bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of IFN-γ or IL-12 Has Different Effects on Experimental Myasthenia Gravis in C57BL/6 Mice
Authors: Karachunski Peter I. , Ostlie Norma S. , Monfardini Cristina , Conti-Fine Bianca M.
Published: April 2014

Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-γ and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-γ and IL-12 knockout mutants (IFN-γ−/−, IL-12−/−) with Torpedo AChR (TAChR). WT and IFN-γ−/− mice developed EMG with similar frequency, IL-12−/−mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-γ−/− mice had significantly less IgG2c, and IL-12−/− mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-γ−/− mice had complement; WT mice had both IgG2b and IgG2c, IFN-γ−/− only IgG2b, and IL-12−/− neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-γ−/− mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12−/− mice secreted less IFN-γ, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12−/− mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-γ−/− mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.
Tolerance to Cardiac Allografts Via Local and Systemic Mechanisms After Adenovirus-Mediated CTLA4Ig Expression
Authors: Guillot Cécile , Mathieu Patrick , Coathalem Hélène , Le Mauff Brigitte , Castro Maria G. , Tesson Laurent , Usal Claire , Laumonier Thomas , Brouard Sophie , Soulillou Jean-Paul , Lowenstein Pedro R. , Cuturi Maria Cristina , Anegon Ignacio
Published: April 2014

Blockade of the CD28/B7 T cell costimulatory pathway prolongs allograft survival and induces tolerance in some animal models. We analyzed the efficacy of a CTLA4Ig-expressing adenovirus in preventing cardiac allorejection in rats, the mechanisms underlying heart transplant acceptance, and whether the effects of CTLA4Ig were restricted to the graft microenvironment or were systemic. CTLA4Ig gene transfer into the myocardium allowed indefinite graft survival (>100 days vs 9 ± 1 days for controls) in 90% of cases, whereas CTLA4Ig protein injected systemically only prolonged cardiac allograft survival (by up to 22 days). CTLA4Ig could be detected in the graft and in the serum for at least 1 year after gene transfer. CTLA4Ig gene transfer induced local intragraft immunomodulation at day 5 after transplantation, as shown by decreased expression of the IL-2R and MHC II Ags; decreased levels of mRNA encoding for IFN-γ, inducible NO synthase, and TGF-β; and inhibited proliferative responses of graft-infiltrating cells. Systemic immune responses were also down-modulated, as shown by the suppression of Ab production against donor alloantigens and cognate Ags, up to at least 120 days after gene transfer. Alloantigenic and mitogenic proliferative responses of graft-infiltrating cells and total splenocytes were inhibited and were not reversed by IL-2. In contrast, lymph node cells and T cells purified from splenocytes showed normal proliferation. Recipients of long-term grafts treated with adenovirus coding for CTLA4Ig showed organ and donor-specific tolerance. These data show that expression of CTLA4Ig was high and long lasting after adenovirus-mediated gene transfer. This expression resulted in down-modulation of responses against cognate Ags, efficient suppression of local and systemic allograft immune responses, and ultimate induction of donor-specific tolerance.
IL-10 Is Required for Prevention of Necrosis in the Small Intestine and Mortality in Both Genetically Resistant BALB/c and Susceptible C57BL/6 Mice Following Peroral Infection withToxoplasma gondii
Authors: Suzuki Yasuhiro , Sher Alan , Yap George , Park Daniel , Neyer Lauri Ellis , Liesenfeld Oliver , Fort Madeline , Kang Hoil , Gufwoli Edgar
Published: April 2014

The role for IL-10 in the immunopathogenesis of acute toxoplasmosis following peroral infection was examined in both genetically susceptible C57BL/6 and resistant BALB/c mice. C57BL/6-background IL-10-targeted mutant (IL-10−/−) mice all died in 2 wk after infection with 20 cysts of the ME49 strain, whereas only 20% of control mice succumbed. Histological studies revealed necrosis in the small and large intestines and livers of infected IL-10−/− mice. The necrosis in the small intestine was the most severe pathologic response and was not observed in control mice. Treatment of infected IL-10−/− mice with either anti-CD4 or anti-IFN-γ mAb prevented intestinal pathology and significantly prolonged time to death. Treatment of these animals with anti-IL-12 mAb also prevented the pathology. Significantly greater amounts of IFN-γ mRNA were detected in the lamina propria lymphocytes obtained from the small intestine of infected IL-10−/− mice than those from infected control mice. In common with C57BL/6-background IL-10−/− mice, BALB/c-background IL-10−/− mice all died developing intestinal pathology after infection. Control BALB/c mice all survived even after infection with 100 cysts and did not develop the intestinal lesions. Treatment with anti-IFN-γ mAb prevented the pathology and prolonged time to death of the infected IL-10−/− mice. These results strongly suggest that IL-10 plays a critical role in down-regulating IFN-γ production in the small intestine following sublethal peroral infection with Toxoplasma gondii and that this down-regulatory effect of IL-10 is required for prevention of development of IFN-γ-mediated intestinal pathology and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice.
Expression of IFN-Inducible T Cell α Chemoattractant by Human Endothelial Cells Is Cyclosporin A-Resistant and Promotes T Cell Adhesion: Implications for Cyclosporin A-Resistant Immune Inflammation
Authors: Mazanet Melissa M. , Neote Kuldeep , Hughes Christopher C. W.
Published: April 2014

