bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellular Mechanism of Intraabdominal Abscess Formation by Bacteroides fragilis
Authors: Gibson Frank C. , Onderdonk Andrew B. , Kasper Dennis L. , Tzianabos Arthur O.
Published: January 2023

We investigated the cellular mechanism by which Bacteroides fragilis promotes the development of intraabdominal abscesses in experimental models of sepsis. B. fragilis, as well as purified capsular polysaccharide complex (CPC) from this organism, adhered to primary murine mesothelial cells (MMCs) in vitro. The binding of CPC to murine peritoneal macrophage stimulated TNF-α production, which when transferred to monolayers of MMCs elicited significant ICAM-1 expression by these cells. This response resulted in enhanced polymorphonuclear leukocyte attachment to MMCs that could be inhibited by Abs specific for TNF-α or ICAM-1. Mice treated with TNF-α- or ICAM-1-specific Abs failed to develop intraabdominal abscesses following challenge with purified CPC. These results illustrated the role of the CPC in promoting adhesion of B. fragilis to the peritoneal wall and coordinating the cellular events leading to the development of abscesses associated with experimental intraabdominal sepsis.
CD4+ and CD8+ T Cell Interactions in IFN-γ and IL-4 Responses to Viral Infections: Requirements for IL-2
Authors: Su Helen C. , Cousens Leslie P. , Fast Loren D. , Slifka Mark K. , Bungiro Richard D. , Ahmed Rafi , Biron Christine A.
Published: January 2023

Cytokine responses to lymphocytic choriomeningitis virus infections were evaluated, and CD8+ T cell, CD4+ T cell, and IL-2 contributions delineated. In immunocompetent mice, lymphocytic choriomeningitis virus induced both IFN-γ and IL-4 as well as IL-2. Experiments in mice either β2-microglobulin-deficient, lacking MHC class I molecules and CD8+ T cells, or Aβb-deficient, lacking MHC class II molecules and CD4+ T cells, demonstrated that mixtures of T cell responses were required for optimal ex vivo cytokine productions. Intracellular cytokine expression analyses of cells from immunocompetent and immunodeficient mice showed that CD8+ T cells were predominant IFN-γ producers, and that expansion of CD8+ T cells primed to make IFN-γ was independent of CD4+ T cells in vivo. Studies in IL-2-deficient mice demonstrated that this cytokine promoted IFN-γ and IL-4 responses, and ex vivo experiments showed that exogenous IL-2 was required to maintain high-level IFN-γ production by in vivo-primed CD8+ T cells. Conditions associated with cytokine decreases were accompanied by reduced detectable plasma Ab responses. The results indicate that, although IL-2-dependent CD8+ T cell proliferation does not require endogenous CD4+ T cells, IL-2 production by the CD4+ T cells may promote continued cytokine release from activated CD8+ T cells. By defining these critical steps in cellular and cytokine interactions for shaping endogenous immune responses, the studies advance understanding of the unique conditions regulating CD8+ T cell responses to viral challenges.
Adenovirus-Mediated Transfer of Viral IL-10 Gene Inhibits Murine Collagen-Induced Arthritis
Authors: Apparailly Florence , Verwaerde Claudie , Jacquet Chantal , Auriault Claude , Sany Jacques , Jorgensen Christian
Published: January 2023

IL-10 is a potent anti-inflammatory cytokine that has received growing attention for its therapeutic potential. We examined the efficiency of adenoviral-mediated gene transfer of IL-10 on the incidence and severity of murine collagen-induced arthritis (CIA). Male DBA1 mice immunized with collagen II were treated by systemic administration of 109 plaque-forming units of replication-defective adenoviral vector expressing viral IL-10 (vIL-10) on day 30, when clinical symptoms of arthritis start. The transgene was shown to inhibit the onset of CIA, to decrease severity, and profoundly suppress the overall joint histopathology of the experimental arthritis. Significant IL-10 concentrations were obtained in the serum of injected animals for 7 days. Inhibition of arthritis was enhanced by administration of increasing doses of adenovirus-vIL-10. In addition, the local immunosuppressive effect of gene-delivered vIL-10 could be neutralized by a monoclonal anti-vIL-10 Ab. The CIA symptoms in the group treated with the same construct expressing inactive vIL-10 (vIL-10 mut) were similar to those in untreated animals. Our data indicate that a single systemic administration of an adenoviral vector encoding vIL-10 may be a good candidate to suppress arthritis.
Generation of an MHC Class II-Restricted T Cell Epitope by Extracellular Processing of Hepatitis δ Antigen
Authors: Accapezzato Daniele , Nisini Roberto , Paroli Marino , Bruno Guglielmo , Bonino Ferruccio , Houghton Michael , Barnaba Vincenzo
Published: January 2023

Hepatitis δ virus is a human pathogen that is responsible for a severe form of hepatitis affecting hepatitis B envelope Ag carriers. We have previously identified a series of hepatitis δ Ag (HDAg) epitopes that are recognized by CD4+ T cell clones isolated from infected subjects. Herein, we show that the presentation of soluble HDAg to CD4+ T cell clones that are specific for the HDAg(106–121) epitope was exceptionally unaffected by the inhibition of the APC-processing machinery when APCs were fixed with glutaraldehyde before Ag pulsing or treated with chloroquine or brefeldin A. Interestingly, 5 h of pulsing was strictly required for the efficient presentation of the HDAg(106–121) epitope by fixed APCs, suggesting that some form of extracellular processing had occurred. Indeed, fixed APCs were able to present HDAg after only 1 h of pulsing when HDAg was preincubated with serum for 5 h. More important, presentation was completely abrogated when Ag was previously incubated in medium containing serum in the presence of a potent inhibitor of trypsin activity such as the secretory leukoprotease inhibitor. These results show that HDAg may undergo extracellular processing and suggest that the generation of immunogenic epitopes directly by serum proteases could play a role in the immune response against hepatitis δ virus during infection.
Autocrine and Paracrine Apoptosis Are Mediated by Differential Regulation of Fas Ligand Activity in Two Distinct Jurkat T Cell Populations
Authors: Su Xiao , Cheng Jianhua , Liu Weimin , Liu Changdan , Wang Zheng , Yang PingAr , Zhou Tong , Mountz John D.
Published: January 2023

