bluuebunny/crossref_metadata_2025 huggingface dataset:
The Inhibitory Receptor IRp60 (CD300a) Is Expressed and Functional on Human Mast Cells
Authors: Bachelet Ido , Munitz Ariel , Moretta Alessandro , Moretta Lorenzo , Levi-Schaffer Francesca
Published: April 2014

Mast cell-mediated responses are likely to be regulated by the cross talk between activatory and inhibitory signals. We have screened human cord blood mast cells for recently characterized inhibitory receptors expressed on NK cells. We found that IRp60, an Ig superfamily member, is expressed on human mast cells. On NK cells, IRp60 cross-linking leads to the inhibition of cytotoxic activity vs target cells in vitro. IRp60 is constitutively expressed on mast cells but is down-regulated in vitro by the eosinophil proteins major basic protein and eosinophil-derived neurotoxin. An immune complex-mediated cross-linking of IRp60 led to inhibition of IgE-induced degranulation and stem cell factor-mediated survival via a mechanism involving tyrosine phosphorylation, phosphatase recruitment, and termination of cellular calcium influx. To evaluate the role of IRp60 in regulation of allergic responses in vivo, a murine model of allergic peritonitis was used in which the murine homolog of IRp60, LMIR1, was neutralized in BALB/c mice by mAbs. This neutralization led to a significantly augmented release of inflammatory mediators and eosinophilic infiltration. These data demonstrate a novel pathway for the regulation of human mast cell function and allergic responses, indicating IRp60 as a candidate target for future treatment of allergic and mast cell-associated diseases.
Disparate Regulation and Function of the Class A Scavenger Receptors SR-AI/II and MARCO
Authors: Józefowski Szczepan , Arredouani Mohamed , Sulahian Timothy , Kobzik Lester
Published: April 2014

The macrophage class A scavenger receptors, macrophage receptor with a collagenous structure (MARCO) and type I/II class A scavenger receptor (SR-AI/II), share structural features and roles in host defense, but little is known about their regulation and signaling properties. Ligation of MARCO on mouse thioglycollate-elicited peritoneal macrophages (PEMs) with immobilized mAb costimulated IL-12 production, in contrast to previously reported inhibition by SR-AI/II. PEMs from MARCO-deficient mice exhibited 2.7 times lower IL-12 production in responses to stimulation with LPS and IFN-γ and lack of significant IL-12 production on stimulation with LPS alone. Conversely, SR-AI/II-deficient PEMs produced 2.4 and 1.8 times more IL-12 than wild-type PEMs in response to LPS or LPS and IFN-γ, respectively. Corresponding differences in regulation of SR-A and MARCO expression were also observed. Th1 adjuvants (LPS, a CpG motif-containing oligodeoxynucleotide (CpG-ODN), IL-12, and GM-CSF) increased, whereas Th2-polarizing factors (IL-4, M-CSF, and non-CpG ODN) decreased expression of MARCO on J774 macrophage-like cells. Expression of SR-A was regulated in the opposite manner to MARCO or not affected. Whereas MARCO was involved in opsonin-independent phagocytosis in CpG-ODN-pretreated but not in IL-4-pretreated J774 cells, anti-SR-A Abs inhibited particle uptake in untreated and IL-4-pretreated but not in CpG-ODN-pretreated cells. SR-A and MARCO are regulated differently and mediate distinct negative and positive effects on IL-12 production in macrophages. These differences may contribute to sustained Th1 or Th2 polarization of ongoing immune responses.
CD11b+/Gr-1+ Immature Myeloid Cells Mediate Suppression of T Cells in Mice Bearing Tumors of IL-1β-Secreting Cells
Authors: Song Xiaoping , Krelin Yakov , Dvorkin Tatyana , Bjorkdahl Olle , Segal Shraga , Dinarello Charles A. , Voronov Elena , Apte Ron N.
Published: April 2014

Tumor cells secreting IL-1β are invasive and metastatic, more than the parental line or control mock-transfected cells, and concomitantly induce in mice general immune suppression of T cell responses. Suppression strongly correlates with accumulation in the peripheral blood and spleen of CD11b+/Gr-1+ immature myeloid cells and hematological alterations, such as splenomegaly, leukocytosis, and anemia. Resection of large tumors of IL-1β-secreting cells restored immune reactivity and hematological alterations within 7–10 days. Treatment of tumor-bearing mice with the physiological inhibitor of IL-1, the IL-1R antagonist, reduced tumor growth and attenuated the hematological alterations. Depletion of CD11b+/Gr-1+ immature myeloid cells from splenocytes of tumor-bearing mice abrogated suppression. Despite tumor-mediated suppression, resection of large tumors of IL-1β-secreting cells, followed by a challenge with the wild-type parental cells, induced resistance in mice; protection was not observed in mice bearing tumors of mock-transfected fibrosarcoma cells. Altogether, we show in this study that tumor-derived IL-1β, in addition to its proinflammatory effects on tumor invasiveness, induces in the host hematological alterations and tumor-mediated suppression. Furthermore, the antitumor effectiveness of the IL-1R antagonist was also shown to encompass restoration of hematological alterations, in addition to its favorable effects on tumor invasiveness and angiogenesis that have previously been described by us.
IL-18 Induces Monocyte Chemotactic Protein-1 Production in Macrophages through the Phosphatidylinositol 3-Kinase/Akt and MEK/ERK1/2 Pathways
Authors: Yoo Jae Kwang , Kwon Hyokjoon , Khil Lee-Young , Zhang Li , Jun Hee-Sook , Yoon Ji-Won
Published: April 2014