IFN-inducible T cell α chemoattractant (I-TAC) is a recently discovered member of the CXC chemokine family. It is a potent T cell chemoattractant expressed by IFN-γ-treated astrocytes, monocytes, keratinocytes, bronchial epithelial cells, and neutrophils. In this study, we show that I-TAC is also expressed by IFN-γ-treated endothelial cells (EC), both at the mRNA and protein levels. Induction of the I-TAC message is rapid and sustained over 24 h. TNF-α does not induce I-TAC mRNA alone, but does act synergistically with IFN-γ. Blocking Abs to I-TAC, or to its receptor, CXCR3, reduce T cell adhesion to EC monolayers demonstrating that the expressed protein is functional. Finally, the expression of I-TAC by EC is resistant to the immunosuppressive drug cyclosporin A, suggesting that I-TAC may contribute to the chronic immune inflammation characteristic of graft arteriosclerosis.
The Inhibition of Apoptosis in Myositis and in Normal Muscle Cells
Authors: Nagaraju Kanneboyina , Casciola-Rosen Livia , Rosen Antony , Thompson Cynthia , Loeffler Lisa , Parker Tomasina , Danning Carol , Rochon Paul J. , Gillespie John , Plotz Paul
Published: April 2014

The mechanism of injury and death of muscle cells in the inflammatory myopathies (dermatomyositis, polymyositis, and inclusion body myositis) remains obscure. We and others have not detected apoptosis in the muscle biopsies from patients with myositis despite clear evidence of cell damage and loss. We provide evidence in this study that Fas ligand (FasL) as well as Fas is present on muscle cells and inflammatory cells in myositis biopsies: Fas is present on most muscle cells and lymphocytes, and FasL is present on degenerating muscle cells and many infiltrating mononuclear cells. The expression of both Fas and FasL in the inflamed tissue makes the absence of apoptosis more striking. To address the mechanisms of this resistance to classical apoptosis in muscle cells, we have investigated the expression of the antiapoptotic molecule FLICE (Fas-associated death domain-like IL-1-converting enzyme)-inhibitory protein (FLIP) in muscle biopsies of myositis patients and in cultured human skeletal muscle cells. Using laser capture microscopy, we have shown that FLIP is expressed in the muscle fibers and on infiltrating lymphocytes of myositis biopsies. Furthermore, we have shown that FLIP, but not Bcl-2, is expressed in cultured human skeletal muscle cells stimulated with proinflammatory cytokines, and inhibition of FLIP with antisense oligonucleotides promotes significant cleavage of poly(ADP-ribose) polymerase autoantigen, a sensitive indicator of apoptosis. These studies strongly suggest that the resistance of muscle to Fas-mediated apoptosis is due to the expression of FLIP in muscle cells in the inflammatory environment in myositis.
Vδ Repertoire During Thymic Ontogeny Suggests Three Novel Waves of γδ TCR Expression
Authors: Weber-Arden Julia , Wilbert Oliver M. , Kabelitz Dieter , Arden Bernhard
Published: April 2014

Taking advantage of a PCR technique that allows amplification of all variable region genes with equal efficiency, we defined three novel waves of TCR δ-chain transcription during thymic ontogeny. The canonical DV101-D2-J2 rearrangement was confined to a narrow window from days 14 to 18 of gestation, indicating that the postulated two consecutive γδ precursor waves bearing this canonical DV101 rearrangement will coincide on day 16. Neonatal δ-chain transcripts used a second wave of diverse Vα gene segments that are exclusively located in the δ locus-proximal gene cluster of intermingled single members of different Vα subfamilies. In the adult, only expression of a clan of three homologous subfamilies, ADV7, DV104, and ADV17, persists. The members of the ADV7 subfamily are also scattered across the α locus, but their usage does not show the position-dependent bias of the other Vα-to-δ rearrangements.
Inosine Inhibits Inflammatory Cytokine Production by a Posttranscriptional Mechanism and Protects Against Endotoxin-Induced Shock
Authors: Haskó György , Kuhel David G. , Németh Zoltán H. , Mabley Jon G. , Stachlewitz Robert F. , Virág László , Lohinai Zsolt , Southan Garry J. , Salzman Andrew L. , Szabó Csaba
Published: April 2014

Extracellular purines, including adenosine and ATP, are potent endogenous immunomodulatory molecules. Inosine, a degradation product of these purines, can reach high concentrations in the extracellular space under conditions associated with cellular metabolic stress such as inflammation or ischemia. In the present study, we investigated whether extracellular inosine can affect inflammatory/immune processes. In immunostimulated macrophages and spleen cells, inosine potently inhibited the production of the proinflammatory cytokines TNF-α, IL-1, IL-12, macrophage-inflammatory protein-1α, and IFN-γ, but failed to alter the production of the anti-inflammatory cytokine IL-10. The effect of inosine did not require cellular uptake by nucleoside transporters and was partially reversed by blockade of adenosine A1 and A2 receptors. Inosine inhibited cytokine production by a posttranscriptional mechanism. The activity of inosine was independent of activation of the p38 and p42/p44 mitogen-activated protein kinases, the phosphorylation of the c-Jun terminal kinase, the degradation of inhibitory factor κB, and elevation of intracellular cAMP. Inosine suppressed proinflammatory cytokine production and mortality in a mouse endotoxemic model. Taken together, inosine has multiple anti-inflammatory effects. These findings, coupled with the fact that inosine has very low toxicity, suggest that this agent may be useful in the treatment of inflammatory/ischemic diseases.
Modulation of Human Neutrophil Responses to CD32 Cross-Linking by Serine/Threonine Phosphatase Inhibitors: Cross-Talk Between Serine/Threonine and Tyrosine Phosphorylation
Authors: Rollet-Labelle Emmanuelle , Gilbert Caroline , Naccache Paul H.
Published: April 2014