Fas ligand (FasL) produced by activated T cells mediates autocrine-induced apoptosis to limit T cell expansion. To investigate the regulation of FasL activity, Jurkat cells were stably transfected with a 2.3-kb fragment of human FasL promoter that controlled the expression of a GFP reporter gene. Two populations of Jurkat cells with different levels of GFP expression were obtained. One population constitutively expressed high levels of GFP (GFP+), while the other population expressed low levels of GFP (GFP−). The level of GFP expression in the two populations correlated with their levels of FasL transcription and its functional activity. Autocrine regulation of apoptosis was demonstrated by increased FasL activity after stimulation of GFP− cells with anti-CD3, phorbyl myristyl acetate plus ionomycin, or Con A. Paracrine regulation of apoptosis was suggested by the induction of apoptosis of GFP− cells after coculture with unstimulated GFP+ cells. GFP+ cells exhibited a decreased sensitivity to FasL-mediated apoptosis compared with GFP− cells. Furthermore, the cell surface expression of Fas and CD4 was lower on GFP+ cells than GFP− cells, whereas the expression of CD45RO was higher. A decreased level of IL-2 was produced by GFP+ cells after phorbyl myristyl acetate and ionomycin stimulation. Our results indicate that a subpopulation of T cells that express low levels of FasL and IL-2, which are responsive to up-regulation of these molecules after activation, can undergo apoptosis either by suicide after activation or by a paracrine pathway mediated by T cells that constitutively express higher levels of FasL.
Topical FK506 Suppresses Cytokine and Costimulatory Molecule Expression in Epidermal and Local Draining Lymph Node Cells During Primary Skin Immune Responses
Authors: Homey Bernhard , Assmann Till , Vohr Hans-Werner , Ulrich Peter , Lauerma Antti I. , Ruzicka Thomas , Lehmann Percy , Schuppe Hans-Christian
Published: January 2023

Recently, it has been shown that the immunosuppressive macrolide lactone, FK506, exerts good therapeutic efficacy in inflammatory skin diseases. The aim of this study was to analyze the influence of topical FK506 on molecular (IL-1α, IL-1β, IL-2, IL-4, IL-12 p35, IL-12 p40, macrophage inflammatory protein-2 (MIP-2), granulocyte-macrophage CSF (GM-CSF), TNF-α, and IFN-γ) and cellular (I-A+/CD80+, I-A+/CD54+, I-A+/CD69+, I-A+/B220+, and CD4+/CD25+) events in epidermal (EC) and local draining lymph node (LNC) cells during primary contact hypersensitivity responses. Cytokine mRNA levels for IL-1α, IL-1β, GM-CSF, TNF-α, MIP-2, and IFN-γ in EC and for IL-2, IL-4, IL-12 p35, IL-12 p40, and IFN-γ in LNC were increased and resulted in significant LNC proliferation during oxazolone-induced contact hypersensitivity. Topical FK506 treatment dose-dependently suppressed oxazolone-induced LNC proliferation. This effect was correlated with decreased IL-1α, IL-1β, GM-CSF, TNF-α, MIP-2, and IFN-γ mRNA expression within the epidermis and decreased IL-12 p35 and p40 mRNA expression in LNC. Further analysis of the LNC cytokine pattern revealed that the production of both Th1 (IFN-γ and IL-2) and Th2 (IL-4) cytokines was dramatically impaired after topical FK506 treatment. Flow cytometric analysis showed that topical FK506 decreased the population of epidermis-infiltrating CD4+ T cells and suppressed the expression of CD54 and CD80 on I-A+ EC and LNC during hapten-induced contact hypersensitivity. Furthermore, topical FK506 profoundly impaired oxazolone-induced up-regulation of CD25 expression on CD4+ LNC and dramatically decreased hapten-induced expansion of I-A+/B220+ and I-A+/CD69+ LNC subsets. In conclusion, these results give new insights into the mechanisms of action of topical FK506 treatment.
Alloreactive T Cells That Do Not Require TCR and CD8 Coengagement Are Present in Naive Mice and Contribute to Graft Rejection
Authors: Smith Pamela A. , Potter Terry A.
Published: January 2023

Class I alloreactive CTL populations have been defined as either CD8 dependent or CD8 independent, based upon their ability to kill target cells in the presence of Ab to CD8. The CD8-dependent population uses CD8 in a coreceptor role with the TCR, and mutations in the class I molecule that destroy the CD8 binding site abrogate CTL killing, even if the target cell expresses other allelic forms of class I molecules with an intact binding site for CD8. The CD8-independent population apparently does not require CD8, as Ab to CD8 has no effect on the ability of these cells to kill appropriate target cells. We have isolated a third population of CTL that is inhibited by the addition of CD8 Ab yet can kill target cells that express the alloantigenic molecule incapable of binding CD8, provided that the target cells also express non antigenic class I molecules that contain an intact binding site for CD8. We refer to these cells as CD8 bystander-dependent CTL. Many (10 of 12) of these CTL were able to kill H-2Kb-expressing transfectants of T2 cells, consistent with the idea that they recognize a peptide-independent determinant that may be expressed at a high density on the cell surface. These CD8 bystander-dependent CTL are only readily detectable in vitro when spleen cells from mice primed in vivo with a skin graft are used.
Role of the Hypothalamic Pituitary Adrenal Axis and IL-6 in Stress-Induced Reactivation of Latent Herpes Simplex Virus Type 1
Authors: Noisakran Sansanee , Halford William P. , Veress Livia , Carr Daniel J. J.
Published: January 2023