Macrophages are activated during an inflammatory response and produce multiple inflammatory cytokines. IL-18 is one of the most important innate cytokines produced from macrophages in the early stages of the inflammatory immune response. Monocyte chemoattractant protein (MCP-1) is expressed in many inflammatory diseases such as multiple sclerosis and rheumatoid arthritis, and its expression is correlated with the severity of the disease. Both IL-18 and MCP-1 have been shown to be involved in inflammatory immune responses. However, it has been unclear whether IL-18 is involved in the induction of MCP-1. This investigation was initiated to determine whether IL-18 can induce MCP-1 production, and if so, by which signal transduction pathways. We found that IL-18 induced the production of MCP-1 in macrophages, which was IL-12-independent and was not mediated by autocrine cytokines such as IFN-γ or TNF-α. We then examined signal transduction pathways involved in IL-18-induced MCP-1 production. We found that IL-18 did not activate the IκB kinase/NF-κB pathway, evidenced by no degradation of IκBα and no translocation of NF-κB p65 to the nucleus in IL-18-stimulated macrophages. Instead, IL-18 activated the PI3K/Akt and MEK/ERK1/2 pathways. Inhibition of either of these pathways attenuated MCP-1 production in macrophages, and inhibition of both signaling pathways resulted in the complete inhibition of MCP-1 production. On the basis of these observations, we conclude that IL-18 induces MCP-1 production through the PI3K/Akt and MEK/ERK1/2 pathways in macrophages.
Topical Superantigen Exposure Induces Epidermal Accumulation of CD8+ T Cells, a Mixed Th1/Th2-Type Dermatitis and Vigorous Production of IgE Antibodies in the Murine Model of Atopic Dermatitis
Authors: Savinko Terhi , Lauerma Antti , Lehtimäki Sari , Gombert Michael , Majuri Marja-Leena , Fyhrquist-Vanni Nanna , Dieu-Nosjean Marie-Caroline , Kemeny Lajos , Wolff Henrik , Homey Bernhard , Alenius Harri
Published: April 2014

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVβ8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-γ protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.
Aberrant Expression of the Autoantigen Heterogeneous Nuclear Ribonucleoprotein-A2 (RA33) and Spontaneous Formation of Rheumatoid Arthritis-Associated Anti-RA33 Autoantibodies in TNF-α Transgenic Mice
Authors: Hayer Silvia , Tohidast-Akrad Makiyeh , Haralambous Silva , Jahn-Schmid Beatrice , Skriner Karl , Trembleau Sylvie , Dumortier Hélène , Pinol-Roma Serafin , Redlich Kurt , Schett Georg , Muller Sylviane , Kollias George , Smolen Josef , Steiner Günter
Published: April 2014

Human TNF-α transgenic (hTNFtg) mice develop erosive arthritis closely resembling rheumatoid arthritis (RA). To investigate mechanisms leading to pathological autoimmune reactions in RA, we examined hTNFtg animals for the presence of RA-associated autoantibodies including Abs to citrullinated epitopes (anti-cyclic citrullinated peptide), heterogeneous nuclear ribonucleoprotein (hnRNP)-A2 (anti-RA33), and heat shock proteins (hsp) (anti-hsp). Although IgM anti-hsp Abs were detected in 40% of hTNFtg and control mice, IgG anti-hsp Abs were rarely seen, and anti-cyclic citrullinated peptide Abs were not seen at all. In contrast, >50% of hTNFtg mice showed IgG anti-RA33 autoantibodies, which became detectable shortly after the onset of arthritis. These Abs were predominantly directed to a short epitope, which was identical with an epitope previously described in MRL/lpr mice. Incidence of anti-RA33 was significantly decreased in mice treated with the osteoclast inhibitor osteoprotegerin and also in c-fos-deficient mice lacking osteoclasts. Pronounced expression of hnRNP-A2 and a smaller splice variant was seen in joints of hTNFtg mice, whereas expression was low in control animals. Although the closely related hnRNP-A1 was also overexpressed, autoantibodies to this protein were infrequently detected. Because expression of hnRNP-A2 in thymus, spleen, brain, and lung was similar in hTNFtg and control mice, aberrant expression appeared to be restricted to the inflamed joint. Finally, immunization of hTNFtg mice with recombinant hnRNP-A2 or a peptide harboring the major B cell epitope aggravated arthritis. These findings suggest that overproduction of TNF-α leads to aberrant expression of hnRNP-A2 in the rheumatoid joint and subsequently to autoimmune reactions, which may enhance the inflammatory and destructive process.
Mcl-1 Is Essential for the Survival of Synovial Fibroblasts in Rheumatoid Arthritis
Authors: Liu Hongtao , Eksarko Polikseni , Temkin Vladislav , Haines G. Kenneth , Perlman Harris , Koch Alisa E. , Thimmapaya Bayar , Pope Richard M.
Published: April 2014

Mcl-1 is a Bcl-2-family, antiapoptotic molecule that is critical for the survival of T and B lymphocytes and macrophages; however, its role in nonhemopoietic cells remains to be fully elucidated. The current study focuses on the role of Mcl-1 in rheumatoid arthritis (RA). Mcl-1 was strongly expressed in the synovial lining and was increased in the sublining fibroblasts of patients with RA, compared with control synovial tissue. The expression of Mcl-1 in sublining fibroblasts correlated with the degree of inflammation and TNF-α, and IL-1β treatment of cultured synovial fibroblasts resulted in the increased expression of Mcl-1 at the mRNA and protein levels. Mcl-1 was critical for the survival of RA synovial fibroblasts, because the forced reduction of Mcl-1 using a Mcl-1 antisense-expressing adenoviral vector induced apoptotic cell death, which was mediated through Bax, Bak, and Bim. These observations document a critical role for Mcl-1 in protecting against apoptosis in RA and suggest that Mc1–1 is a potential therapeutic target in this disease.
CD8+ T Cell-Mediated HLA-A*0201-Restricted Cytotoxicity to Transaldolase Peptide 168–176 in Patients with Multiple Sclerosis
Authors: Niland Brian , Banki Katalin , Biddison William E. , Perl Andras
Published: April 2014

Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes and targeted by autoreactive T cells of patients with multiple sclerosis (MS). Among 14 TAL peptides with predicted HLA-A2 binding, TAL 168–176 (LLFSFAQAV, TALpep) exhibited high affinity for HLA-A2. Prevalence of HLA-A2-restricted CD8+ T cells specific for TALpep was increased in PBMC of HLA-A2+ MS patients, as compared with HLA-A2− MS patients, HLA-A2+ other neurological disease patients, and HLA-A2+ healthy donors. HLA-A*0201/TALpep tetramers detected increased frequency of TAL-specific CD8+ T cells, and precursor frequency of TAL-specific IFN-γ-producing T cells was increased in each of seven HLA-A2+ MS patients tested. Stimulation by TALpep or rTAL of PBMC from HLA-A2+ MS patients elicited killing of TALpep-pulsed HLA-A2-transfected HmyA2.1 lymphoma cells, but not HLA-A3-transfected control HmyA3.1 targets. Without peptide pulsing of targets, HLA-A2-transfected, but not control MO3.13 oligodendroglial cells, expressing high levels of endogenous TAL, were also killed by CD8+ CTL of MS patients, indicating recognition of endogenously processed TAL. TCR Vβ repertoire analysis revealed use of the TCR Vβ14 gene by T cell lines (TCL) of MS patients generated via stimulation by TAL- or TALpep-pulsed APCs. All TAL-specific TCL-binding HLA-A*0201/TALpep tetramers expressed TCR Vβ14 on the cell surface. Moreover, Ab to TCR Vβ14 abrogated cytotoxicity by HLA-A2-restricted TAL-specific TCL. Therefore, TAL-specific CTL may serve as a novel target for therapeutic intervention in patients with MS.
Simultaneous Targeting of CD3 on T Cells and CD40 on B or Dendritic Cells Augments the Antitumor Reactivity of Tumor-Primed Lymph Node Cells
Authors: Li Qiao , Grover Amelia C. , Donald Elizabeth J. , Carr Abbey , Yu Jiyun , Whitfield Joel , Nelson Mark , Takeshita Nobuhiro , Chang Alfred E.
Published: April 2014

To date, molecular targets chosen for Ab activation to generate antitumor effector cells have been confined on T cells, such as TCR/CD3, CD28, CD137 (4-1BB), CD134 (OX40), and inducible costimulator. In this report we investigated the immune function of murine tumor-draining lymph node (TDLN) cells after simultaneous Ab targeting of CD3 on T cells and CD40 on APCs. Anti-CD3 plus anti-CD40-activated TDLN cells secreted significantly higher amounts of IFN-γ, but less IL-10, compared with anti-CD3-activated cells. In adoptive immunotherapy, ligation of CD3 and CD40 resulted in the generation of more potent effector cells in mediating tumor regression. Freshly harvested TDLN cells were composed of ∼60% CD3+ T cells, 30–35% CD19+ B cells, 5% CD11c+ dendritic cells (DC), and few CD14+ or NK cells (each <3%). CD40 was distributed predominantly on B cells and DCs. Cell depletion indicated that simultaneous targeting was toward CD3 on T cells and CD40 on APCs, respectively. Elimination of APCs completely abrogated the augmented antitumor responses induced by anti-CD40. Either B cell or DC removal partially, but significantly, reduced the therapeutic efficacy conferred by CD40 engagement. Furthermore, the immunomodulation function of anti-CD40 was associated with its capability to increase IL-12 secretion while inhibiting IL-4 production. Our study establishes a role for CD40 expressed on B cells or DCs in the costimulation of TDLN cells. Eliciting antitumor activity via simultaneous targeting of CD3 on T cells and CD40 on APCs is relevant for the design of effective T cell-based cancer immunotherapy.
Quantum Proteolytic Activation of Chemokine CCL15 by Neutrophil Granulocytes Modulates Mononuclear Cell Adhesiveness
Authors: Richter Rudolf , Bistrian Roxana , Escher Sylvia , Forssmann Wolf-Georg , Vakili Jalal , Henschler Reinhard , Spodsberg Nikolaj , Frimpong-Boateng Adjoa , Forssmann Ulf
Published: April 2014

Monocyte infiltration into inflammatory sites is generally preceded by neutrophils. We show here that neutrophils may support this process by activation of CCL15, a human chemokine circulating in blood plasma. Neutrophils were found to release CCL15 proteolytic activity in the course of hemofiltration of blood from renal insufficiency patients. Processing of CCL15 immunoreactivity (IR) in the pericellular space is suggested by a lack of proteolytic activity in blood and blood filtrate, but a shift of the retention time (tR) of CCL15-IR, detected by chromatographic separation of CCL15-IR in blood and hemofiltrate. CCL15 molecules with N-terminal deletions of 23 (Δ23) and 26 (Δ26) aa were identified as main proteolytic products in hemofiltrate. Neutrophil cathepsin G was identified as the principal protease to produce Δ23 and Δ26 CCL15. Also, elastase displays CCL15 proteolytic activity and produces a Δ21 isoform. Compared with full-length CCL15, Δ23 and Δ26 isoforms displayed a significantly increased potency to induce calcium fluxes and chemotactic activity on monocytes and to induce adhesiveness of mononuclear cells to fibronectin. Thus, our findings indicate that activation of monocytes by neutrophils is at least in part induced by quantum proteolytic processing of circulating or endothelium-bound CCL15 by neutrophil cathepsin G.
CpG-Containing Oligodeoxynucleotides Act through TLR9 to Enhance the NK Cell Cytokine Response to Antibody-Coated Tumor Cells
Authors: Roda Julie M. , Parihar Robin , Carson William E.
Published: April 2014

