine, with a similar pattern of expression in inflamed colon. In contrast, there is little hBD-2 expression by the epithelium of normal colon, but abundant hBD-2 expression by the epithelium of inflamed colon. hBD-1 and hBD-2 may be integral components of epithelial innate immunity in the intestine, with each occupying a distinct functional niche in intestinal mucosal defense.
Reduced Ultraviolet-Induced Carcinogenesis in Mice with a Functional Disruption in B7-Mediated Costimulation
Authors: Beissert Stefan , Bluestone Jeffrey A. , Mindt Iris , Voskort Maik , Metze Dieter , Mehling Annette , Luger Thomas A. , Schwarz Thomas , Grabbe Stephan
Published: December 2022

Immunosuppression by UV light contributes significantly to the induction of skin cancer by suppressing the cell-mediated immune responses which control the development of carcinogenesis. The B7/CD28-CTLA-4 signaling pathway provides costimulatory signals essential for Ag-specific T cell activation. To investigate the role of this pathway in photocarcinogenesis, we utilized transgenic (Tg) mice which constitutively express CTLA-4Ig, a high-affinity CD28/CTLA-4 antagonist that binds to both B7-1 and B7-2. The transgene is driven by a skin-specific promoter yielding high levels of CTLA-4Ig in the skin and serum. Chronic UV exposure of CTLA-4Ig Tg mice resulted in significantly reduced numbers of skin tumors, when compared to control mice. In addition, Tg mice were resistant to UV-induced suppression of delayed-type hypersensitivity responses to alloantigens. Most importantly, upon stimulation with mitogens and alloantigens, T cells isolated from CTLA-4Ig Tg mice produced significantly less IL-4 but more IFN-γ compared to control T cells, suggesting an impaired Th2 response and a relative increase of Th1-type immunity. Together, these data show that overall B7 engagement directs immune responses toward the Th2 pathway. Moreover, they point out the crucial role of Th1 immune reactions in the protection against photocarcinogenesis.
Vaccinia Virus Inhibits the Maturation of Human Dendritic Cells: A Novel Mechanism of Immune Evasion
Authors: Engelmayer Jose , Larsson Marie , Subklewe Marion , Chahroudi Ann , Cox William I. , Steinman Ralph M. , Bhardwaj Nina
Published: December 2022

Vaccinia virus employs multiple mechanisms to evade the immune system, yet is highly immunogenic. We studied the interaction between vaccinia and human dendritic cells (DCs), potent APCs. DCs develop from precursor cells in two stages: an immature stage in which Ag uptake and processing occur, and a mature stage in which there is up-regulation of costimulatory and HLA molecules and efficient T cell activation. Vaccinia virus undergoes an abortive replication in both stages of DCs and induces apoptotic cell death. Furthermore, maturation of immature DCs and consequently T cell activation are inhibited. Obstruction of DC maturation may constitute a novel mechanism by which vaccinia attempts to evade the immune response.