The interplay between serine/threonine and tyrosine phosphorylation was studied in human neutrophils. The direct effects of calyculin and okadaic acid, potent inhibitors of PP1 and PP2A serine/threonine phosphatases, on the patterns of neutrophil phosphorylation, and their effects on the responses of neutrophils to CD32 cross-linking were monitored. After a 2-min incubation with 10−6 M calyculin, a transient tyrosine phosphorylation of a subset of proteins, among which Cbl and Syk, was observed. After a longer incubation (>5 min) with calyculin, concomitant with an accumulation of serine and threonine phosphorylation, neutrophil responses to CD32 cross-linking were selectively altered. Tyrosine phosphorylation of Cbl in response to CD32 cross-linking was inhibited by calyculin, and this inhibition was linked with a slower electrophoretic mobility of Cbl as a consequence of its phosphorylation on serine/threonine residues. However, tyrosine phosphorylation of Syk and of the receptor itself were not affected. Furthermore, the mobilization of intracellular calcium stimulated by CD32 cross-linking was totally abrogated by calyculin. Finally, the stimulation of superoxide production observed in response to CD32 cross-linking was enhanced in calyculin-treated cells. These results suggest that serine/threonine phosphorylation events regulate the signaling pathways activated by CD32 cross-linking in neutrophils and identify a novel mechanism of modulation of the functional responsiveness of human neutrophils to CD32 cross-linking.
Cyclooxygenase-2-Derived Prostaglandin E2 and Lipoxin A4 Accelerate Resolution of Allergic Edema inAngiostrongylus costaricensis-Infected Rats: Relationship with Concurrent Eosinophilia
Authors: Bandeira-Melo Christianne , Serra Magda F. , Diaz Bruno L. , Cordeiro Renato S. B. , Silva Patricia M. R. , Lenzi Henrique L. , Bakhle Y. S. , Serhan Charles N. , Martins Marco A.
Published: April 2014

In noninfected rats, challenge with allergen following local IgE sensitization induced a pleurisy marked by intense protein exudation that plateaued from 30 min to 4 h after challenge, reducing thereafter. Infection of rats with Angiostrongylus costaricensis induced a 5-fold increase in blood eosinophil numbers by 25 days postinfection, whereas the numbers of eosinophils in the pleural cavity ranged from normal to a weak increase. In infected rats, identically sensitized, challenge with Ag induced a much shorter duration of pleural edema with complete resolution by 4 h, but no change in the early edema response. In parallel, infection increased the number of eosinophils recovered from the pleural cavity at 4 h, but not at 30 min, following allergen challenge. Pretreatment with IL-5 (100 IU/kg, i.v.) also increased eosinophil numbers in blood and, after allergen challenge, shortened the duration of the pleural edema and increased pleural eosinophil numbers. There were increases in the levels of both PGE2 and lipoxin A4 (LXA4) in pleural exudate. Selective cyclooxygenase (COX)-2 inhibitors, NS-398, meloxicam, and SC-236, did not alter pleural eosinophilia, but reversed the curtailment of the edema in either infected or IL-5-pretreated rats. Pretreatment of noninfected animals with the PGE analogue, misoprostol, or two stable LXA4 analogues did not alter the magnitude of pleural exudation response, but clearly shortened its duration. These results indicate that the early resolution of allergic pleural edema observed during A. costaricensis infection coincided with a selective local eosinophilia and seemed to be mediated by COX-2-derived PGE2 and LXA4.
Macrophage Inflammatory Protein-2 Is a Mediator of Polymorphonuclear Neutrophil Influx in Ocular Bacterial Infection
Authors: Kernacki Karen A. , Barrett Ronald P. , Hobden Jeffery A. , Hazlett Linda D.
Published: April 2014

Polymorphonuclear neutrophils (PMN) in Pseudomonas aeruginosa-infected cornea are required to clear bacteria from affected tissue, yet their persistence may contribute to irreversible tissue destruction. This study examined the role of C-X-C chemokines in PMN infiltration into P. aeruginosa-infected cornea and the contribution of these mediators to disease pathology. After P. aeruginosa challenge, corneal PMN number and macrophage inflammatory protein-2 (MIP-2) and KC levels were compared in mice that are susceptible (cornea perforates) or resistant (cornea heals) to P. aeruginosa infection. While corneal PMN myeloperoxidase activity (indicator of PMN number) was similar in both groups of mice at 1 and 3 days postinfection, by 5–7 days postinfection corneas of susceptible mice contained a significantly greater number of inflammatory cells. Corneal MIP-2, but not KC, levels correlated with persistence of PMN in the cornea of susceptible mice. To test the biological relevance of these data, resistant mice were treated systemically with rMIP-2. This treatment resulted in increased corneal PMN number and significantly exacerbated corneal disease. Conversely, administration of neutralizing MIP-2 pAb to susceptible mice reduced both PMN infiltration and corneal destruction. Collectively, these findings support an important role for MIP-2 in recruitment of PMN to P. aeruginosa-infected cornea. These data also strongly suggest that a timely down-regulation of the host inflammatory response is critical for resolution of infection.
Activation of Peroxisome Proliferator-Activated Receptor γ Does Not Inhibit IL-6 or TNF-α Responses of Macrophages to Lipopolysaccharide In Vitro or In Vivo
Authors: Thieringer Rolf , Fenyk-Melody Judy E. , Le Grand Cheryl B. , Shelton Beverly A. , Detmers Patricia A. , Somers Elizabeth P. , Carbin Linda , Moller David E. , Wright Samuel D. , Berger Joel
Published: April 2014