Hyperthermic stress induces reactivation of herpes simplex virus type 1 (HSV-1) in latently infected mice and also stimulates corticosterone release from the adrenals via activation of the hypothalamic pituitary adrenal axis. In the present study, we tested the hypothesis that stress-induced elevation of corticosterone potentiates HSV-1 reactivation in latently infected mice. Because of the putative role of IL-6 in facilitating HSV-1 reactivation in mice, the effect of hyperthermic stress and cyanoketone treatment on IL-6 expression in the trigeminal ganglion was also measured. Preadministration of cyanoketone, a glucocorticoid synthesis inhibitor, blocked the stress-induced elevation of corticosterone in a dose-dependent manner. Furthermore, inhibition of corticosterone synthesis was correlated with reduced levels of HSV-1 reactivation in latently infected mice. Hyperthermic stress elicited a transient rise in IL-6 mRNA levels in the trigeminal ganglion, but not other cytokine transcripts investigated. In addition, there was a significant reduction in MAC-3+, CD8+, and DX5+ (NK cell marker) cells in the trigeminal ganglion of latent HSV-1-infected mice 24 h after stress. Cyanoketone blocked the stress-induced rise in IL-6 mRNA and protein expression in the trigeminal ganglion latently infected with HSV-1. Collectively, the results indicate that the activation of the hypothalamic pituitary adrenal axis plays an important role in stimulating IL-6 expression and HSV-1 reactivation in the trigeminal ganglion following hyperthermic stress of mice.
The Role of CD14 in Signaling Mediated by Outer Membrane Lipoproteins ofBorrelia burgdorferi
Authors: Wooten R. Mark , Morrison Tom B. , Weis John H. , Wright Samuel D. , Thieringer Rolf , Weis Janis J.
Published: January 2023

Borrelia burgdorferi possesses membrane lipoproteins that exhibit stimulatory properties and, consequently, have been implicated in the pathology related to Lyme disease. As CD14 has been shown to mediate signaling by a number of lipid-modified bacterial products, the involvement of CD14 in signaling mediated by two B. burgdorferi lipoproteins, outer surface protein A (OspA) and OspC, was determined. Lipoprotein-mediated induction of nuclear factor-κB nuclear translocation and production of IL-8 and IL-6 in HUVEC was enhanced in the presence of serum or soluble rCD14. CD14-specific Abs that block LPS-mediated signaling also inhibited lipoprotein-dependent signaling in HUVEC and neutrophils. The formation of stable complexes between OspA and CD14 was demonstrated by native gel electrophoresis. LPS was found to compete with OspA for binding with CD14, suggesting that LPS and OspA bind similar regions on CD14. The similarity in binding was further supported by the finding that a mutant soluble CD14, lacking the LPS binding site, did not facilitate lipoprotein signaling, nor did it form a complex with OspA. Binding of OspA to CD14 was dependent on the lipid modification, as unlipidated OspA did not form a complex with CD14 or stimulate cells. In contrast, the lipopeptide remaining after proteinase K digestion both formed a complex with CD14 and retained stimulatory properties. These findings indicate that CD14 facilitates bacterial lipoprotein signaling in mammalian cells.
Differential Requirement for CD4 Help in the Development of an Antigen-Specific CD8+ T Cell Response Depending on the Route of Immunization
Authors: Bour Hélène , Horvath Clotilde , Lurquin Christophe , Cerottini Jean-Charles , MacDonald H. Robson
Published: January 2023

Previous studies in our laboratory have shown that DBA/2 mice injected i.p. with syngeneic P815 tumor cells transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8+CD62L− T cells expressing exclusively the Vβ10 segment. We have used this model to study the regulatory mechanisms involved in the development of the CW3-specific CD8+ response, with respect to different routes of immunization. Whereas both intradermal (i.d.) and i.p. immunization of DBA/2 mice with P815-CW3 cells led to a strong expansion of CD8+CD62L−Vβ10+ cells, only the i.d. route allowed this expansion after immunization with P815 cells transfected with a minigene coding for the antigenic epitope CW3 170–179 (P815 miniCW3). Furthermore, depletion of CD4+ T cells in vivo completely abolished the specific response of CD8+CD62L−Vβ10+ cells and prevented the rejection of P815-CW3 tumor cells injected i.p, whereas it did not affect CD8+CD62L−Vβ10+ cell expansion after i.d. immunization with either P815-CW3 or P815 miniCW3. Finally, the CW3-specific CD8+ memory response was identical whether or not CD4+ T cells were depleted during the primary response. Collectively, these results suggest that the CD8+ T cell response to P815-CW3 tumor cells injected i.p. is strictly dependent upon recognition of a helper epitope by CD4+ T cells, whereas no such requirement is observed for i.d. injection.
Mast Cells Enhance Eosinophil Survival In Vitro: Role of TNF-α and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Levi-Schaffer Francesca , Temkin Vladislav , Malamud Vivian , Feld Sari , Zilberman Yael
Published: January 2023

Mast cell-eosinophil interactions in allergy have not yet been completely defined. To determine whether mast cells influence eosinophil survival, human peripheral blood eosinophils were incubated with rat peritoneal mast cell sonicate. After 3 days, viable eosinophils in medium were 21.3% compared with 44% with mast cell sonicate. Like sonicate, supernatants of compound 48/80-activated mast cells enhanced eosinophil survival, demonstrating that the factor(s) involved is stored preformed and rapidly released. Increased eosinophil survival was due to an inhibition of apoptosis (morphologic analysis; annexin V/PI). Neutralizing Abs to granulocyte-macrophage CSF (GM-CSF), but not to IL-3 or IL-5, decreased by 61.7% the enhancing effect on eosinophil viability. Eosinophils are the source of GM-CSF since its release in the culture medium was inhibited by their incubation with the mast cell sonicate together with dexamethasone. In addition, eosinophils incubated with the sonicate expressed mRNA for GM-CSF. To partially characterize the mast cell-derived factor(s) increasing eosinophil survival, the sonicate was heated (56°C/30 min or 100°C/10 min) or preincubated with antihistamines or with anti-TNF-α-neutralizing Abs. Most of the activity was heat labile. TNF-α was found to be predominantly (70%) responsible, while histamine had no role. Mast cell sonicate also caused eosinophils to release eosinophil peroxidase and to display morphologic signs of activation. In conclusion, we have demonstrated that mast cells enhance eosinophil survival in part through their activation to produce and release the autocrine survival cytokine GM-CSF.
Coengagement of ICAM-3 and Fc Receptors Induces Chemokine Secretion and Spreading by Myeloid Leukocytes
Authors: Kessel Julie M. , Hayflick Joel , Weyrich Andrew S. , Hoffman Patricia A. , Gallatin Michael , McIntyre Thomas M. , Prescott Stephen M. , Zimmerman Guy A.
Published: January 2023