Bacterial DNA contains a high frequency of unmethylated CpG motifs that stimulate immune cells via TLR9. NK cells express a low-affinity activating receptor for the Fc portion of IgG (FcγRIIIa), but were not thought to express TLR9 protein. The direct response of NK cells to CpG oligodeoxynucleotides (ODN) in the presence of FcR stimulation was investigated. Human NK cells cultured in the presence of CpG ODN plus immobilized IgG or Ab-coated tumor cells secreted large amounts of IFN-γ (>2000 pg/ml), whereas cells stimulated with Ab alone, CpG ODN alone, or Ab and control ODN produced negligible amounts. Enhanced secretion of IL-8, macrophage-derived chemokine, and MIP-1α was also observed after costimulation. NK cell cytokine production was not the result of interactions with APCs or their cytokine products. Flow cytometric analysis revealed that 36 ± 3.5% of human NK cells expressed basal levels of TLR9. TLR9 expression in human NK cells was confirmed by immunoblot analysis. Only TLR9-expressing NK cells responded to CpG ODN and Ab, because cytokine production was not observed in NK cells from TLR9-deficient mice. Mice receiving CpG ODN and HER2/neu-positive tumor cells treated with an anti-HER2 Ab exhibited enhanced systemic levels of IFN-γ compared with mice receiving either agent alone. TLR9−/− animals reconstituted with TLR9+/+ NK cells secreted IFN-γ in response to CpG ODN and Ab-coated tumor cells. These findings indicate that CpG ODN can directly enhance the NK cell cytokine response to Ab-coated targets via activation of TLR9.
Roles for T and NK Cells in the Innate Immune Response to Shigella flexneri
Authors: Le-Barillec Karine , Magalhaes Joao Gamelas , Corcuff Erwan , Thuizat Audrey , Sansonetti Philippe J. , Phalipon Armelle , Di Santo James P.
Published: April 2014

Shigella flexneri, an enteroinvasive Gram-negative bacterium, is responsible for the worldwide endemic form of bacillary dysentery. The host response to primary infection is characterized by the induction of an acute inflammation, which is accompanied by polymorphonuclear cell (PMN) infiltration, resulting in massive destruction of the colonic mucosa. However, PMN play a major role in the recovery from primary infection, by restricting the bacterial infection at the intestinal mucosa. In this study, we assessed the roles for T and NK cells in the control of primary S. flexneri infection, using an alymphoid mouse strain (Rag°γc°) devoid of B, T, and NK cells. Using the mouse pulmonary model of Shigella infection, we showed that alymphoid Rag°γc° mice were highly susceptible to S. flexneri infection in comparison with wild-type (wt) mice. Whereas PMN recruitment upon infection was similar, macrophage recruitment and production of proinflammatory cytokines were significantly decreased in Rag°γc° mice compared with wt mice. Upon selective engraftment of Rag°γc° mice with polyclonal αβ T cells, but not with αβ T cells from IFN-γ°, S. flexneri infection could be subsequently controlled. Rag° mice devoid of B and T cells but harboring NK cells could control infection. Local IFN-γ production by T and NK cells recruited to the lung was demonstrated in S. flexneri-infected wt mice. These data demonstrate that both αβ T cells and NK cells contribute to the early control of S. flexneri infection through amplification of an inflammatory response. This cellular lymphocyte redundancy assures IFN-γ production, which is central to innate immunity against Shigella infection.
Opposing Effects of CXCR3 and CCR5 Deficiency on CD8+ T Cell-Mediated Inflammation in the Central Nervous System of Virus-Infected Mice
Authors: de Lemos Carina , Christensen Jeanette Erbo , Nansen Anneline , Moos Torben , Lu Bao , Gerard Craig , Christensen Jan Pravsgaard , Thomsen Allan Randrup
Published: April 2014

T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice were generated and infected intracerebrally with noncytolytic lymphocytic choriomeningitis virus. Because these chemokine receptors are mostly expressed by overlapping subsets of activated CD8+ T cells, it was expected that absence of both receptors would synergistically impair effector T cell invasion and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double-deficient mice were more susceptible to intracerebral infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8+ T cell response in CXCR3/CCR5 double-deficient mice. Furthermore, while the accumulation of CD8+ T cells in the neural parenchyma was significantly delayed in both CXCR3- and CXCR3/CCR5-deficient mice, more CD8+ T cells were found in the parenchyma of double-deficient mice when these were analyzed around the time when the difference in clinical outcome becomes manifest. Taken together, these results indicate that while CXCR3 plays an important role in controlling CNS inflammation, other receptors but not CCR5 also contribute significantly. Additionally, our results suggest that CCR5 primarily functions as a negative regulator of the antiviral CD8+ T cell response.
The TLR7 Agonist Imiquimod Enhances the Anti-Melanoma Effects of a Recombinant Listeria monocytogenes Vaccine
Authors: Craft Noah , Bruhn Kevin W. , Nguyen Bidong D. , Prins Robert , Lin Jia Wei , Liau Linda M. , Miller Jeffery F.
Published: April 2014

Activation of innate immune cells through TLR triggers immunomodulating events that enhance cell-mediated immunity, raising the possibility that ligands to these receptors might act as adjuvants in conjunction with T cell activating vaccines. In this report, topical imiquimod, a synthetic TLR7 agonist, significantly enhanced the protective antitumor effects of a live, recombinant listeria vaccine against murine melanoma. This tumor protective effect was not dependent on direct application to the tumor and was associated with an increase in tumor-associated and splenic dendritic cells. Additionally, the combination of imiquimod treatment with prior vaccination led to development of localized vitiligo. These findings indicate that activation of the innate immune system with TLR ligands stimulates dendritic cell activity resulting in a bypass of peripheral tolerance and enhanced antitumor activity. The results of these studies have broad implications for future designs of immunotherapeutic vaccines against tumors and the treatment of metastatic melanoma.
Intestinal Fibroblast-Derived IL-10 Increases Survival of Mucosal T Cells by Inhibiting Growth Factor Deprivation- and Fas-Mediated Apoptosis
Authors: Ina Kenji , Kusugami Kazuo , Kawano Yasushi , Nishiwaki Tsuyoshi , Wen Zhonghui , Musso Alessandro , West Gail A. , Ohta Michio , Goto Hidemi , Fiocchi Claudio
Published: April 2014