We have investigated the potential use of peroxisome proliferator-activated receptor γ (PPARγ) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARγ agonists. Although 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-α and IL-6, four other high affinity PPARγ ligands failed to affect cytokine production. Similar results were obtained when the monocytes were allowed to differentiate in culture into macrophages that expressed significantly higher levels of PPARγ or when the murine macrophage cell line RAW 264.7 was used. Furthermore, saturating concentrations of a potent PPARγ ligand not only failed to block cytokine production, but also were unable to block the inhibitory activity of 15d-PGJ2. Thus, activation of PPARγ does not appear to inhibit the production of cytokines by either monocytes or macrophages, and the inhibitory effect observed with 15d-PGJ2 is most likely mediated by a PPARγ-independent mechanism. To examine the anti-inflammatory activity of PPARγ agonists in vivo, db/db mice were treated with a potent thiazolidinedione that lowered their elevated blood glucose and triglyceride levels as expected. When thiazolidinedione-treated mice were challenged with LPS, they displayed no suppression of cytokine production. Rather, their blood levels of TNF-α and IL-6 were elevated beyond the levels observed in control db/db mice challenged with LPS. Comparable results were obtained with the corresponding lean mice. Our data suggest that compounds capable of activating PPARγ in leukocytes will not be useful for the treatment of acute inflammation.
Molecular Analysis of CCR-3 Events in Eosinophilic Cells
Authors: Zimmermann Nives , Daugherty Bruce L. , Stark James M. , Rothenberg Marc E.
Published: April 2014

CCR-3 is a major receptor involved in regulating eosinophil trafficking. Initial analysis of chemokine receptors has demonstrated unique receptor events in different cell types, indicating the importance of investigating CCR-3 events in eosinophilic cell lines. We now report that the eosinophilic cell line, acute myelogenous leukemia (AML) 14.3D10, expresses eosinophil granule proteins and eotaxin, but has no detectable expression of eosinophil chemokine receptors. Treatment of the cell line with butyric acid and IL-5 results in a dose-dependent synergistic induction of CCR-3 and, to a lesser extent, CCR-1 and CCR-5. Interestingly, using a luciferase reporter construct under the control of the hCCR-3 promoter, the uninduced and induced cells display high, but comparable, levels of promoter activity. Differentiated AML cells developed enhanced functional activation, as indicated by adhesion to respiratory epithelial cells and chemokine-induced transepithelial migration. Chemokine signaling did not inhibit adenylate cyclase activity even though calcium transients were blocked by pertussis toxin. Additionally, chemokine-induced calcium transients were inhibited by pretreatment with PMA, but not forskolin. Eotaxin treatment of differentiated AML cells resulted in marked down-modulation of CCR-3 expression for at least 18 h. Receptor internalization was not dependent upon chronic ligand exposure and was not accompanied by receptor degradation. Thus, CCR-3 is a late differentiation marker on AML cells and uses a signal transduction pathway involving rapid and prolonged receptor internalization, calcium transients inhibitable by protein kinase C but not protein kinase A, and the paradoxical lack of inhibition of adenylate cyclase activity.
A Codominant Role of FcγRI/III and C5aR in the Reverse Arthus Reaction
Authors: Baumann Ulrich , Köhl Jörg , Tschernig Thomas , Schwerter-Strumpf Kirsten , Verbeek J. Sjef , Schmidt Reinhold E. , Gessner J. Engelbert
Published: April 2014

Recent attempts to specify the relative contribution of FcR and complement in various experimental systems of immune complex disease have led to opposing conclusions. As concluded in IgG FcRγ−/− mice, manifestation of disease is almost exclusively determined by FcγR on effector cells, arguing for a minor role of complement. In contrast, data obtained with C5aR−/− mice suggested that, dependent on the tissue site, complement is more important than FcγR. In this paper, we demonstrate that, in response to IgG immune complex formation, FcγRI/III- and C5aR-mediated pathways are both necessary and only together are they sufficient to trigger the full expression of inflammation in skin and lung. Moreover, both effector systems are not entirely independent, suggesting an interaction between FcγR and C5aR. Therefore, FcγR-mediated responses can be integrated through C5aR activation, which may explain why these two receptor pathways have previously been considered to dominate each other.
Glomerular Deposition of Immune Complexes Made with IgG2a Monoclonal Antibodies
Authors: Gonzalez Melva L. , Waxman Frank J.
Published: April 2014

The factors that determine whether immune complexes (IC) are cleared safely from the circulation or are deposited in vulnerable tissues such as glomeruli are not well defined. To better understand how IC are handled, the present study examined the fate in vivo of three model IC preparations with different immunochemical characteristics. Radiolabeled IC were constructed with murine IgG1, IgG2a, or IgG3 anti-DNP mAbs bound to DNP-BSA, designated IgG1 IC, IgG2a IC, and IgG3 IC, respectively. The IC were infused i.v. into BALB/c mice, and clearance and tissue localization of the three IC probes were compared. The results indicate that the major portion of each IC preparation was cleared from the circulation by the liver. However, compared with the other two probes, IgG2a IC were preferentially deposited in the kidney. Histologic examination revealed the presence of IgG2a IC in glomeruli. The enhanced renal uptake of IgG2a IC could not be attributed solely to such characteristics as IC size, Ag/Ab ratio, Ab charge, or affinity. However, the preferential renal deposition of IgG2a IC was abrogated by complement depletion. Thus, enhanced renal uptake in normal mice was complement dependent. These data suggest that interactions between IC and the complement system can influence the propensity of IC to deposit in tissues susceptible to IC-mediated injury.
Decay-Accelerating Factor in Guinea Pig Stomachs Following Ischemia Reperfusion Stress
Authors: Oshima Tadayuki , Okada Noriko , Joh Takashi , Sasaki Makoto , Tada Toyohiro , Matsukawa Noriyuki , Nomura Tomoyuki , Ohara Hirotaka , Itoh Makoto , Okada Hidechika
Published: April 2014