ICAM-3 is expressed at high levels on myeloid leukocytes, but its function on these cells is unknown. We tested the hypothesis that it transduces outside-in proinflammatory signals using immobilized mAbs to engage ICAM-3 on freshly isolated human monocytes and neutrophils. Two immobilized Abs that recognize epitopes in the extracellular domain 1 of ICAM-3, which is critical for recognition by the αL/β2 integrin, potently induced secretion of MIP-1α, IL-8, and MCP-1 by monocytes and triggered IL-8 secretion by neutrophils. These chemokines are products of immediate-early genes that are induced when myeloid cells are activated. Chemokine secretion induced by “triggering” Abs was greater than that induced by isotype-matched immobilized Abs against ICAM-1, ICAM-2, PECAM-1, control Igs, or immobilized control proteins. Coengagement of ICAM-3 and Fc receptors (FcγRI or FcγRII) was required for maximal chemokine secretion by monocytes. Microscopy documented that there is also dramatic spreading of monocytes when surface ICAM-3 is engaged by immobilized Abs. Spreading was induced by Fab and F(ab′)2 fragments of triggering anti-ICAM-3 mAb, demonstrating direct outside-in signaling, but was not required for chemokine secretion. These experiments indicate that ICAM-3 may transmit outside-in signals when it is engaged by β2 integrins during myeloid cell-cell interactions in inflammatory lesions. Binding of Fc receptors by Ig in the local environment can amplify the responses.
Interaction of Vaccinia Virus Complement Control Protein with Human Complement Proteins: Factor I-Mediated Degradation of C3b to iC3b1 Inactivates the Alternative Complement Pathway
Authors: Sahu Arvind , Isaacs Stuart N. , Soulika Athena M. , Lambris John D.
Published: January 2023

Vaccinia virus complement control protein (VCP) is a virulence determinant of vaccinia virus that helps protect the virus from the complement attack of the host. To characterize the interaction of VCP with C3 and C4 and understand the mechanism by which VCP inactivates complement, we have expressed VCP in a yeast expression system and compared the biologic activity of the purified protein to that of human factor H and complement receptor 1 (CR1). Recombinant VCP bound to C3 and the proteolytically cleaved form of C3 (C3b), but not to the 135,300-m.w. fragment of C3 generated using elastase (C3c) and the 35,000-m.w. fragment of C3 generated using elastase (C3d) and inhibited both the classical and alternative pathways of complement activation. Although rVCP was less effective at inhibiting the alternative pathway than factor H or CR1, it was more effective than factor H at inhibiting the classical pathway. Unlike factor H, rVCP was unable discriminate between alternative pathway-mediated lysis of rabbit and sheep E. A comparison of the cofactor activity in factor I-mediated cleavage of C3b suggested that in contrast to factor H and CR1, which displayed cofactor activity for the three sites, rVCP displayed cofactor activity primarily for the first site, leading to generation of C3b cleaved by factor I between Arg1281-Ser1282 (iC3b1). Its cofactor activity for C4b cleavages was similar to that of soluble complement receptor type 1. Purification and functional analysis of iC3b1 showed that it was unable to interact with factor B to form the alternative pathway C3 convertase, C3b,Bb. These results suggest that the interaction of VCP with C3 is different from that of factor H and CR1 and that VCP-supported first cleavage of C3b by factor I is sufficient to render C3b nonfunctional.
Potassium Leakage During the Apoptotic Degradation Phase
Authors: Dallaporta Bruno , Hirsch Tamara , Susin Santos A. , Zamzami Naoufal , Larochette Nathanael , Brenner Catherine , Marzo Isabel , Kroemer Guido
Published: January 2023

The subcellular compartmentalization of ions is perturbed during the process of apoptosis. In this work, we investigated the impact of K+ on the apoptotic process in thymocytes and T cell hybridoma cells. Irrespective of the death-inducing stimulus (glucocorticoids, topoisomerase inhibition, or Fas-crosslinking), a significant K+ outflow was observed during apoptosis, as determined on the single-cell level by means of the K+-sensitive fluorochrome, benzofuran isophtalate. This loss of cytosolic K+ only occurs in cells that have completely disrupted their inner mitochondrial transmembrane potential. Inhibition of this mitochondrial transmembrane potential loss by Bcl-2 or by specific inhibitors acting on the mitochondrial permeability transition pore (bongkrekic acid, cyclosporin A) prevents K+ leakage. K+ drops at the same stage at which cells expose phosphatidylserine residues on the outer leaflet of the membrane and reduce the levels of nonoxidized glutathione, but before they hyperproduce reactive oxygen species, undergo massive Ca2+ influx, shrink, and lyse. In a cell-free system of apoptosis, isolated nuclei exposed to the supernatant of mitochondria that have undergone permeability transition only manifest chromatinolysis when the K+ concentration is lowered from physiologic to apoptotic levels. Accordingly, massive DNA fragmentation causing subdiploidy is confined to cells that have undergone K+ leakage. Together, these data point to the step-wise acquisition of membrane dysfunction in apoptosis and indicate an important role for the disruption of normal K+ homeostasis in apoptotic degradation. Derepression of endonucleases due to low K+ concentrations may be a decisive prerequisite for end-stage DNA fragmentation.
IL-4-Induced Eosinophil Accumulation in Rat Skin Is Dependent on Endogenous TNF-α and α4 Integrin/VCAM-1 Adhesion Pathways
Authors: Sanz Maria-Jesus , Marinova-Mutafchieva Lilia , Green Patricia , Lobb Roy R. , Feldmann Marc , Nourshargh Sussan
Published: January 2023