Mucosal T cells are essential to immune tolerance in the intestine, an organ constantly exposed to large amounts of dietary and bacterial Ags. We investigated whether local fibroblasts affect mucosal T cell survival, which is critical for maintenance of immune tolerance. Coculture with autologous fibroblasts significantly increased viability of mucosal T cells by inhibiting IL-2 deprivation- and Fas-mediated apoptosis, an effect that was both contact- and secreted product-dependent. Investigation of antiapoptotic factors in the fibroblast-conditioned medium (FCM) revealed the presence of IL-10 and PGE2, but not IFN-β, IL-2, or IL-15. Although recombinant IFN-β, but not PGE2, effectively prevented T cell apoptosis, neutralizing Ab studies showed that only IL-10 blockade significantly increased T cells apoptosis, whereas neutralizing IFN-β or IFN-α failed to inhibit the antiapoptotic effect of FCM. To confirm that fibroblast-derived IL-10 was responsible for preserving mucosal T cell viability, IL-10 mRNA was demonstrated in fibroblasts by Southern blotting and RT-PCR. When FCM was submitted to HPLC fractionation, only the peak matching rIL-10 contained the antiapoptotic activity, and this was eliminated by treatment with an IL-10-neutralizing Ab. Finally, when fibroblasts were transiently transfected with IL-10 antisense oligonucleotides, the conditioned medium lost its T cell antiapoptotic effect, whereas medium from fibroblasts transfected with IFN-β antisense oligonucleotides displayed the same antiapoptotic activity of medium from untransfected fibroblasts. These results indicate that local fibroblast-derived IL-10 is critically involved in the survival of mucosal T cells, underscoring the crucial importance of studying organ-specific cells and products to define the mechanisms of immune homeostasis in specialized tissue microenvironments like the intestinal mucosa.
Cutting Edge: Chemoattractant Receptor-Homologous Molecule Expressed on TH2 Cells Plays a Restricting Role on IL-5 Production and Eosinophil Recruitment
Authors: Chevalier Eric , Stock Jeff , Fisher Tim , Dupont Mathieu , Fric Magali , Fargeau Helene , Leport Murielle , Soler Sylvain , Fabien Schmidlin , Pruniaux Marie-Pierre , Fink Michel , Bertrand Claude P. , McNeish John , Li Baiyong
Published: April 2014

PGs play key regulatory roles in inflammation and immunity. PGD2, released from mast cells and Th2 cells during allergic responses, has recently been shown to target a novel receptor, chemoattractant receptor-homologous molecule expressed TH2 cells (CRTH2), in addition to the classic PGD (DP) receptor. CRTH2 is expressed on Th2 cells and eosinophils and mediates chemotaxis of these cells to PGD2. Thus, CRTH2 is thought to be a key receptor mediating eosinophil and Th2 cell recruitment during allergic responses. To examine the role of CRTH2 in this context in vivo, we generated CRTH2 knockout mice. Surprisingly, in an allergic inflammatory model of asthma, CRTH2 knockout mice showed enhanced eosinophil recruitment into the lung compared with wild-type littermate mice. This is consistent with our observation that CRTH2 knockout cells produce significantly higher amounts of IL-5 and IL-3 in vitro. These results suggest a nonredundant role of CRTH2 in restricting eosinophilia and allergic response in vivo.
IL-21 Influences the Frequency, Phenotype, and Affinity of the Antigen-Specific CD8 T Cell Response
Authors: Li Yongqing , Bleakley Marie , Yee Cassian
Published: April 2014

IL-21, a newly described cytokine belonging to the IL-2 γ-chain receptor cytokine family (that includes IL-2, IL-7, and IL-15), has been described as an important regulator of the cellular immune response. In this study, the role of IL-21 in the generation of a human Ag-specific CD8+ T cell response is characterized by tracking a rare, but measurable population of self-Ag-specific T cells in vitro. Autologous dendritic cells pulsed with the melanoma antigen recognized T cells 1 self-peptide were used to stimulate CD8+ T cells from HLA-A2+ healthy donors and melanoma patients. We demonstrate that exposure to IL-21 increased the total number of MART-1-specific CD8+ T cells that could be elicited by >20-fold and, at the clonal level, enriched for a population of high-affinity CD8+ T cells with a peptide dose requirement more than 1 log10–fold less than their untreated counterparts. Phenotypic analysis of T cells from IL-21-treated cultures revealed a unique population of CD45RO+CD28highCD8+ T cells, a phenotype that was stable for at least 4 wk after IL-21 exposure. These CD28highCD8+ T cells produced IL-2 upon Ag stimulation and represent potential helper-independent CTLs. Our studies demonstrate a significant role for IL-21 in the primary Ag-specific human CTL response and support the use of IL-21 in the ex vivo generation of potent Ag-specific CTLs for adoptive therapy or as an adjuvant cytokine during in vivo immunization against tumor Ags.
Expression and Function of Glial Cell Line-Derived Neurotrophic Factor Family Ligands and Their Receptors on Human Immune Cells
Authors: Vargas-Leal Vivian , Bruno Roxana , Derfuss Tobias , Krumbholz Markus , Hohlfeld Reinhard , Meinl Edgar
Published: April 2014