A complement regulatory protein, decay-accelerating factor (DAF, CD55), is known to protect host tissues from autologous complement activation. DAF is present on the apical side of human gastric epithelial cells, and its expression increases during gastritis. To develop an animal model for analysis of DAF expression on gastric cells, a mAb to guinea pig DAF was successfully used. Although DAF expression in the mucosal epithelium of the stomach is weak, as judged by immunohistochemical staining with the mAb, it was temporarily up-regulated at 12 and 24 h, and at 3 days after ischemia reperfusion (I/R) (p < 0.05). The DAF mRNA level in gastric tissues was determined by Northern blot analysis and found to be highest at 6 h after I/R, returning to the baseline at 24 h. Strong DAF mRNA expression was observed in the cytoplasm of cells beneath the eroded tissues 6 h after I/R. In guinea pigs, alternative splicing of DAF mRNA generates both GPI-anchored types and transmembrane types of DAF. RT-PCR analysis revealed that mRNAs of the transmembrane types had become significantly dominant by 6 h after I/R, whereas levels for the GPI-anchored types remained unchanged. In guinea pigs depleted of complement by cobra venom factor treatment, the area of erosion and the up-regulation of DAF expression in gastric epithelial cells after I/R were significantly limited compared with the normocomplementemic group, indicating that DAF may be up-regulated by an inflammatory stress.
Soluble IL-4 Receptor Inhibits Airway Inflammation Following Allergen Challenge in a Mouse Model of Asthma
Authors: Henderson William R. , Chi Emil Y. , Maliszewski Charles R.
Published: April 2014

In vitro and in vivo studies, in both animal models and human asthmatics, have implicated IL-4 as an important inflammatory mediator in asthma. In a murine asthma model, we examined the anti-inflammatory activities of soluble IL-4R (sIL-4R). In this model, mice sensitized to OVA by i.p. and intranasal (i.n.) routes are challenged with the allergen by i.n. administration. The OVA challenge elicits an eosinophil infiltration into the lungs, with widespread mucus occlusion of the airways, and results in bronchial hyperreactivity. sIL-4R (0.1–100 μg) was administered by either i.n. or i.p. routes before OVA challenge in OVA-sensitized mice. Both blood and bronchoalveolar lavage fluid levels of sIL-4R were significantly elevated compared with controls by i.n. delivery of 100 μg sIL-4R; i.p. delivery of 100 μg sIL-4R only raised blood levels of sIL-4R. The i.n. administration of 100 μg sIL-4R before allergen challenge significantly reduced late phase pulmonary inflammation, blocking airway eosinophil infiltration, VCAM-1 expression, and mucus hypersecretion. In contrast, i.p. delivery of 100 μg sIL-4R inhibited only the influx of eosinophils into the lungs, but not airway mucus release. Furthermore, sIL-4R treatment by either i.n. or i.p. routes did not reduce airway hyperreactivity in response to methacholine challenge. Thus, elevating airway levels of sIL-4R through the administration of exogenous sIL-4R is effective in blocking the late phase pulmonary inflammation that occurs in this murine allergen-challenge asthma model. These results suggest that sIL-4R may have beneficial anti-inflammatory effects in asthmatic patients.
Differential Roles of IL-18 in Allergic Airway Disease: Induction of Eotaxin by Resident Cell Populations Exacerbates Eosinophil Accumulation
Authors: Campbell Emma , Kunkel Steven L. , Strieter Robert M. , Lukacs Nicholas W.
Published: April 2014

Cytokine regulation during an allergic response can dictate the severity of the inflammation and resulting injury. In the present study, we have examined the systemic and local effects of IL-18, a Th1-associated cytokine, on a cockroach allergen-induced airway response. In initial studies, temporal increases in IL-18 levels were observed within the lungs. When IL-18 was neutralized systemically the allergen-associated eosinophil accumulation was significantly accelerated 5-fold by 8 h postchallenge, suggesting a regulatory role for IL-18. Recombinant IL-18 (200 ng) was instilled into the airway at the time of allergen challenge to examine whether a direct impact on local eosinophil accumulation could be induced. When IL-18 was instilled, a significant increase in peribronchial eosinophil accumulation was observed in allergic mice as well as in nonallergic mice. A possible mechanism was observed in a significant increase in eotaxin, but not other eosinophil chemotactic factors, in bronchoalveolar lavage fluid after IL-18 instillation. The role of eotaxin was confirmed using eotaxin −/− mice, which demonstrated significantly less eosinophil accumulation compared with littermate controls. IL-18 was subsequently shown to induce eotaxin production from bronchial epithelial cells and isolated macrophages in in vitro assays. The clinical relevance of these findings was determined in treated mice and demonstrated that neutralization of IL-18 exacerbated, whereas exogenous IL-18 had no effect on airway hyperreactivity. Altogether, these data demonstrate that IL-18 may have multiple functions during an immune response that differ depending upon the local or systemic effects.
Myelin-Associated Oligodendrocytic Basic Protein: Identification of an Encephalitogenic Epitope and Association with Multiple Sclerosis
Authors: Holz Andreas , Bielekova Bibiana , Martin Roland , Oldstone Michael B. A.
Published: April 2014

Myelin-associated oligodendrocytic basic protein (MOBP) is an abundant myelin constituent expressed exclusively by oligodendrocytes, the myelin-forming cells of the CNS. We report that MOBP causes experimental allergic encephalomyelitis and is associated with multiple sclerosis. First, we note that purified recombinant MOBP inoculated into SJL/J mice produces CNS disease. Tests of overlapping peptides spanning the murine MOBP molecule map the encephalitogenic site to amino acids 37–60. MOBP-induced experimental allergic encephalomyelitis shows a severe clinical course and is characterized by a prominent CD4+ T lymphocyte infiltration and a lesser presence of CD8+ T cells and microglia/macrophages around vessels and in the white matter of the CNS. Second, PBL obtained from patients with relapsing/remitting multiple sclerosis mount a proliferative response to human MOBP, especially at amino acids 21–39. This response equals or exceeds the response to myelin basic protein and an influenza virus hemagglutinin peptide, both serving as internal controls. Thus, a novel myelin Ag, MOBP aa 37–60, plays a role in rodent autoimmune CNS disease, and its human MOBP counterpart is associated with the human demyelinating disease multiple sclerosis.
Rheumatoid Arthritis Synovial Stromal Cells Inhibit Apoptosis and Up-Regulate Bcl-xL Expression by B Cells in a CD49/CD29-CD106-Dependent Mechanism
Authors: Hayashida Kenji , Shimaoka Yasunori , Ochi Takahiro , Lipsky Peter E
Published: April 2014