IL-4 has been implicated in the pathogenesis of a number of allergic inflammatory disease states where the accumulation of eosinophils is a prominant feature. The aim of the present study was to use an isotopic in vivo model to investigate the ability of recombinant rat IL-4 in inducing eosinophil accumulation in rat skin. 111In-eosinophil accumulation in response to intradermally injected IL-4 was measured during 0 to 4 h, 24 to 28 h, and 48 to 52 h. Accumulation was detected during the first two periods, but not at the later time point. The accumulation during 24 to 28 h, which was dose dependent, was investigated in detail. Administration i.v. of an anti-rat VCAM-1 mAb, but not an anti-rat ICAM-1 mAb, inhibited the accumulation of 111In-eosinophils induced by IL-4 (maximum inhibition, 80%). Further, when the 111In-eosinophils were pretreated in vitro with an anti-β2 integrin mAb, an anti-α4 integrin mAb, or a combination of both mAbs, before their injection into recipient rats, the IL-4-induced cell accumulation was inhibited by 63, 60, and 74%, respectively. Finally, coadministration of IL-4 with the soluble TNF receptor (p55)-IgG fusion protein significantly reduced the 111In-eosinophil accumulation induced by the cytokine, and TNF-α was detected in IL-4-injected skin sites by both immunostaining and bioassay. Our results demonstrate that IL-4 is a potent inducer of eosinophil accumulation in vivo, the response being dependent on the endogenous generation of TNF-α, β2 integrins, and α4 integrin/VCAM-1 interactions.
The Effect of Deletion of the V3 Loop of gp120 on Cytotoxic T Cell Responses and HIV gp120-Mediated Pathogenesis
Authors: Kmieciak Dariusz , Wasik Thomas J. , Teppler Hedy , Pientka Janet , Hsu Susan H. , Takahashi Hidemi , Okumura Ko , Kaneko Yutaro , Kozbor Danuta
Published: January 2023

New strategies for improving the efficacy of HIV vaccines are of significant importance. In this study, we analyzed the effect of deletion of the hypervariable V3 loop of gp120 on envelope (env)-specific CTL responses in PBMC of HIV-infected individuals. We showed increased CTL activities against conserved epitopes of the env glycoprotein in cultures induced with the ΔV3 mutant compared with those stimulated with the full-length env gene products. In contrast to the wild-type env, the ΔV3 mutant-expressing cells were resistant to Ab-dependent cell-mediated cytotoxicity, formed no syncytia, and neither underwent nor induced apoptosis in CD4+ cells. Thus, the ΔV3 mutant may redirect immune responses toward conserved epitopes of gp160, has longer expression time due to increased resistance to Ab-dependent cell-mediated cytotoxicity, and does not trigger cytopathic effects associated with apoptosis and syncytium formation. This approach may apply to other Ags of HIV, where deletions of highly variable or immunosuppressive epitopes may improve the efficacy of HIV vaccines.
Targeting the IL-15 Receptor with an Antagonist IL-15 Mutant/Fcγ2a Protein Blocks Delayed-Type Hypersensitivity
Authors: Kim Yon Su , Maslinski Wlodzimierz , Zheng Xin Xiao , Stevens A. Christopher , Li Xian Chang , Tesch Gregory H. , Kelley Vicki R. , Strom Terry B.
Published: January 2023

Owing to shared receptor components, the biologic activities of IL-15 are similar to those of IL-2. However, the patterns of tissue expression of IL-2/IL-2Rα and IL-15/IL-15Rα differ. The development of agents targeting the receptor and signaling elements of IL-15 may provide a new perspective for treatment of diseases associated with expression of IL-15/IL-15R. We designed, genetically constructed, and expressed a receptor site-specific IL-15 antagonist by mutating glutamine residues within the C terminus of IL-15 to aspartic acid and genetically linked this mutant IL-15 to murine Fcγ2a. These mutant IL-15 proteins specifically bind to the IL-15R, competitively inhibit IL-15-triggered cell proliferation, and do not activate the STAT-signaling pathway. Because the receptor site-specific antagonist IL-15 mutant/Fcγ2a fusion proteins had a prolonged t1/2 in vivo and the potential for destruction of IL-15R+ leukocytes, we examined the immunosuppressive activity of this agent. An IL-15 mutant/Fcγ2a fusion protein markedly attenuated Ag-specific delayed-type hypersensitivity responses and decreased leukocyte infiltration within the delayed-type hypersensitivity sites. These findings suggest that 1) IL-15/IL-15R+ cells are crucial to these T cell-dependent immune responses, and 2) treatment with IL-15 mutant/Fcγ2a protein may ameliorate T cell-dependent immune/inflammatory diseases.
Expanding Dendritic Cells In Vivo Enhances the Induction of Oral Tolerance
Authors: Viney Joanne L. , Mowat Allan M. , O’Malley Jamie M. , Williamson Eilidh , Fanger Neil A.
Published: January 2023

The intestine is under perpetual challenge from both pathogens and essential nutrients, yet the mucosal immune system is able to discriminate effectively between harmful and innocuous Ags. It is likely that this selective immunoregulation is dependent on the nature of the APC at sites where gut Ags are processed and presented. Dendritic cells (DC) are considered the most potent of APC and are renowned for their immunostimulatory role in the initiation of immune responses. To investigate the role of DC in regulating the homeostatic balance between mucosal immunity and tolerance, we treated mice with Flt3 ligand (Flt3L), a growth factor that expands DC in vivo, and assessed subsequent systemic immune responsiveness using mouse models of oral tolerance. Surprisingly, mice treated with Flt3L to expand DC exhibited more profound systemic tolerance after they were fed soluble Ag. Most notably, tolerance could be induced in Flt3L-treated mice using very low doses of Ag that were ineffective in control animals. These findings contrast with the generally accepted view of DC as immunostimulatory APC and furthermore suggest a pivotal role for DC during the induction of tolerance following mucosal administration of Ag.
High Frequency Apoptosis of Recent Thymic Emigrants in the Liver of Lymphopenic Diabetes-Prone BioBreeding Rats
Authors: Iwakoshi Neal N. , Goldschneider Irving , Tausche Frances , Mordes John P. , Rossini Aldo A. , Greiner Dale L.
Published: January 2023