There is increasing evidence that factors originally identified due to their neurotrophic activity also function within the immune system. This study focused on the related molecules glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) as well as their receptors. GDNF and NTN signaling is mediated by a two-component receptor: a signal-transducing component, RET, which is shared by both ligands, and a ligand-specific binding component, GFRα-1 (higher GDNF affinity) or GFRα-2 (higher NTN affinity). We report that human T cells, B cells, and monocytes produce NTN but not GDNF, as seen by RT-PCR and immunocytochemistry. RET was expressed by B cells, T cells, and monocytes. Exons 2–5 of RET encoding the cadherin-like domains 1–3 in the extracellular part and exons 16–19 encoding a section of the second tyrosine kinase domain were transcribed in CD4+ T cells, CD8+ T cells, B cells, and monocytes. Different splice variants encoding the C-terminal intracellular part (exons 19–21) of RET were detected. The ligand-binding receptors GFRα-1 and GFRα-2 were transcribed in all immune cell subsets. Quantitative PCR showed that GFRα-2 is by far the dominant ligand binding chain in T cells, B cells, and monocytes. Addition of GDNF or NTN to activated PBMCs reduced the amount of detectable TNF protein without altering its transcription. Together, this suggests that immune cells communicate with each other via NTN. Production of NTN by immune cells might also contribute to the neuroprotective immunity in the CNS observed in different model systems.
Exacerbation of Experimental Allergic Asthma by Augmented Th2 Responses in WSX-1-Deficient Mice
Authors: Miyazaki Yoshiyuki , Inoue Hiromasa , Matsumura Mikiko , Matsumoto Koichiro , Nakano Takako , Tsuda Miyuki , Hamano Shinjiro , Yoshimura Akihiko , Yoshida Hiroki
Published: April 2014

WSX-1 (IL-27R) is a class I cytokine receptor with homology to gp130 and IL-12 receptors and is typically expressed on CD4+ T lymphocytes. Although previous reports have clarified that IL-27/WSX-1 signaling plays critical roles in both Th1 differentiation and attenuation of cell activation and proinflammatory cytokine production during some bacterial or protozoan infections, little is known about the importance of WSX-1 in cytokine-mediated diseases of allergic origin. To this aim, we took advantage of WSX-1-deficient (WSX-1−/−) mice and induced experimental asthma, in which Th2 cytokines are central modulators of the pathology. OVA-challenged WSX-1−/− mice showed marked enhancement of airway responsiveness with goblet cell hyperplasia, pulmonary eosinophil infiltration, and increased serum IgE levels compared with wild-type mice. Production of Th2 cytokines, which are largely responsible for the pathogenesis of asthma, was augmented in the lung or in the culture supernatants of peribronchial lymph node CD4+ T cells from WSX-1−/− mice compared with those from wild-type mice. Surprisingly, IFN-γ production was also enhanced in WSX-1−/− mice, albeit at a low concentration. The cytokine overproduction, thus, seems independent from the Th1-promoting property of WSX-1. These results demonstrated that IL-27/WSX-1 also plays an important role in the down-regulation of airway hyper-reactivity and lung inflammation during the development of allergic asthma through its suppressive effect on cytokine production.
Heat Shock Protein-70 Mediates the Cytoprotective Effect of Carbon Monoxide: Involvement of p38β MAPK and Heat Shock Factor-1
Authors: Kim Hong Pyo , Wang Xue , Zhang Jinglan , Suh Gee Young , Benjamin Ivor J. , Ryter Stefan W. , Choi Augustine M. K.
Published: April 2014

Carbon monoxide (CO), a product of heme oxygenase activity, exerts antiapoptotic and anti-inflammatory effects in vitro and in vivo. The anti-inflammatory effects of CO involve the inhibition of TNF-α expression and the enhancement of IL-10 production, resulting in reduced mortality after endotoxin challenge. In this study we demonstrate for the first time that the protective effects of CO involve the increased expression of the 70-kDa inducible heat shock protein (Hsp70) in murine lung endothelial cells and fibroblasts. The p38β MAPK mediated the effects of CO on cytoprotection and Hsp70 regulation. Suppression of Hsp70 expression and/or genetic deletion of heat shock factor-1, the principle transcriptional regulator of Hsp70, attenuated the cytoprotective and immunomodulatory effects of CO in mouse lung cells and in vivo. These data provide a novel mechanism for the protective effects of CO and underscore a potential application of this gaseous molecule in anti-inflammatory therapies.
Altered Cytokine Responses of Dengue-Specific CD4+ T Cells to Heterologous Serotypes
Authors: Mangada Maloy M. , Rothman Alan L.
Published: April 2014

The interplay of different inflammatory cytokines induced during a dengue (DEN) virus infection plays a role in either protection or increased disease severity. We measured the frequencies and characterized the cytokine responses of DEN virus-specific memory CD4+ T cells in PBMC of six volunteers who received experimental live attenuated monovalent DEN vaccines. IFN-γ and TNF-α responses to inactivated DEN Ags were detected in up to 0.54 and 1.17% of total circulating CD4+ T cells, respectively. Ags from the homologous serotype elicited the highest IFN-γ response. The ratio of TNF-α- to IFN-γ-producing CD4+ T cells was higher after stimulation with Ags from heterologous DEN serotypes. Peptide-specific CD4+ T cell frequencies of up to 0.089% were detected by direct staining using HLA class II tetramers. IFN-γ and TNF-α responses to individual HLA class II-restricted peptide epitopes were detected in up to 0.05 and 0.27% of CD4+ T cells, respectively. Peptide sequences from the homologous serotype elicited a variety of cytokine response patterns. TNF-α- to IFN-γ-positive CD4+ T cell ratios varied between peptides, but the ratio of the sum of responses was highest against heterologous serotypes. These results demonstrate epitope sequence-specific differences in T cell effector function. These patterns of effector responses may play a role in the immunopathogenesis of DEN hemorrhagic fever.
Spontaneous Immunity against Bcl-xL in Cancer Patients
Authors: Andersen Mads Hald , Reker Sine , Kvistborg Pia , Becker Jürgen C. , Straten Per thor
Published: April 2014