Inflammatory sites, such as rheumatoid arthritis (RA) synovial tissue, contain large numbers of activated B cells and plasma cells. However, the mechanisms maintaining B cell viability and promoting their differentiation are not known, but interactions with stromal cells may play a role. To examine this, purified human peripheral B cells were cultured with a stromal cell line (SCL) derived from RA synovial tissue, and the effects on apoptosis and expression of Bcl-2-related proteins were analyzed. As a control, B cells were also cultured with SCL from osteoarthritis synovium or skin fibroblasts. B cells cultured with medium alone underwent spontaneous apoptosis. However, B cells cultured with RA SCL cells exhibited less apoptosis and greater viability. Although SCL from osteoarthritis synovium and skin fibroblasts also rescued B cells from apoptosis, they were less effective than RA SCL. B cell expression of Bcl-xL was markedly increased by RA SCL in a contact-dependent manner, whereas B cell expression of Bcl-2 was unaffected. Protection of B cells from apoptosis and up-regulation of Bcl-xL by RA SCL were both blocked by mAbs to CD106 (VCAM-1), but not CD54 (ICAM-1). Furthermore, cross-linking of CD49d/CD29 (very late Ag-4) on the surface of B cells rescued them from apoptosis and up-regulated Bcl-xL expression. These results indicate that SCL derived from RA synovial tissue play a role in promoting B cell survival by inducing Bcl-xL expression and blocking B cell apoptosis in a CD49d/CD29-CD106-dependent manner.
Therapeutic Potential of Phosphodiesterase-4 and -3 Inhibitors in Th1-Mediated Autoimmune Diseases
Authors: Bielekova Bibiana , Lincoln Anne , McFarland Henry , Martin Roland
Published: April 2014

Phosphodiesterase-4 (PDE4) inhibitors have the potential to modulate immune responses from the Th1 toward the Th2 phenotype and are considered candidate therapies for Th1-mediated autoimmune disorders. However, depending on the model and cell types employed, studies of atopic individuals have come to the opposite conclusion, i.e., that PDE inhibitors may be beneficial in asthma. Using in vitro immunopharmacologic techniques we analyzed the effects of PDE4 and PDE3 inhibitors on human immune cells to address these discrepancies and broaden our understanding of their mechanism of action. Our results indicate that PDE inhibitors have complex inhibitory effects within in vivo achievable concentration ranges on Th1-mediated immunity, whereas Th2-mediated responses are mostly unaffected or enhanced. The Th2 skewing of the developing immune response is explained by the effects of PDE inhibitors on several factors contributing to T cell priming: the cytokine milieu; the type of costimulatory signal, i.e., up-regulation of CD86 and down-regulation of CD80; and the Ag avidity. The combination of PDE4 and PDE3 inhibitors expresses synergistic effects and may broaden the therapeutic window. Finally, we observed a differential sensitivity to PDE inhibition in autoreactive vs foreign Ag-specific T cells and cells derived from multiple sclerosis patients vs those derived from healthy donors. This suggests that PDE inhibition weakens the strength of the T cell stimulus and corrects the underlying disease-associated cytokine skew in T cell-mediated autoimmune disorders. These new findings broaden the understanding of the immunomodulatory actions of PDE inhibitors and underscore their promising drug profile for the treatment of autoimmune disorders.
Induction of Potent Antitumor CTL Responses by Recombinant Vaccinia Encoding a Melan-A Peptide Analogue
Authors: Valmori Danila , Lévy Frederic , Miconnet Isabelle , Zajac Paul , Spagnoli Giulio C. , Rimoldi Donata , Liénard Danielle , Cerundolo Vincenzo , Cerottini Jean-Charles , Romero Pedro
Published: April 2014

There is considerable interest in the development of vaccination strategies that would elicit strong tumor-specific CTL responses in cancer patients. One strategy consists of using recombinant viruses encoding amino acid sequences corresponding to natural CTL-defined peptide from tumor Ags as immunogens. However, studies with synthetic tumor antigenic peptides have demonstrated that introduction of single amino acid substitutions may dramatically increase their immunogenicity. In this study we have used a well-defined human melanoma tumor Ag system to test the possibility of translating the immunological potency of synthetic tumor antigenic peptide analogues into recombinant vaccinia viruses carrying constructs with the appropriate nucleotide substitutions. Our results indicate that the use of a mutated minigene construct directing the expression of a modified melanoma tumor Ag leads to improved Ag recognition and, more importantly, to enhanced immunogenicity. Thus, recombinant vaccinia viruses containing mutated minigene sequences may lead to new strategies for the induction of strong tumor-specific CTL responses in cancer patients.
Cutting Edge: Cationic Antimicrobial Peptides Block the Binding of Lipopolysaccharide (LPS) to LPS Binding Protein
Authors: Scott Monisha G. , Vreugdenhil Anita C. E. , Buurman Wim A. , Hancock Robert E. W. , Gold Michael R.
Published: April 2014