Diabetes-prone (DP) BioBreeding (BB) rats develop spontaneous autoimmune diabetes. DP-BB thymocyte export is reduced, and most thymic emigrants disappear rapidly from peripheral lymphoid tissues. DP-BB rats are consequently lymphopenic and circulate severely reduced numbers of T cells. Peripheral T cells present are phenotypically immature (Thy1+) and appear activated. We hypothesized that DP-BB recent thymic emigrants have a shortened life span and disappear by apoptosis. The percentage of T cells with an αβTCRlowB220+CD4−CD8− phenotype was increased in DP peripheral lymphoid tissues when compared with normal, nonlymphopenic diabetes-resistant (DR) BB rat tissues. There was no evidence of DNA fragmentation in freshly isolated DP- or DR-BB rat cells, but, after 24 h of culture, a higher proportion of DP- than DR-BB splenic T cells underwent apoptosis. We then tested the hypothesis that BB rat T cells with the αβTCRlowB220+CD4−CD8− phenotype accumulate and undergo apoptosis in the liver. Such cells were observed undergoing apoptosis in both DP- and DR-BB rats, but comprised ∼80% of intrahepatic T cells in DP vs ∼20% in DR-BB rats. Most αβTCRlowB220+CD4−CD8− cells in the liver were also Thy1+. The data suggest that T cell apoptosis in the DP-BB rat is underway in peripheral lymphoid tissues and is completed in the liver. Increased intrahepatic apoptosis of recent thymic emigrants appears in part responsible for lymphopenia in DP-BB rats and the concomitant predisposition of these animals to autoimmunity.
Exogenous and Endogenous IL-10 Regulate IFN-α Production by Peripheral Blood Mononuclear Cells in Response to Viral Stimulation
Authors: Payvandi Faribourz , Amrute Sheela , Fitzgerald-Bocarsly Patricia
Published: January 2023

IL-10 is an important regulator of the production of proinflammatory cytokines. Its effect on IFN-α production, however, has not been reported. In this study, PBMC from healthy donors were stimulated with virus in the presence of IL-10. Human IL-10 (hIL-10) caused reductions in both the frequency of IFN-α-producing cells (IPC) and bulk IFN in response to herpes simplex virus type-1 (HSV-1), Sendai virus, Newcastle disease virus, and vesicular stomatitis virus. The inhibitory effect occurred when IL-10 was added 2 or 4 h before, or 2 h poststimulation with HSV or Sendai virus, but not when added 4 h postinduction. Unlike IL-10, IL-4 did not affect the IFN-α response to HSV. However, when PBMC were induced with Sendai virus, IFN-α production was also reduced by IL-4. IL-10 treatment of PBMC resulted in strong reductions in the steady state levels of both HSV- and Sendai virus-induced IFN-α1, -α2, and -β mRNA as determined by RT-PCR. IFN-α production to Sendai virus occurs predominantly by monocytes, whereas most enveloped viruses stimulate low frequency “natural IFN-producing cells (NIPC),” which are thought to be dendritic cells. Peripheral blood dendritic cells were found to express the IL-10 receptor, suggesting that IL-10 may directly act on the dendritic IPC. Addition of monoclonal anti-IL-10 to PBMC resulted in a significant increase in both the frequency of IPC and the amount of secreted IFN-α in response to HSV but not Sendai virus. We conclude that human IL-10 can serve as both an endogenous and exogenous regulator of IFN-α production.
A Pathogenic Role of Th2 Cells and Their Cytokine Products on the Pulmonary Metastasis of Murine B16 Melanoma
Authors: Kobayashi Makiko , Kobayashi Hiroyuki , Pollard Richard B. , Suzuki Fujio
Published: January 2023

The role of Th2 cells and the cytokines produced by these cells on experimental pulmonary metastasis of B16 melanoma was investigated in a murine model implanted with high metastatic (B16F10) or low metastatic (B16F1) melanoma cells. An average of 250 colonies of metastasis in the lungs was counted in mice (BF10 mice) at 14 days after the inoculation of 2 × 105 B16F10 cells/mouse, while <20 colonies were detected in mice (BF1 mice) inoculated with the same number of B16F1 cells. CD4+CD11b+TCR-αβ+ T cells (BF10-Th2 cells) were produced in the spleens of BF10 mice, while these cells were not detected in BF1 mice. The BF10-Th2 cells produced IL-4 and IL-10 into culture fluids when stimulated in vitro with anti-CD3 mAb. However, IL-2 and IFN-γ were not produced. The level of a pulmonary metastasis in BF1 mice increased to the level observed in BF10 mice, when BF10-Th2 cells were adoptively transferred to BF1 mice. Also, an increase in the number of pulmonary melanoma was demonstrated in BF1 mice treated with 10 μg/kg murine rIL-4. The level of pulmonary metastasis in BF10 mice or in BF1 mice inoculated with BF10-Th2 cells decreased to the level observed in BF1 mice when mice were treated with an anti-IL-4 mAb at a dose of 250 μg/kg on days 1, 3, and 5 after tumor inoculation. These results suggest that the severity of pulmonary metastasis in mice receiving B16 melanoma cells is strongly influenced by the IL-4 released from tumor-associated Th2 cells.
CpG Oligodeoxyribonucleotides Rescue Mature Spleen B Cells from Spontaneous Apoptosis and Promote Cell Cycle Entry
Authors: Yi Ae-Kyung , Chang Ming , Peckham David W. , Krieg Arthur M. , Ashman Robert F.
Published: January 2023