It is well-established that peptide epitopes derived from human tumor-associated Ags can be recognized by CTL in the context of the MHC molecule. However, the vast majority of Ags described are not vital for survival and growth of the tumor cells, and immunoselection of Ag-loss variants during immunotherapy has been demonstrated in several cases. Malfunctions in death pathways observed in human cancers are often due to overexpression of antiapoptotic proteins in the Bcl-2 protein family, i.e., Bcl-2, Mcl-1, and Bcl-xL. These antiapoptotic proteins are implicated in cancer development, tumor progression, and drug resistance. The general overexpression of the antiapoptotic members of the Bcl-2 family in cancer and the fact that down-regulation or loss of expression of these proteins as a means of immune escape would impair sustained tumor growth makes them very attractive targets for anticancer immunotherapy. Recently, we identified spontaneous T cell responses against Bcl-2- and Mcl-1-derived peptides in patients suffering from cancers of different origin. In this study, we demonstrate that Bcl-xL is a target for T cell recognition in cancer patients. Thus, we describe spontaneous HLA-A2-restricted cytotoxic T cell responses against peptide epitopes derived from Bcl-xL by means of ELISPOT and flow cytometry stainings, whereas no responses were detected against any of the Bcl-xL epitopes in any healthy controls. Moreover, Bcl-xL-specific T cells are cytotoxic against HLA-matched cancer cells of different origin. Thus, cellular immune responses against apoptosis inhibitors like the Bcl-2 family proteins appear to represent a general feature in cancer.
TNF Inhibition Rapidly Down-Regulates Multiple Proinflammatory Pathways in Psoriasis Plaques
Authors: Gottlieb Alice B. , Chamian Francesca , Masud Salman , Cardinale Irma , Abello Maria Veronica , Lowes Michelle A. , Chen Fei , Magliocco Melissa , Krueger James G.
Published: April 2014

The mechanisms of action of marketed TNF-blocking drugs in lesional tissues are still incompletely understood. Because psoriasis plaques are accessible to repeat biopsy, the effect of TNF/lymphotoxin blockade with etanercept (soluble TNFR) was studied in ten psoriasis patients treated for 6 months. Histological response, inflammatory gene expression, and cellular infiltration in psoriasis plaques were evaluated. There was a rapid and complete reduction of IL-1 and IL-8 (immediate/early genes), followed by progressive reductions in many other inflammation-related genes, and finally somewhat slower reductions in infiltrating myeloid cells (CD11c+ cells) and T lymphocytes. The observed decreases in IL-8, IFN-γ-inducible protein-10 (CXCL10), and MIP-3α (CCL20) mRNA expression may account for decreased infiltration of neutrophils, T cells, and dendritic cells (DCs), respectively. DCs may be less activated with therapy, as suggested by decreased IL-23 mRNA and inducible NO synthase mRNA and protein. Decreases in T cell-inflammatory gene expression (IFN-γ, STAT-1, granzyme B) and T cell numbers may be due to a reduction in DC-mediated T cell activation. Thus, etanercept-induced TNF/lymphotoxin blockade may break the potentially self-sustaining cycle of DC activation and maturation, subsequent T cell activation, and cytokine, growth factor, and chemokine production by multiple cell types including lymphocytes, neutrophils, DCs, and keratinocytes. This results in reversal of the epidermal hyperplasia and cutaneous inflammation characteristic of psoriatic plaques.
Neutrophils Accelerate Macrophage-Mediated Digestion of Apoptotic Cells In Vivo as Well as In Vitro
Authors: Iyoda Takuya , Nagata Kisaburo , Akashi Makoto , Kobayashi Yoshiro
Published: April 2014

It is generally believed that the clearance of apoptotic cells does not lead to inflammation. In contrast, we previously found that injection of apoptotic cells into the peritoneal cavity induced the expression of an inflammatory chemokine, MIP-2, and infiltration of neutrophils, and that anti-MIP-2 Abs suppressed the infiltration significantly. Because our previous study showed that whole-body x-irradiation caused neutrophil infiltration into the thymus along with T cell apoptosis, we examined the role of neutrophils in apoptotic cell clearance. Neutrophil infiltration reached a peak 12 h after irradiation with 1 Gy of x-rays. Immunohistological analysis revealed that apoptotic cells disappeared dramatically from 10.5 to 12 h after x-irradiation. As neutrophils moved from an inner area of the cortex to the periphery, apoptotic cells disappeared concomitantly. Either anti-MIP-2 or anti-CXCR2 Abs suppressed neutrophil infiltration significantly, and the suppression of neutrophil infiltration by anti-MIP-2 Abs delayed the disappearance of apoptotic cells. Moreover, macrophage-mediated digestion of apoptotic thymocytes was accelerated in vitro on coculturing with neutrophils, even if neutrophils were separated from macrophages. These results suggest that neutrophils are recruited to the thymus mainly by MIP-2 after whole-body x-irradiation and that such neutrophils may not induce inflammation but rather accelerate complete digestion of apoptotic cells by macrophages.
Crk-Associated Substrate Lymphocyte Type Is Required for Lymphocyte Trafficking and Marginal Zone B Cell Maintenance
Authors: Seo Sachiko , Asai Takashi , Saito Toshiki , Suzuki Takahiro , Morishita Yasuyuki , Nakamoto Tetsuya , Ichikawa Motoshi , Yamamoto Go , Kawazu Masahito , Yamagata Tetsuya , Sakai Ryuichi , Mitani Kinuko , Ogawa Seishi , Kurokawa Mineo , Chiba Shigeru , Hirai Hisamaru
Published: April 2014