We investigated the mechanism by which cationic antimicrobial peptides block the activation of macrophages by LPS. The initial step in LPS signaling is the transfer of LPS to CD14 by LPS binding protein (LBP). Because many cationic antimicrobial peptides bind LPS, we asked whether these peptides block the binding of LPS to LBP. Using an assay that measures the binding of LPS to immobilized LBP, we show for the first time that a variety of structurally diverse cationic antimicrobial peptides block the interaction of LPS with LBP. The relative ability of different cationic peptides to block the binding of LPS to LBP correlated with their ability to block LPS-induced TNF-α production by the RAW 264.7 macrophage cell line.
Cutting Edge: Preferentially the R-Stereoisomer of the Mycoplasmal Lipopeptide Macrophage-Activating Lipopeptide-2 Activates Immune Cells Through a Toll-Like Receptor 2- and MyD88-Dependent Signaling Pathway
Authors: Takeuchi Osamu , Kaufmann Andreas , Grote Karsten , Kawai Taro , Hoshino Katsuaki , Morr Michael , Mühlradt Peter F. , Akira Shizuo
Published: April 2014

Mycoplasmas and their membranes are potent activators of macrophages, the active principle being lipoproteins and lipopeptides. Two stereoisomers of the mycoplasmal lipopeptide macrophage-activating lipopeptide-2 (MALP-2) differing in the configuration of the lipid moiety were synthesized and compared in their macrophage-activating potential, the R-MALP being >100 times more active than the S-MALP in stimulating the release of cytokines, chemokines, and NO. To assess the role of the Toll-like receptor (TLR) family in mycoplasmal lipopeptide signaling, the MALP-2-mediated responses were analyzed using macrophages from wild-type, TLR2-, TLR4-, and MyD88-deficient mice. TLR2- and MyD88-deficient cells showed severely impaired cytokine productions in response to R- and S-MALP. The MALP-induced activation of intracellular signaling molecules was fully dependent on both TLR2 and MyD88. There was a strong preference for the R-MALP in the recognition by its functional receptor, TLR2.
Cutting Edge: Heat Shock Protein 60 Is a Putative Endogenous Ligand of the Toll-Like Receptor-4 Complex
Authors: Ohashi Koji , Burkart Volker , Flohé Stefanie , Kolb Hubert
Published: April 2014

Human heat shock protein 60 (hsp60) elicits a potent proinflammatory response in cells of the innate immune system and therefore has been proposed as a danger signal of stressed or damaged cells. We report here that macrophages of C3H/HeJ mice, carrying a mutant Toll-like-receptor (Tlr) 4 are nonresponsive to hsp60. Both the induction of TNF-α and NO formation were found dependent on a functional Tlr4 whereas stimulation of macrophages by CpG DNA was Tlr4 independent. We conclude that Tlr4 mediates hsp60 signaling. This is the first report of a putative endogenous ligand of the Tlr4 complex.
Cutting Edge: CD4+ T Cell Control of CD8+ T Cell Reactivity to a Model Tumor Antigen
Authors: Surman Deborah R. , Dudley Mark E. , Overwijk Willem W. , Restifo Nicholas P.
Published: April 2014

Neoantigens resulting from the inherent genomic instability of tumor cells generally do not trigger immune recognition. Similarly, transfection of tumors with model Ags often fails to elicit CD8+ T cell responses or alter a tumor’s growth rate or lethality. We report here that the adoptive transfer of activated Th1-type CD4+ T cells specific for a model tumor Ag results in the de novo generation of CD8+ T cells with specificity to that Ag and concomitant tumor destruction. The anti-tumor effects of the CD4+ T cells required the presence of both MHC class I and class II on host cells, as evidenced by experiments in knockout mice, suggesting that CD4+ T cells enhanced the ability of host APC to activate endogenous CD8+ T cells. These results indicate that the apparent inability of tumor cells expressing highly immunogenic epitopes to activate tumor-specific CD8+ T cells can be altered by activated CD4+ T cells.
IFN-α-Expressing Tumor Cells Enhance Generation and Promote Survival of Tumor-Specific CTLs
Authors: Hiroishi Kazumasa , Tüting Thomas , Lotze Michael T.
Published: April 2014

IFN-α gene therapy has been successfully applied in several tumor models. Our studies involving the murine colorectal adenocarcinoma cell line MC38 confirm that IFN-α transduction of a poorly immunogenic tumor cell reduces tumorigenicity and leads to long-lasting tumor immunity. To investigate the effect of IFN-α transduction on the development of antitumor immune responses, we restimulated splenocytes from MC38-immune mice in vitro. Detection of MC38-specific cytotoxicity was markedly enhanced when murine IFN-α2-transduced MC38 (MC38-IFNα) or CD80-transduced MC38 (MC38-CD80) was used for restimulation compared with wild type (MC38-WT) or neomycin resistance gene-transduced MC38 (MC38-Neo) cells. MC38-specific CD8+ CTL line and clone were established from splenocytes of mouse immunized with MC38-IFNα. Stimulation with MC38-IFNα as well as MC38-CD80 enhanced the proliferation of MC38-specific CTLs in vitro much more effectively than stimulation with WT or MC38-Neo (p < 0.05). Coincubation of MC38-specific CTLs with MC38-IFNα or MC38-CD80 resulted in significantly less DNA fragmentation (8.0% and 12.8%, respectively) compared with coincubation of the CTLs with MC38-WT (43.5%; p < 0.001) or MC38-Neo cells (38.1%; p < 0.003). These results suggest that prevention of apoptotic cell death in tumor-specific CTLs may be one mechanism by which IFN-α-expressing tumor cells can promote the generation of antitumor immunity. The effect of IFN-α on CTLs appears to be similar to that of CD80, which also prevents apoptotic cell death after stimulation of T lymphocytes.
Presentation of IFN-γ to Nitric Oxide-Producing Cells: A Novel Function for Mast Cells
Authors: Brooks Bernadette , Briggs David M. , Eastmond Nigel C. , Fernig David G. , Coleman John W.
Published: April 2014