Isolated murine splenic B cells undergo spontaneous apoptosis. Motifs containing unmethylated CpG dinucleotides in bacterial DNA or in synthetic oligodeoxynucleotides (ODN) are known to activate murine B cells. Now we show that ODN that induce spleen B cell cycle entry also inhibit spontaneous apoptosis in a sequence-specific fashion. Reversal of the CG to GC abolished activity. Methylation of the central cytosine decreased activity. When CpG is preceded by a cytosine or followed by a guanine, activity was abolished. Other substitutions at the same positions had no effect. Dose-response curves for apoptosis protection and G1 entry suggested that a uniform population of ODN recognition sites controlled downstream ODN effects. A CpG ODN with a nuclease-resistant phosphorothioate backbone (S-ODN) was also active, and increased the levels of c-myc, egr-1,c-jun, bclXL, and bax mRNA and c-Myc, c-Jun, Bax, and BclXL protein in spleen B cells. Levels of c-myb, myn, c-Ki-ras, and bcl2 mRNA remained unchanged. When protein synthesis was inhibited, at 16 h ODN-induced cell cycle entry was abolished and apoptosis protection was partially preserved. Under these conditions, c-Myc was still present, but c-Jun and BclXL were not detected. Our results suggest that CpG containing ODN motifs provide signals for both survival and cell cycle entry. Single base changes determine whether this signal proceeds through a rate-limiting step governing at least two steps in apoptosis (plasma membrane transition, DNA cleavage) and two phases of the cell cycle (G1 and S phase entry). This biologic action is associated with increased c-Myc, c-Jun, and BclXL expression.
The Regulation of Murine H-2Dd Expression by Activation Transcription Factor 1 and cAMP Response Element Binding Protein
Authors: Ishiguro Nobuhisa , Brown G. Dalon , Ishizu Akihiro , Meruelo Daniel
Published: January 2023

Resistance to radiation leukemia virus (RadLV)-induced leukemia is correlated with an increase in H-2Dd expression on the thymocyte surface. It has been shown that elevated H-2Dd expression on infected thymocytes is a result of elevated mRNA transcription and that the transcriptional increase is correlated with elevated levels of a DNA binding activity, H-2 binding factor 1 (H-2 BF1), which recognizes the 5′-flanking sequence (5′-TGACGCG-3′) of the H-2Dd gene. Recently, it has been shown that the activation transcription factor 1 (ATF-1) homodimer is one form of the H-2 BF1 complex. Here we demonstrate that the cAMP response element binding protein (CREB) homodimer and the heterodimer of CREB/ATF-1 also recognize the cis regulatory motif and are two additional forms of the H-2 BF1 complex. The levels of mRNA encoding ATF-1 and CREB were both increased in RadLV-infected thymocytes that showed increased levels of H-2 mRNA. Also, all three H-2 BF1 binding activities, ATF-1 homodimer, CREB homodimer, and ATF-1/CREB heterodimer, were increased in RadLV-infected thymocytes that expressed high levels of H-2Dd Ag on the cell surface. Transfection experiments demonstrated that ATF-1 and CREB activated a reporter plasmid containing the H-2 BF1 motif. These observations strongly suggest that both ATF-1 and CREB are involved in the regulation of H-2 gene expression following RadLV infection of mouse thymocytes.
The Effect of γδ T Cell Depletion on Cytokine Gene Expression in Experimental Allergic Encephalomyelitis
Authors: Rajan Alice J. , Klein Jonathan D. S. , Brosnan Celia F.
Published: January 2023

In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, we showed previously that depletion of γδ T cells using the mAb GL3 immediately before disease onset, or during the chronic phase, significantly ameliorated clinical severity. We now report on the effect of γδ T cell depletion on expression of five cytokine genes, IL-1, IL-6, TNF, lymphotoxin, and IFN-γ in spinal cords of mice during the pre-onset, onset, height, and recovery phases of EAE, and on expression of type II nitric oxide synthase. In control animals, the mRNAs for IL-1 and IL-6 rose dramatically at disease onset and peaked before disease height, whereas the mRNAs for TNF, lymphotoxin, and IFN-γ rose more slowly and peaked with peak of disease. In GL3-treated animals, a dramatic reduction in all five cytokines was noted at disease onset, but only IFN-γ remained significantly reduced at a time point equivalent to height of disease in control animals. ELISA data confirmed the reduced levels of IL-1 and IL-6 at disease onset in GL3-treated animals, and pathologic analysis demonstrated a marked reduction in meningeal infiltrates at the same time point. Studies of type II NOS also demonstrated a significant reduction in both mRNA and protein expression at the height of disease in GL3-treated animals. These results suggest that γδ T cells contribute to the pathogenesis of EAE by regulating the influx of inflammatory cells into the spinal cord and by augmenting the proinflammatory cytokine profile of the inflammatory infiltrates.
Role of Mucosal Immunity in Herpes Simplex Virus Infection
Authors: Kuklin Nelly A. , Daheshia Massoud , Chun Sangjun , Rouse Barry T.
Published: January 2023

This study evaluates whether the vaginal mucosal surface of immunized mice can prevent invasion by herpes simplex virus (HSV) and aims to identify immune components that affect immunity after challenge at the vaginal mucosa. Despite the induction of both IgA and IgG vaginal Ab following immunization with recombinant vaccinia virus vectors expressing either glycoproteins B or D, viral infection occurred in most animals even after minimal viral dose challenge. Challenged immune animals, including those genetically unable to generate anti-HSV Ab, survived and showed few if any clinical signs of infection. Experiments with T cell subtype knockout animals and depletion with T cell subset-specific MAb indicated that immunity following vaginal challenge was principally dependent on the function of CD4+ T cells. Our results indicate that anti-HSV vaccines may not provide barrier immunity at the vaginal mucosal site but may be adequate to minimize clinical expression of disease.
Mucosal Addressin Is Required for the Development of Diabetes in Nonobese Diabetic Mice
Authors: Hänninen Arno , Jaakkola Ilkka , Jalkanen Sirpa
Published: January 2023