The lymphocyte-specific Cas family protein Cas-L (Crk-associated substrate lymphocyte type) has been implicated to function in lymphocyte movement, mediated mainly by integrin signaling. However, its physiological role is poorly understood. In this study we analyzed the function of Cas-L in lymphocytes using gene-targeted mice. The mutant mice showed a deficit of marginal zone B (MZB) cells and a decrease of cell number in secondary lymphoid organs. An insufficient chemotactic response and perturbed cell adhesion were observed in Cas-L-deficient lymphocytes, suggesting that the aberrant localization was responsible for the deficit of MZB cells. Moreover, we found that lymphocyte trafficking was altered in Cas-L-deficient mice, which gave a potential reason for contraction of secondary lymphoid tissues. Thus, Cas-L affects homeostasis of MZB cells and peripheral lymphoid organs, which is considered to be relevant to impaired lymphocyte migration and adhesion.
Influence of Xenogeneic β2-Microglobulin on Functional Recognition of H-2Kb by the NK Cell Inhibitory Receptor Ly49C
Authors: Benoit Loralyn A. , Shannon John , Chamberlain John W. , Miller Richard G.
Published: April 2014

NK cells maintain self-tolerance through expression of inhibitory receptors that bind MHC class I (MHC-I) molecules. MHC-I can exist on the cell surface in several different forms, including “peptide-receptive” or PR-MHC-I that can bind exogenous peptide. PR-MHC-I molecules are short lived and, for H-2Kb, comprise ∼10% of total MHC-I. In the present study, we confirm that signaling through the mouse NK inhibitory receptor Ly49C requires the presence of PR-Kb and that this signaling is prevented when PR-Kb is ablated by pulsing with a peptide that can bind to it with high affinity. Although crystallographic data indicate that Ly49C can engage H-2Kb loaded with high-affinity peptide, our data suggest that this interaction does not generate an inhibitory signal. We also show that no signaling occurs when the PR-Kb complex has mouse β2-microglobulin (β2m) replaced with human β2m, although replacement with bovine β2m has no effect. Furthermore, we show that β2m exchange occurs preferentially in the PR-Kb component of total H-2Kb. These conclusions were reached in studies modulating the sensitivity to lysis of both NK-resistant syngeneic lymphoblasts and NK-sensitive RMA-S tumor cells. We also show, using an in vivo model of lymphocyte recirculation, that engrafted lymphocytes are unable to survive NK attack when otherwise syngeneic lymphocytes express human β2m. These findings suggest a qualitative extension of the “missing self” hypothesis to include NK inhibitory receptors that are restricted to the recognition of unstable forms of MHC-I, thus enabling NK cells to respond more quickly to events that decrease MHC-I synthesis.
Enhanced Oral Tolerance in Transgenic Mice with Hepatocyte Secretion of IL-10
Authors: Safadi Rifaat , Alvarez Carlos E. , Ohta Masayuki , Brimnes Jens , Kraus Thomas , Mehal Wajahat , Bromberg Jonathan , Mayer Lloyd , Friedman Scott L.
Published: April 2014

Several cytokines derived from Th3 and Tr1 cells, including IL-10, are believed to regulate oral tolerance, but direct evidence is lacking. We have explored the potential role of IL-10 by generating transgenic (TG) mice with sustained hepatocyte-specific expression of rat IL-10. TG mice expressed rat IL-10 downstream of a transthyretin promoter, which led to serum levels that were increased 10- to 100-fold compared with normal animals. Animals were orally administered 1 mg of whole OVA for 5 consecutive days, with control animals receiving PBS. There were six animal groups: Either OVA or PBS were fed orally to rat IL-10 TG mice, non-TG wild-type mice without IL-10 administration, and non-TG wild-type mice administered rat IL-10 systemically. On day 8, all mice were immunized with two injections of OVA, and then analyzed on day 18. T cell proliferation responses were reduced by 65.8 ± 14.3% after feeding of OVA in rIL-10 TG animals, compared with 39.4 ± 15.6% in the non-TG mice (p = 0.02). Anti-OVA titers were expressed as fold increase over naive non-TG mice. After feeding, titers decreased by ∼33% (from 3- to 2-fold) in TG animals and, to a lesser extent, in non-TG animals. IFN-γ secretion by cultured popliteal lymphocytes decreased in TG animals by 83% after feeding and by 69% in non-TG animals. IL-4 secretion increased 4-fold in TG-fed mice, but did not significantly change in non-TG OVA-fed animals. In contrast to hepatic TG expression of rIL-10, systemic administration of rIL-10 had only a modest effect on tolerance. IL-10, when transgenically expressed in the liver enhances mucosal tolerance to an oral Ag.
Interplay of Cytokines and Microbial Signals in Regulation of CD1d Expression and NKT Cell Activation
Authors: Sköld Markus , Xiong Xiaowei , Illarionov Petr A. , Besra Gurdyal S. , Behar Samuel M.
Published: April 2014

In this study we show that like MHC class I and class II molecules, cell surface CD1d expression on APC is regulated and affects T cell activation under physiological conditions. Although IFN-γ alone is sufficient for optimum expression of MHC, CD1d requires two signals, one provided by IFN-γ and a second mediated by microbial products or by the proinflammatory cytokine TNF. IFN-γ-dependent CD1d up-regulation occurs on macrophages following infection with live bacteria or exposure to microbial products in vitro and in vivo. APC expressing higher CD1d levels more efficiently activate NKT cell hybridomas and primary NKT cells independently of whether the CD1d-restricted TCR recognizes foreign or self-lipid Ags. Our findings support a model in which CD1d induction regulates NKT cell activation.