We report that mast cells can bind and present IFN-γ in a functionally active form to macrophages. Flow-cytometric analysis revealed that biotinylated IFN-γ bound equally well to purified peritoneal mast cells from both IFN-γR knockout and wild-type mice, indicating a non-IFN-γR binding site. Purified peritoneal mast cells, loaded with IFN-γ for 30 min and washed, were able to induce NO synthesis by peritoneal macrophages. This response required cell contact and expression of IFN-γR on the responding macrophages, but not the mast cells. Human HMC-1 mast cells were also able to present IFN-γ to mouse macrophages. Enzyme treatment of mouse mast cells revealed that binding of IFN-γ was predominantly to chondroitin sulfate B (dermatan sulfate). Binding of IFN-γ to dermatan sulfate was confirmed by inhibition ELISA. This study demonstrates for the first time that mast cells can present IFN-γ to other cells via glycosaminoglycans. Mast cells may act as a reservoir of surface-stored functionally active cytokines.
Modulation of Th2 Responses by Peptide Analogues in a Murine Model of Allergic Asthma: Amelioration or Deterioration of the Disease Process Depends on the Th1 or Th2 Skewing Characteristics of the Therapeutic Peptide
Authors: Janssen Edith M. , van Oosterhout Antoon J. M. , van Rensen Annemiek J. M. L. , van Eden Willem , Nijkamp Frans P. , Wauben Marca H. M.
Published: April 2014

Allergen-specific CD4+ Th2 cells play an important role in the immunological processes of allergic asthma. Previously we have shown that, by using the immunodominant epitope OVA323–339, peptide immunotherapy in a murine model of OVA induced allergic asthma, stimulated OVA-specific Th2 cells, and deteriorated airway hyperresponsiveness and eosinophilia. In the present study, we defined four modulatory peptide analogues of OVA323–339 with comparable MHC class II binding affinity. These peptide analogues were used for immunotherapy by s.c. injection in OVA-sensitized mice before OVA challenge. Compared with vehicle-treated mice, treatment with the Th2-skewing wild-type peptide and a Th2-skewing partial agonistic peptide (335N-A) dramatically increased airway eosinophilia upon OVA challenge. In contrast, treatment with a Th1-skewing peptide analogue (336E-A) resulted in a significant decrease in airway eosinophilia and OVA-specific IL-4 and IL-5 production. Our data show for the first time that a Th1-skewing peptide analogue of a dominant allergen epitope can modulate allergen-specific Th2 effector cells in an allergic response in vivo. Furthermore, these data suggest that the use of Th1-skewing peptides instead of wild-type peptide may improve peptide immunotherapy and may contribute to the development of a successful and safe immunotherapy for allergic patients.
HIV-1 gp120 Induces IL-4 and IL-13 Release from Human FcεRI+ Cells Through Interaction with the VH3 Region of IgE
Authors: Patella Vincenzo , Florio Giovanni , Petraroli Angelica , Marone Gianni
Published: April 2014

HIV-1 glycoprotein (gp) 120 from different clades is a potent stimulus for IL-4 and IL-13 release from basophils purified from healthy individuals seronegative for Abs to HIV-1 and HIV-2. IL-4 mRNA, constitutively present in basophils, was increased after stimulation by gp120 and was inhibited cyclosporin A and tacrolimus. IL-4 and IL-13 secretion from basophils activated by gp120 was not correlated. There was a correlation between the maximum gp120- and anti-IgE-induced IL-4 release from basophils. The average t1/2 gp120-induced IL-4 release was lower than for IL-13 release. Basophils from which IgE had been dissociated by brief exposure to lactic acid no longer released IL-4 in response to gp120 or to anti-IgE. The response to a mAb cross-linking the α-chain of high-affinity receptor for IgE (FcεRI) was unaffected by this treatment. Three human VH3+ monoclonal IgM inhibited gp120-induced secretion of IL-4 from basophils. In contrast, VH6+ monoclonal IgM did not inhibit the release of IL-4 induced by gp120. Synthetic peptides distant from the NH2 and COOH termini of gp120MN inhibited the activating property of gp120MN. These results indicate that gp120, which acts as a viral superantigen, interacts with the VH3 region of IgE to induce the release of IL-4 and IL-13 from human FcεRI+ cells.
The Use of HLA A2.1/p53 Peptide Tetramers to Visualize the Impact of Self Tolerance on the TCR Repertoire
Authors: Hernández Javier , Lee Peter P. , Davis Mark M. , Sherman Linda A.
Published: April 2014

p53 is an attractive target for cancer immunotherapy since it is overexpressed in half of all tumors. However, it is also expressed in normal lymphoid tissue, and self tolerance leaves a p53-specific repertoire purged of high avidity CTL. To better understand the mechanism of tolerance and the basis for such low avidity interaction, p53-specific CTL from p53 deficient (p53−) and sufficient (p53+) A2.1/Kb transgenic mice were compared with respect to their ability to bind HLA-A2.1 tetramers containing cognate murine p53 peptide Ag, p53 261–269. Since the murine CD8 molecule cannot interact with human HLA-A2.1, this tests the ability of the TCR to bind the A2.1/peptide complex tetramer. CTL from p53− mice demonstrated strong binding of such A2.1/p53 261–269 tetramers; however, the CTL from tolerant p53+ mice were devoid of tetramer-binding CD8+ T cells. Examination of TCR expression at the clonal level revealed that CTL from p53+ and p53− mice each expressed comparable levels of the p53-specific TCR. These results indicate that normal expression of p53 promotes elimination of T cells expressing TCRs with sufficient affinity to achieve stable binding of the A2.1/p53 261–269 tetramers.