Immune responses are best initiated in the environment of lymphoid tissues wherein circulating lymphocytes enter by interacting with endothelial adhesion molecules. In type 1 diabetes, immune responses against pancreatic islets develop, but the environment in which this occurs remains unidentified. To determine whether lymphocyte homing to lymphoid organs is involved in the pathogenesis of diabetes in nonobese diabetic (NOD) mice, we blocked the function of the mucosal addressin cell adhesion molecule-1 (MAdCAM-1), which is a vascular addressin-mediating lymphocyte homing into mucosal lymphoid tissues, in these mice. While ineffective if started later, a blockade started at 3 wk of age reduced the incidence of diabetes from 50% to 9% (p < 0.01). This finding is associated with Peyer’s patch atrophy, a marked decrease of naive (CD44lowCD45RBhigh) T lymphocytes, and a reduction in the relative numbers of memory (CD44high) T lymphocytes in the spleen. The potential of these spleen cells to cause diabetes was diminished. Anti-MAdCAM-1 treatment also inhibited both lymphocyte entry into the pancreas and diabetes development in NOD/SCID recipients after the transfer of lymphocytes derived from the mesenteric lymph nodes of young, but not of diabetic, NOD donors. Therefore, MAdCAM-1 may be required during two distinct steps in an early phase of diabetes development: for the entry of naive lymphocytes into the lymphoid tissues in which diabetes-causing lymphocytes are originally primed, and for the subsequent homing of these lymphocytes into the pancreas. The role of MAdCAM-1 as a mucosal vascular addressin suggests that mucosal lymphoid tissues are involved in the initiation of pathologic immune responses in NOD mice.
The Antibody Response to Fungal Melanin in Mice
Authors: Nosanchuk Joshua D. , Rosas Angel L. , Casadevall Arturo
Published: January 2023

Melanins are associated with virulence in several important human pathogens, but little is known about the immune response to this ubiquitous biologic compound. We hypothesized that melanin produced by the fungus Cryptococcus neoformans was immunogenic. C. neoformans melanin was purified from melanized fungal cells and was used to immunize C57BL/6, BALB/c, and T cell-deficient (nude) BALB/c mice. The Ab response was evaluated by ELISA, immunofluorescence, and agglutination. The results demonstrate that melanin can be immunogenic, and the humoral immune response is T cell independent. Furthermore, the experiments demonstrate 1) a sensitive ELISA for the measurement of Ab to melanin, 2) that mice mount an intense Ab response to fungal melanin that includes Abs of IgM and IgG isotypes, 3) that melanins from different sources have cross-reactive epitopes, and 4) melanin in the cell wall of melanized yeast cells reacts with Abs raised to l-dopa C. neoformans melanin. The biologic significance of Ab to melanin remains to be determined, but the development of Ab suggests that this amorphous insoluble polymer can stimulate the immune system. The serologic techniques described here may prove useful for the evaluation of Ab responses to melanin in a variety of diseases.
Clostridium difficile Toxin A Stimulates Macrophage- Inflammatory Protein-2 Production in Rat Intestinal Epithelial Cells
Authors: Castagliuolo Ignazio , Keates Andrew C. , Wang Chi Chung , Pasha Asiya , Valenick Leyla , Kelly Ciaran P. , Nikulasson Sigfus T. , LaMont J. Thomas , Pothoulakis Charalabos
Published: January 2023

Neutrophil infiltration of the colonic mucosa is a hallmark of Clostridium difficile toxin A-mediated enterocolitis. Macrophage-inflammatory protein-2 (MIP-2) is a potent neutrophil chemoattractant secreted by rat macrophages and epithelial cells in response to inflammatory stimuli. In this work, we report that administration of toxin A into rat ileal loops increased mucosal levels of MIP-2 before the onset of fluid secretion and mucosal neutrophil infiltration. Administration of rabbit anti-MIP-2 IgG, but not control IgG, reduced toxin A-mediated secretion (by 58%), mucosal permeability (by 80%), and myeloperoxidase activity (by 85%). Immunohistochemical analysis demonstrated increased MIP-2 expression in intestinal epithelial and lamina propria cells 1 h after toxin A administration. Intestinal epithelial cells purified from toxin A-exposed ileal loops also showed increased MIP-2 mRNA expression and MIP-2 protein release that was inhibited by pretreatment of rats with the transcriptional inhibitor actinomycin D. These results indicate that C. difficile toxin A induces MIP-2 release from intestinal epithelial cells and that MIP-2 contributes to neutrophil mucosal influx during toxin A enteritis.
Th1 Cells Specific for a Secreted Protein ofListeria monocytogenesAre Protective In Vivo
Authors: Geginat Gernot , Lalic Mio , Kretschmar Marianne , Goebel Werner , Hof Herbert , Palm Dieter , Bubert Andreas
Published: January 2023

In the present study we have investigated the role of the secreted p60 protein from Listeria monocytogenes as an Ag for CD4 T cells. The p60 protein is an abundant extracellular protein that is highly conserved within the members of the genus Listeria. Our results show that L. monocytogenes infection induces a potent p60-specific Th1 immune response. Remarkably, we found that p60-specific Th1 clones mediate significant protection against L. monocytogenes infection. For one p60-specific clone, the peptide epitope was defined. This clone recognized p60 301-312 (EAAKPAPAPSTN) in the context of the H-2Ad molecule. Despite the fact that acquired immunity against L. monocytogenes is primarily mediated by cytotoxic CD8 T lymphocytes, our data clearly demonstrate that secreted bacterial proteins are important CD4 T cell Ags and that Th1 clones specific for a secreted bacterial protein can contribute to the protection against an intracellular pathogen such as L. monocytogenes.